Effects of Source and Concentration on Relative Oral Bioavailability of

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Effects of Source and Concentration on Relative Oral Bioavailability of Benzo(a)pyrene from Soil Stephen M. Roberts, John W. Munson, Michael V. Ruby, and Yvette W. Lowney Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01534 • Publication Date (Web): 12 Sep 2016 Downloaded from http://pubs.acs.org on September 14, 2016

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Environmental Science & Technology

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Effects of Source and Concentration on Relative Oral Bioavailability of Benzo(a)pyrene from Soil

10 Stephen M. Roberts, *† John W. Munson,† Michael V. Ruby,§ and Yvette Lowney††

11 12 13 14 15 16

†

Center for Environmental & Human Toxicology, University of Florida, Gainesville, FL 32611, United States; §

Integral Consulting, Inc., Louisville, CO 80027, United States; and ††

Alloy, LLC, Boulder, CO 80302, United Stated

17 18 19 20

* Corresponding author email: [email protected]; phone: (352) 294-4514; FAX: (352) 392-4707

21

ACS Paragon Plus Environment

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Abstract

23

The objective of this study was to examine the influence of soil composition, PAH

24

concentration, and source material type on PAH bioavailability using an approach

25

capable of measuring uptake at low, environmentally relevant PAH concentrations (down

26

to 1 ppm). Contaminated soil samples were constructed using PAHs from three source

27

materials —solvent, soot, and fuel oil— to which 3H-benzo(a)pyrene (3H-BaP; total BaP

28

concentrations of 1, 10, and 100 ppm) was added in a mixture of PAHs. The soils were

29

weathered for eight weeks using weekly wet-dry cycles. Each soil was administered as

30

a single dose to rats, and blood samples were taken over six days.

31

bioavailability (RBA) of the BaP from soil was estimated by comparing the area under

32

the curve (AUC) for 3H concentration versus time in blood with the AUC observed from

33

the same PAH mixture dosed in a food matrix. The extent to which BaP RBA was

34

diminished in soil versus food varied among the source materials, but little or no

35

difference was observed among the soil types examined unless carbon amendments

36

were added. These results suggest that the type of PAH source material can have a

37

strong influence on PAH oral bioavailability.

Relative oral

38


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Introduction

40

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and

41

commonly identified as chemicals of concern in soil at contaminated sites. Several

42

PAHs are considered potential human carcinogens,1-3 and estimated risk of cancer from

43

PAH exposure typically drives cleanup decisions for these sites. In current exposure

44

models for human contact with soil, the dominant pathway for contaminant intake is

45

through incidental ingestion. In laboratory animals given a PAH such as benzo(a)pyrene

46

(BaP) orally, tumors occur at a variety of sites, including sites away from the

47

gastrointestinal tract.4

48

systemically, and that the risk of cancer is therefore related to the amount of PAH that is

49

absorbed after ingestion.

This indicates that PAHs are capable of producing cancer

Several studies have shown that the absorption of PAHs from soil is incomplete.5-

50 51

11

Although there are limitations in the approaches used in a number of studies,12 there is

52

ample evidence that determining the bioavailability from soil is important for accurate

53

estimation of potential human exposure.

54

determining the extent to which PAH absorption from a specific contaminated soil is

55

reduced compared with the absorption that occurred under the conditions of the critical

56

study used to determine the oral cancer potency of PAHs. Currently, the cancer potency

57

estimates used by the U.S. EPA for all carcinogenic PAHs are derived from the cancer

58

potency estimate for the “index” carcinogenic PAH, BaP.13 The BaP cancer potency

59

estimate, in turn, is based on tumor responses observed in two chronic rodent bioassays

60

in which BaP was given in the diet.14-15 Thus, the information needed to adjust risk

61

estimates for carcinogenic PAHs in soil is the bioavailability of the PAH from soil relative

62

to its bioavailability from food, i.e., its relative bioavailability (RBA).

The specific issue for risk assessment is

63

There are a number of studies that have estimated the RBA of carcinogenic

64

PAHs such as BaP in soil, including studies that investigated the effect of PAH sources

65

and soil characteristics (see Ruby et al. for a recent review).12 However, an important

66

limitation of all is the use of animal models that require PAH concentrations in the tens of

67

ppm (or higher) to be able to measure bioavailability. This issue is described more fully

68

in Ruby et al 2016,12 and is illustrated by a recent PAH bioavailability study where RBA

69

was not measureable in animals using contaminated site soils (BaP up to 300 mg/kg),

70

and only quantifiable in spiked soils containing BaP concentrations in the range of 1000

71

or 10,000 mg/kg.16 In contrast, risk-based cleanup goals for carcinogenic PAHs are


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often 1 ppm or less.

73

concentrations orders of magnitude higher than concentrations for which regulatory

74

decisions must be made. There is currently no validation that RBA results obtained from

75

these highly contaminated soils are applicable to more common, environmentally

76

relevant concentrations.

As a consequence, RBA measurements are made at PAH

77

The ability to obtain estimates of PAH RBA at low, environmentally relevant

78

concentrations could conceivably be achieved with a predictive in vitro bioaccessibility

79

test, and an in vitro approach would have additional benefits of providing RBA

80

information more rapidly and at lower cost than an RBA measurement in vivo. However,

81

assessment of the performance of candidate in vitro tests requires a series of soil

82

samples for which the RBA has been determined through in vivo measurement. This

83

suite of soil samples should ideally include different soil types and PAHs in different

84

concentrations from different source materials. The typical approach to assembling a

85

suite of soil samples for this purpose is to collect samples from a limited number of

86

contaminated sites and measure RBA in vivo through conventional means.

87

disadvantage of this approach is that soil samples are usually selected largely by

88

convenience, limiting the ability to examine the influence of key variables on

89

bioavailability. Additionally, in the case of PAHs, RBA values can only be obtained for

90

soils with relatively high PAH concentrations.

The

91

As an initial step in the development of an in vitro PAH RBA estimation method, a

92

different approach was taken in this study, using constructed rather field soils from

93

contaminated sites. This study focused on the index carcinogenic PAH, BaP, and test

94

soils were created to differ independently with respect to three variables potentially

95

affecting BaP bioavailability from soil, viz. BaP concentration, BaP source material, and

96

soil composition. To enable measurement of BaP RBA at concentrations extending

97

down into the range commonly encountered at contaminated sites (i.e., to 1 ppm), a

98

radiolabeled BaP tracer was included. These soils were weathered to simulate

99

conditions that occur in the environment, and RBA was measured relative to food using

100

a rat model. Table 1 shows the “matrix” of factors incorporated into the design of the soil

101

study substrates. In addition to creating benchmark soil samples for in vitro method

102

development, this study provides new information on the effect of BaP source and

103

concentration on its bioavailability from soil. It also raises interesting questions about


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how RBA information should be applied to PAH concentrations measured at

105

contaminated sites using conventional solvent extraction sample preparation.

106

Materials and Methods

107

Animal Model.

108

radiolabeled BaP was incorporated into the test soils prior to weathering. Tritium was

109

used as the radiolabel because this material provides a high specific activity allowing

110

bioavailability measurements of BaP in ingested material down to the sub-ppm

111

concentration range.17 The use of radiolabel also allows both parent BaP and

112

metabolites appearing in blood to be quantified in a straightforward manner. Systemic

113

absorption was determined by measuring the area under the curve (AUC) for the blood

114

concentration versus time profile.

115

systemic oral bioavailability, the AUC approach avoids potential problems in estimation

116

of absorption of PAHs that occur with other methods such as measuring PAH

117

metabolites in urine.12 A critical assumption in relative bioavailability determinations is

118

that the clearance of the substance is the same for the doses that are being compared.

119

BaP and other PAHs are capable of inducing their own metabolism, making this

120

assumption difficult to meet with repeated doses.12 To avoid this problem, relative oral

121

bioavailability was determined after a single BaP dose. As noted in the Introduction, the

122

cancer potency estimate for BaP developed by the USEPA is based upon studies in

123

which BaP was administered in food. The appropriate basis for determining RBA of BaP

124

in soil was therefore comparison with bioavailability from food, specifically rodent chow

125

as used in the BaP cancer studies.

In order to be able to measure BaP bioavailability at low doses,

Considered the classical method of measuring

126

The rat was selected as the animal model for this study for a number of reasons.

127

The rat is well established as model for bioavailability studies. In fact, for determining

128

bioavailability of organic compounds in drug development, the rat is second only to

129

humans in frequency of use,18 and is one of the two species from which the BaP cancer

130

slope factor was derived. The rat is large enough to provide several blood samples of

131

reasonable volume over time from which a blood concentration versus time profile can

132

be obtained, yet small enough so that large amounts of radiolabel are not required and

133

radioactive waste from excreta can be easily managed.

134

Male Sprague-Dawley rats (approx. 300 g bw) were obtained from Harlan

135

Laboratories (Fredrick, MD). The animals were purchased from the vendor with


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surgically placed jugular catheters to facilitate blood collection. A polyurethane catheter

137

had been placed in the right jugular vein under ketamine/xylazine anesthesia, and the

138

catheter was tunneled subcutaneously and exteriorized dorsally between the scapulae.

139

The catheter was locked with sterile heparin glycerol solution so as to remain patent.

140

Upon receipt, the animals were housed individually in polycarbonate cages with

141

controlled light/dark cycles of 12 hours (08:00-20:00) in temperature- and humidity-

142

controlled animal quarters. Animals were fed a standard diet (Teklad Rodent Diet 8406,

143

Harlan Laboratories) and had free access to water. This study was approved by the

144

Institutional Animal Care and Use Committee and animals were treated according to

145

criteria in the NIH Guide for the Care and Use of Laboratory Animals.

146

Chemicals.

147

Radiolabeled Chemicals Inc. (St. Louis, MO).

148

verified by radiochromatography and was found to be 99%.

149

purchased from Sigma-Aldrich (St. Louis, MO). Other polycyclic aromatic hydrocarbons

150

were obtained from Ultra Scientific (Kingstown, RI). Soluene 350®, ethanol, hydrogen

151

peroxide (30%), sodium sulfate, dichloromethane, methanol, acetone, ScintiVerse®

152

scintillation cocktail, soot, and charcoal were purchased from Fisher Scientific

153

(Pittsburgh, PA). Fuel oil (Fuel Oil No. 6) was provided by Chevron (San Ramon, CA),

154

kaolinite and montmorillonite clay were from ACROS Organics (through Fisher Scientific,

155

Pittsburgh, PA), sand (SIL-CO-SIL®) was obtained from U.S. Silica (Frederick, MD),

156

peat was from Miracle-Gro (Marysville, OH), and humus was from Organic Valley (La

157

Farge, WI).

158

Food and soil sample preparation. Soils were constructed consistent with an ASTM

159

standard soil developed for the testing of toxicity to terrestrial organisms (ASTM E1676-

160

12). This “Baseline Synthetic Soil (BSS)” consisted of 70% sand, 20% kaolinite clay,

161

and 10% peat. Some samples were modified to reduce peat or clay content, or to add

162

charcoal or replace peat with humus (Table 1).

163

pulverizing rodent chow pellets to a fine powder. Each constructed soil was thoroughly

164

mixed, air dried, and sieved to 150 µm. This particle size range was recently identified

165

as the most appropriate soil fraction to assess in understanding human exposures to

166

PAHs in soil.19 A mixture of PAHs, including BaP, was added to each soil and to food to

167

create a consistent profile, i.e., in constant proportions among individual PAHs. Target

168

PAH concentrations in test soils spanned two orders of magnitude, with BaP

3

H-Benzo(a)pyrene

(20

Ci/mmol)

was

obtained

from

American

Purity of the radiolabeled BaP was BaP (unlabeled) was

Food material was prepared by


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concentrations set at 1, 10, and 100 ppm (Table S1). PAHs were added to soil in a

170

variety of “source materials,” including solvent (dichloromethane), soot, or fuel oil, and in

171

solvent to food. The soot and fuel oil naturally contained PAHs (Table S2), and these

172

concentrations were taken into account in determining the amounts of various PAHs

173

added to food and soil to achieve the target concentrations. Some of the added BaP

174

was labeled with tritium (3H) such that each soil and food sample had an initial

175

concentration of 3H-BaP of 50 µCi/g.

176

Addition of the PAHs to soil and food was as follows. For soils with a solvent

177

source, mixtures of PAHs as shown in Table S1 were prepared in 10 ml

178

dichloromethane and added to a 100 ml glass bottle. The liquid was swirled gently so

179

that the wall of vessel was coated with liquid. Soil (20 g) was immediately added, and

180

the bottle was capped and placed on a roller for 24 hours. The bottle was then opened

181

and the soil was allowed to air dry for 24 hours at room temperature. The soil was

182

disaggregated with a spatula until it appeared homogenous and then mixed on a roller

183

for 24 hours. The same procedure was followed for addition of PAHs to food. For soils

184

where soot served as the source of PAHs, a mixture of PAHs in dichloromethane was

185

added to soot, and a slurry containing 0.1, 1, or 10 g of soot was gently swirled in a 100

186

ml glass bottle, followed by addition of the soil to a total weight of 20 g. The bottle was

187

then capped, rolled, and vented, and the soil air dried, as described for PAH addition in

188

solvent.

189

concentrations of PAHs already present in the soot (Table S2) such that the target

190

concentrations in Table S1 were achieved, including BaP concentrations of 1, 10, or 100

191

ppm from addition of 0.1, 1, or 10 g of soot, respectively, to soil. For PAHs added to soil

192

in fuel oil, a similar procedure was followed in which the PAH mixture in dichloromethane

193

was first added to fuel oil, and 0.01, 0.1, or 1.0 g of fuel oil with added PAHs were placed

194

in a glass bottle to which the sand component was added. The bottle was capped, rolled,

195

vented and then after 24 hours, the rest of the soil components added. The bottle was

196

again capped, rolled, and vented with the rest of the processing of the soil samples

197

identical to PAHs added in solvent or soot.

198

Soil weathering.

199

consisting of weekly wet/dry cycles. To weather the soils, each week deionized water

200

was added in an amount equal to the water holding capacity of the soil (56% by weight).

201

During the weathering process, soils were kept in open wide-mouthed bottles at room

The amounts of the PAHs added to soot were determined based upon the

Each test soil was subjected to an 8-week weathering process


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temperature.

203

aliquots of soil were taken for assessment of 3H-BaP content. Coefficients of variation in

204

radioactivity in triplicate samples from each of the soils used in the study were less than

205

10%, and all but two were less than 4%, indicating good homogeneity.

206

Determining the tritium content of soils. Tritium content of soils after weathering was

207

determined using liquid scintillation counting in two ways. Solvent-extractable tritium

208

content was determined using a modified version of EPA Method 3550. A 200 mg

209

aliquot from each test soil was added to a 20 ml borosilicate glass scintillation vial, along

210

with 6 ml of a 1:1 acetone-dichloromethane mix, and the vial was pulse-sonicated

211

(alternating 30 sec on and 30 sec off at full power) for 3 min on ice. The sample was

212

centrifuged at 1,000 x g for 5 min at room temperature. The supernatant extract was

213

transferred to a 13 x 100 mm silanized borosilicate glass tube. The soil pellet was re-

214

extracted with another 3 ml acetone-dichloromethane solvent, and after point sonication

215

and centrifugation, the supernatant extract was combined with the original extract then

216

mixed and the volume measured. A 200 µl aliquot of the combined soil extract was

217

added to scintillation fluid (15 ml) and the vials were vortex mixed and allowed to stand

218

for 24 before scintillation counting.

219

determined following acid digestion. One hundred µl of concentrated nitric acid was

220

added to a 25 mg aliquot of soil in a 20 ml scintillation vial. The vial was capped and

221

allowed to stand for 24 hours. Scintillation fluid (15 ml) was then added, and after

222

another 24 h the sample was counted. In each approach, the amount of radioactivity (in

223

nCi) was determined from dpm with quench correction.

224

Radiochromatography of weathered soil. After weathering, soils were evaluated for

225

the integrity of the tritium label on BaP. An aliquot from each test soil was extracted

226

using the modified Method 3550 procedure described above.

227

dichloromethane extract was heated at 50° C under nitrogen to near dryness. Samples

228

were brought up to 1 ml with methanol, passed through a 0.22 micron filter, and

229

analyzed by HPLC.

230

fluorescence detection and fraction collection (Agilent, Santa Clara, CA). A C18 column

231

(5 µm packing, 150 mm x 4.6 mm; Grace Corp., Columbia, MD) was used with a

232

programmed change in mobile phase from 50:50 water:methanol to 100% methanol

233

between 3 and 15 min. Fluorescence detection used an excitation wavelength of 260

234

nm and an emission wavelength of 430 nm. Injection of a BaP standard was used to

At the conclusion of the weathering process, were homogenized and

Separately, total tritium content of the soil was

The acetone-

HPLC separation used an Agilent 1100 HPLC system with


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235

determine the BaP retention time. Eluent samples were collected into 6 ml scintillation

236

vials every 30 s for 20 min. Five ml of scintillation fluid was added to each vial, vials

237

were vortexed and allowed to stand for 24 hours, and radioactivity was determined using

238

liquid scintillation counting with quench correction.

239

sample was constructed from radioactivity eluted over each 30 s interval.

240

Dosing and blood sampling. Animals were fasted overnight prior to food or soil dosing.

241

Treatment groups consisted of five animals.

A radiochromatogram for each

Each rat was administered 0.5 g

3

242

(containing 25 µCi

243

administration, the food or soil dose was prepared as a slurry by adding 1.5 ml deionized

244

water and mixed until the food or soil was dispersed uniformly.

245

material remaining in the dead space of the gavage tube after administration was

246

collected by flushing with 150 µl deionized water and radioactivity was measured by

247

scintillation counting. This was subtracted from the nominal 25 µCi dose to determine the

248

actual administered dose for each animal. Two hours after administration of the dose,

249

access to food was restored.

H-BaP) of food or soil by gavage.

Immediately prior to The residual dose

250

A blood sample (300 µl) was taken via the jugular catheter immediately prior to

251

the dose and 2, 4, 8, 12, 24, 48, 72, 96, 120, and 144 h after the dose. Blood samples

252

were placed into a 20 ml borosilicate glass scintillation vial and stored at 4° C for up 48 h

253

before determination of radioactive content. One ml of a 1:1 Soluene 350®- ethanol

254

mixture was added to each vial, and the vial was capped and vortexed for 60 s. The

255

capped vials were incubated for 2 h at 60° C and allowed to cool to room temperature.

256

One ml of hydrogen peroxide (30%) was then added drop-wise to each vial, and the

257

vials were again incubated at 60° C for one h and allowed to return to room temperature.

258

Fifteen ml of scintillation cocktail were added and the vials were vortexed for 60 s and

259

allowed to stand for 24 h before counting. Radioactivity in each vial was determined

260

using liquid scintillation counting with quench correction.

261

Relative bioavailability measurement.

262

RBA estimates were based upon the concentration versus time profile for BaP and

263

metabolites in blood quantified by measuring the 3H- label in sequential samples over

264

time. Blood concentrations were expressed as nCi/ml, and the AUC was calculated

265

using the trapezoidal rule. As described above, all soils and food were prepared such

266

that a 0.5 g dose would include 25 µCi, irrespective of the concentration of BaP.

267

Therefore, although the soil concentration and administered dose of BaP varied across


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268

the soils, the dose of tritium was held constant.

269

was the same from both food and soil (25 µCi), there were slight differences in doses

270

actually administered, e.g., from dose solution retention in the gavage tube.

271

eliminate this source of error, AUC values for each animal were corrected based on

272

administered dose using the following relationship:

Although the nominal dose of tritium

= ×

To

25 μ (1)

273 274

As described above, in this study design it is important that each animal be “naive” with

275

regard to PAH exposure to ensure that induced metabolism of the PAHs did not result in

276

differential effects on the AUC for each dose. Consequently, each subject received a

277

single dosing substrate (food or soil containing 3H-BaP) on a single occasion, and as a

278

result RBA estimates could not be derived on an individual animal basis. Instead, RBA

279

estimates for each soil sample were obtained from the mean AUC observed in treated

280

animals and the mean AUC from animals given BaP in food: =

,"# (2) $ ,"#

281 282

Results

283

Eighteen constructed soils were created that varied in terms of total BaP

284

concentration (1, 10, and 100 mg/kg), PAH source material (solvent, soot, or fuel oil),

285

and soil characteristics (BSS with 70% sand, 20% clay, and 10% peat; soil with reduced

286

proportions of clay or peat, or the addition of charcoal fines) (Table 1). Although the

287

focus of the study was measuring the relative bioavailability of BaP, soils were created

288

with a mixture of PAHs typical of environmental samples, and each soil sample was

289

subjected to weekly wet/dry cycles for eight weeks before assessment of relative oral

290

bioavailability to simulate natural weathering processes.

291

Soil Weathering. It was important to verify that the weathering process did not result in

292

degradation of BaP or loss of the tritium label from the BaP molecule. At the completion

293

of weathering, an aliquot was removed from each soil sample and the BaP content was

294

evaluated.

295

single peak with an elution time corresponding to BaP in 15 of the 18 soils (Figure 1),

Radiochromatography of solvent extracts of the soil samples showed a


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296

indicating that the radiolabel in the sample was present on BaP. Three of the weathered

297

soils (the soils with montmorillonite instead of kaolinite, humus instead of peat, and

298

reduced peat with PAHs added from solvent) showed evidence of BaP degradation. The

299

height of the BaP peak in the radiochromatogram was diminished in these soils, and

300

other tritium peaks with somewhat earlier retention times were observed (Figure 1).

301

Although no attempt was made to identify the labeled compounds responsible for these

302

earlier peaks, their increased hydrophilicity relative to BaP would be consistent with BaP

303

metabolites. Because inclusion of both labeled BaP and BaP degradation products in a

304

soil dose would confound measurement of BaP bioavailability, these three soil samples

305

were not carried forward in the study.

306

The amount of remaining radioactivity in the soil was initially measured using

307

solvent extraction similar to a standard method used for measurement of PAHs in soil

308

(EPA Method 3550C). In addition to the radiochromatographic analysis described above,

309

extract using this method was added to scintillation fluid and radioactivity was counted

310

directly. Concentrations of radioactivity in the extracts were variable among soils and, in

311

most cases, corresponded to amounts substantially less than what was added before

312

weathering (Table 2). The discrepancy between the amount of radioactivity added to

313

soil before weathering and what was extracted and measured after weathering could be

314

due to loss of 3H-BaP from the soil or to incomplete solvent extraction. To distinguish

315

between these possibilities, additional aliquots of soil were subjected to complete

316

digestion in strong acid. Although this process resulted in destruction of the soil matrix

317

[and BaP], it was possible to determine the total content of radiolabel present (Table 2).

318

The least amount of radioactivity recovered after digestion relative to what was added

319

originally was observed in the three soils with evidence of degradation by

320

radiochromatography. For the other 15 soils, essentially all of the radioactivity that was

321

initially spiked to the soil remained in the soil after weathering (99.8 ± 15.8%; mean ±

322

SD), with variability in results probably within the range of experimental error for this

323

analytical method. To determine the role of weathering on the poor recovery of BaP

324

radiolabel from soil using solvent extraction, a limited experiment was also conducted in

325

which 3H-BaP was added to soil and dried, but not weathered. This soil was extracted

326

and radioactivity was counted after a few days, with minimal opportunity for degradation

327

or decomposition. Under these conditions, extraction of spiked BaP was essentially

328

complete (data not shown), indicating that the lower recovery of BaP in weathered soils


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329

was likely due to the formation of sequestered, non-extractable BaP residues associated

330

with the weathering process.

331

Constant proportionality between BaP dose and AUC of radiolabel in blood.

332

Relative bioavailability studies are most straightforward to conduct and interpret when

333

the measurement endpoint for absorbed dose (in this case, AUC in blood) is directly

334

proportional to dose over the dose range examined. In a preliminary experiment, rats

335

were given a single oral dose of BaP in food ranging from 0.1 to 100 µg per animal (N=3

336

per group). Food was chosen as the dosing medium for this experiment because BaP

337

was expected to have minimal interactions with a food matrix that might confound

338

interpretation of results.

339

concentrations in the food of 0.2, 2, 20, and 200 ppm BaP. To facilitate comparison

340

among treatment groups, the specific activity of the 3H-BaP in food was adjusted so that

341

the animals received a consistent dose of 25 µCi 3H-BaP while the total BaP dose was

342

varied. Using this approach, the fraction of dose absorbed could be compared across

343

treatment groups directly from comparison of radioactivity in blood without adjustment. If

344

the fraction of total BaP absorbed was constant among the various BaP doses, the

345

AUCs for the radiolabeled portion of the dose would be the same. If the fraction BaP

346

absorbed was different, the extent of difference would be reflected quantitatively in the

347

radiolabel AUCs.

As administered, these BaP doses correspond to

348

Following a single gavage dose of food containing BaP to rats fasted overnight,

349

the concentrations of radioactivity in blood, representing BaP and metabolites in the

350

radiolabeled portion of the dose, were followed for six days (Figure 1).

351

significant differences in the AUCs for radioactivity in blood among any of the BaP doses,

352

indicating consistent absorption regardless the amount of BaP given within the dose

353

range of 0.1 to 100 µg (0.1 µg BaP, AUC = 3598 ± 1019 nCi-hr/ml; 1 µg BaP, 3172 ± 18

354

nCi-hr/ml; 10 µg; BaP, 2835 ± 979 nCi-hr/ml; BaP 100 µg, 3194 ± 101 nCi-hr/ml; p >

355

0.05 by ANOVA). The consistent fractional proportionality between dose and blood

356

concentrations and AUCs is indicative of linear pharmacokinetics for the BaP doses and

357

soil and food concentration ranges included in the study.

358

Effect of BaP concentration and source material on RBA. To establish comparison

359

or benchmark AUCs, BaP was added to food to achieve concentrations of 1, 10, or 100

360

ppm. These concentrations were selected to match the total BaP concentrations in the

361

test soils. These concentrations corresponded to administered total BaP doses of 0.5, 5,


There were no

12

Page 13 of 29


362

and 50 µg. Although the BaP doses (radioactive and total) were somewhat different

363

from those used in the preliminary experiment presented above, the results were the

364

same in that no significant differences were observed in the AUC for radiolabeled BaP

365

among the three doses (800 ± 227, 800 ± 68, and 850 ± 92 nCi-hr/ml; N=5 per treatment

366

group; p > 0.05 by ANOVA). In the absence of differences in BaP absorption from food

367

with different BaP doses, data were combined for graphical presentation of blood

368

concentrations over time for BaP from food and the mean of the pooled AUCs from 1, 10

369

and 100 ppm BaP in food (0.5, 5, and 50 µg total BaP, respectively) (817± 28 nCi-hr/ml,

370

N=15) was used for comparison with AUCs from BaP in soil to derive RBA values.

371

RBA values were estimated for each of the 15 soils that did not show evidence of

372

3

373

profiles for BaP in food compared with BaP [solvent source] in soil and in soil with 2%

374

charcoal. The AUCs for radioactivity in blood for each test soil and for food, and the

375

associated RBA values are summarized in Table 3. RBA values were generally

376

independent of BaP dose when the source was solvent. When the source was soot,

377

BaP RBA was decreased at the highest BaP dose, while RBA values generally

378

increased with BaP dose when fuel oil was the source.

379

Effect of soil characteristics on BaP RBA. The elimination of three soils from the

380

study because of BaP degradation during weathering greatly reduced the comparisons

381

that could be made in BaP RBA from soils with different compositions. The addition of

382

charcoal to soil decreased BaP RBA by two-thirds or more regardless the PAH source.

383

Reducing the clay or peat content of the soil had little or no effect on BaP RBA.

H-BaP decomposition.

Figure 2 shows example blood concentration versus time

384 385

Discussion

386

This study differs from previous efforts to assess the oral bioavailability of a PAH

387

in soil in that it includes examination of potential influences of PAH concentration, source

388

material, and soil characteristics on BaP RBA at lower, arguably more environmentally

389

relevant, soil concentrations.

390

influence on oral bioavailability of these potential key factors could be examined in a

391

controlled, systematic manner, and the use of a radiolabeled tracer allowed

392

measurement of bioavailability at soil concentrations much lower than previously

393

possible.

Through the use of constructed, weathered soils, the


13


Page 14 of 29

394

Effect of BaP concentration and source material on RBA. An important observation

395

made possible by this approach is that the absorption of BaP was linear over the wide

396

range of BaP concentrations and doses of interest in this study. When BaP was added

397

to food in concentrations that resulted in doses of BaP spanning three orders of

398

magnitude (0.1 to 100 µg), the fraction of BaP reaching the systemic circulation, as

399

reflected in the AUC, was unchanged (i.e., the amount absorbed was directly

400

proportional to the dose across the dose range). From a biological perspective, this

401

suggests that there are no capacity-limited or saturable uptake processes affecting

402

absorption of accessible BaP from the GI tract for doses of greatest interest for risk

403

assessment.

404

Similar RBA values were also observed when BaP (in combination with other

405

PAHs) was added in solvent to soil in doses of 0.5, 5, and 50 µg (resulting in

406

concentrations of 1, 10, or 100 ppm) — 0.56, 0.51, and 0.52, respectively. This indicates

407

that, at least for this baseline soil, both biological and geochemical processes affecting

408

bioavailability were linear throughout this BaP concentration range. While other studies

409

have reported RBA values in this approximate range for spiked soils, the study design

410

used in this other research did not directly assess the effect of varying concentrations for

411

the same soil type.21 When the source was soot, the RBA of BaP was decreased at the

412

highest BaP concentration, while RBA values generally increased with BaP

413

concentration when fuel oil was the source. In interpreting these observations, it is

414

important to note that increasing concentrations of BaP in the test soils were

415

accompanied by increased concentrations of the source materials. For example, the

416

100 ppm BaP soil from fuel oil was created by adding 10-times more fuel oil to soil than

417

the 10 ppm BaP soil, and 100-times as much as the 1 ppm soil. Consequently, the fuel

418

oil test soil with the highest BaP concentration also has the highest concentrations of

419

other constituents of fuel oil, some of which may act to enhance the bioavailability of

420

BaP from soil.

421

more soot than the test soils with lower BaP concentrations, and in fact was

422

approximately 50% soot by weight. Black carbon materials like soot have a high affinity

423

for PAHs,20 which may act to diminish the bioavailability of BaP from soil. Thus, with

424

increasing BaP concentration, the higher mass of carbon in soot could serve as a

425

sorption matrix for the BaP. This is confirmed by the results from soil to which charcoal,

426

another form of black carbon, was added; the presence of charcoal in the soil

427

substantially decreased the RBA of 10 ppm BaP soil, whether the BaP was from solvent,

Similarly, the test soil with 100 ppm BaP from soot has substantially


14

Page 15 of 29


428

soot, or fuel oil (Table 3). The point illustrated by these observations is that the source

429

of BaP and the nature of the geosorbents within the soil can potentially have a strong

430

effect on its RBA. Stated another way, the RBA for BaP in soil at a site could be

431

markedly different depending upon how the BaP got there, independent of site-specific

432

soil characteristics. This observation on the effect of source material and black carbon

433

on RBA is consistent with observation of bioavailability to aquatic organisms (e.g.,

434

24

435

which established that the source material has a major impact on the partitioning

436

behavior of PAHs in soil.

437

Effect of soil characteristics on BaP RBA. Our investigation also included a limited

438

evaluation of the potential effect of soil characteristics on the RBA of BaP. Unfortunately,

439

BaP degradation during weathering precluded the inclusion of soils with montmorillonite

440

instead of kaolinite, humus instead of peat, and reduced peat (with BaP addition in

441

solvent) in the study. The reason why degradation occurred in these soils and not the

442

others is unknown. The constructed soils were not sterilized, and the differences could

443

reflect differences in microflora introduced into the various soils. The modifications to

444

soil composition that remained in the study, elimination of clay or peat from the soil,

445

produced little change in the observed RBA.

446

Solvent extractability of BaP and its implications for estimating and using RBA

447

values. The RBA values derived in this study are based on the total BaP radioactivity

448

present in the soil doses given to the rats. At the conclusion of the weathering of soil, it

449

was found that some radioactivity present in the soil was intractable to extraction using

450

an acetone-dichloromethane with a modified Method 3550C. This method was used in

451

this study because it is among the most common for preparation of environmental soil

452

samples for PAH analysis. Among the soils for which RBA was measured, there was no

453

evidence from radiochromatography of biodegradation of the BaP or loss of the label;

454

essentially all of the radioactivity in extracted material was confined to a single peak

455

corresponding to BaP. This led to the conclusion that the radioactivity remaining in

456

weathered soil after solvent extraction was sequestered, non-extractable BaP. The

457

formation of non-extractable bound residues is a well-documented process during the

458

weathering and ageing of PAHs27 and other organic compounds in soils28

20, 22-

), and investigations into the soil-chemical interactions between soil and PAHs,25,25

459

The sequestration of BaP and other PAHs that occurs during weathering raises

460

the question of the most relevant way to quantify administered dose — the total amount


15


Page 16 of 29

461

present in soil that is ingested or only the amount liberated by standard EPA methods?

462

The RBA values presented in Table 3 were derived based upon total BaP present in soil,

463

which could be directly measured in this study because radiolabeled BaP was used.

464

Other methods that fail to capture a sequestered fraction would result in different

465

findings. For example, as shown in Table 2, the solvent (acetone-dichloromethane)

466

extractable fraction of total BaP in the test soils was as low as 30%. Other studies have

467

also shown variable and, in some cases, poor extraction using solvent extraction with

468

sonication for PAHs in soil (see Lau et al. for a review).29 Jonker and Koelmans30

469

compared several solvents for extraction of PAHs from soot and sediment and found

470

dichloromethane to have the worst recovery, with as little as 16% recovered compared

471

with other solvents.

472

If the solvent-extractable fractions in Table 2 are used as the basis for

473

determining the administered BaP doses, and these BaP doses are used with the AUCs

474

presented in Table 3 to calculate RBA values, substantially higher RBA values are

475

obtained. In fact, this approach would give the improbable result that over 50% of the

476

test soils in this study have an RBA of 1.0 or more, with some as high as 2.1. A

477

plausible explanation for these RBA values is that the relevant dose is, or is much closer

478

to, the total BaP content. These observations also suggest that for at least some of the

479

test soils the extent of gastrointestinal extraction of BaP by the animals is more complete

480

than a modified Method 3550C solvent extraction.

481

The study here demonstrates that the RBA of BaP in soil can be significantly

482

lower than the default assumption of 100%, and that the specific RBA will depend

483

significantly on the source of the BaP in the soil, and to some extent soil characteristics,

484

especially the content of black carbon. Clearly, development of in vitro methods to

485

estimate the RBA of BaP and other PAHs from soil will need to ensure that these

486

influences are accurately reflected in any proposed test. Despite the many advantages

487

of using laboratory weathered, constructed soil samples with radiolabeled BaP in

488

exploring critical factors influence bioavailability, it is possible that some differences from

489

contaminated site soils may exist. Thus, it will be important to confirm these findings, to

490

extent possible, with PAH contaminated site soils.

491

The results also illustrate that consideration of bioavailability needs to be

492

addressed in concert with other aspects of site characterization and risk assessment,

493

particularly illustrated by the fact that different methods for soil sample preparation and


16

Page 17 of 29


494

analysis can yield very different estimates of PAH concentrations in soil. Therefore,

495

understanding the implications of the different analytical methods for characterizing PAH

496

contaminated soil is relevant to accurate determination of the bioavailable concentration

497

or bioavailable fraction.

498

implications as well.

Additional research is warranted to better address these


17


Page 18 of 29

499

Acknowledgements

500

This research was supported in part by a grant from the Strategic Environmental

501

Research and Development Program (SERDP Project ER-1743). Dr. Stephen Roberts

502

and John Munson declare no competing interests. Yvette W. Lowney and Michael Ruby

503

work for scientific consulting firms that provide risk assessment services to private and

504

public-sector clients.

505 506

Supporting Information Available

507

This information is available free of charge via the Internet at http://pubs.acs.org. One

508

table showing the total PAH composition of the test soils at the three benzo(a)pyrene

509

target concentrations (1, 10, and 100 ppm); one table with the PAH composition of the

510

soot and fuel oil source materials; and one figure showing solvent extract

511

radiochromatograms from 18 test soils after 8 weeks of weathering.

512 513


18

Page 19 of 29


514

References

515

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516 517

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Evaluation of Carcinogenic Risks to Humans. Volume 92. Some Non-heterocyclic

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Polycylic Aromatic Hydrocarbons and Some Related Exposures. World Health

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Organization, 2010.

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Research Triangle Park, NC: U.S. Department of Health and Human Services,

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Kroese, E.D.; Muller, J.J.A.; Mohn, G.R.;, Dortant, P.M.; Wester, P.W. Tumorigenic

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effects in Wistar rats orally administered benzo(a)pyrene for two years (gavage

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Public Health and the Environment (RIVM), 2001.

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DNA binding of coal tar components in B6C3F1 mice following ingestion. Chem.

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van Schooten, F.J.; Moonen, E.J.C.; van der Wal, L.; Levels, P.; Kleinjans, J.C.S.

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Koganti, A.; Spina, D.A.; Rozett, K.; Ma, B.-L.; Weyand, E.H.; Taylor, B.B., Mauro,

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tar-contaminated soils. Environ. Sci. Technol. 1998, 32 (20), 3104-3112.

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Studies on the applicability of biomarkers in estimating the systemic

Reeves, W.R.; Barhoumi, R.; Burghardt, R.C.; Lemke, S.L.; Mayura, K.; McDonald,

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T.J.; Phillips, T.D.; Donnelly, K.C. Evaluation of methods for predicting the toxicity

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James, K.; Peters, R.E.; Laird, B.D.; Ma, W.K.; Wickstrom, M.; Stephenson, G.L.;

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Siciliano, S.D. (2011). Human exposure assessment: A case study of 8 PAH

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contaminated soils using in vitro digestors and the juvenile swine model. Environ.

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Sci. Technol. 2011, 45, 4586-4593.

548

10.

Duan, L.; Palanisami, T.; Liu, Y.; Dong, A.; Mallavarapu, M.; Kuchel, T.; Semple,

549

KT.; Naidu, R. Effects of ageing and soil properties on the oral bioavailability of

550

benzo[a]pyrene using a swine model. Environ. Int. 2014, 70, 192-202.

551

11.

Ramesh, A.; Walker, S.; Hood, D.; Guillen, M.D.; Schneider, K.; Weyand, E.H.

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Bioavailability and risk assessment of orally ingested polycyclic aromatic

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hydrocarbons. Int. J. Toxicol. 2004, 23, 301-333

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Ruby, M.V.; Gomez-Eyles, J.L.; Ghosh, U.; Roberts, S.M.; Tomlinson, P.; Menzie,

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C.; Kissel, J.C.; Bunge, A.L.; Lowney, Y.W.

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absorption of PAHs from soil – State of the science. Environ. Sci. Technol. 2016,

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50 (5), 2151-2164

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Oral bioavailability and dermal

USEPA (U.S. Environmental Protection Agency) Development of a Relative

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Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH)

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Mixtures. EPA/635/R-08/912A, Washington, D.C., 2010.

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Brune, H.; Deutsch-Wenzel, R.P.; Habs, M.; Ivankovic, S.; Schmahl, D.

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Investigation of the tumorigenic response to benzo(a)pyrene in aqueous caffeine

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solution applied orally to Sprague-Dawley rats. J. Cancer Res. Clin. Oncol. 1981,

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Neal, J.; Rigdon, R.H. Gastric tumors in mice fed benzo(a)pyrene: A quantitative study. Tex. Rep. Biol. Med. 1967, 25, 553-557.

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Peters, R.E.; James, K.; Cave, M.; Wickstrom M.; Siciliano, S.D. Is received dose

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from ingested soil independent of soil PAH concentrations: Animal model results.

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Environ. Toxicol. Chem. 2016, 35 (9), 2261-2269.

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Munson, J.W.; Lowney, Y.W.; Ruby, M.V.; Roberts, S.M. Estimating the relative

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Logan, C. Use of animals for the determination of absorption and bioavailability.

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Ruby, M.V.; Lowney, Y. Selective soil particle adherence to hands: implications

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Cornelissen, G.; Gustafsson, O.; Bucheli, T. D.; Jonker, M. T. O.; Koelmans, A. A.;

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Van Noort, P. C. M., Extensive sorption of organic compounds to black carbon,

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coal, and kerogen in sediments and soils: Mechanisms and consequences for

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Duan, L.; Naidu, R.; Liu, Y.; Dong, Z.; Mallavarapu, M.; Herde, P.; Kuchel, T.;

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Semple K.T. Comparison of oral bioavailability of benzo[a]pyrene in soils using rat

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Hong, L.; Ghosh, U.; Mahajan, T.; Zare, R.M.; Luthy, R.G. PAH sorption

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mechanism and partitioning behavior in lampblack impacted soils from former oil-

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Ghosh,, U.; Zimmerman, J.R.; Luty, R.G.;. PCB, PAH speciationoamong particle

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Environ Sci. Technol. 2003, 37 (10), 2209-2217.

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596 597

carbon and PAH source. Environ. Sci. Technol. 2004, 38 (7), 2029-2037. 25.

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Thorsen, W.A.; Cope W.G.; Shea, D. Bioavailability of PAHs: effects of soot

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Crampon, M.; Bodilis, J.; LeDerf, F.

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Northcott, G. L.; Jones, K. C., Partitioning, extractability, and formation of

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nonextractable PAH residues in soil. 1. Compound differences in aging and

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sequestration. Environ. Sci. Technol. 2001, 35 (6), 1103-1110.

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Gevao, B., Mordaunt, C., Semple, K.T., Piearce, T.G., Jones, K.C. Bioavailability of

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2001, 35 (3), 501-507.

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Lau, E.V.; Gan, S.; Ng, H.K. Extraction techniques for polycyclic aromatic

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hydrocarbons in soils. Int. J. Anal. Chem. 2010, Vol. 2010, article ID 398381, 9

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Jonker, M.T.O.; Koelmans, A.A. Extraction of polycyclic aromatic hydrocarbons

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mechanism. Environ. Sci. Technol. 2002, 36 (19), 4107-4113.


22

Page 23 of 29


Table 1. Test Soil Matrix: PAH Sources, Soil Characteristics, and Benzo(a)pyrene Concentrations PAH Source

Solvent

Baseline a Synthetic Soil

Synthetic Soil Added Charcoal

Synthetic Soil c Reduced Peat

Synthetic Soil d Reduced Clay

Synthetic Soil Montmorillonite in place of kaolinite

Synthetic Soil Humus in place of peat

1, 10, 100 ppm

10 ppm BaP

10 ppm BaP*

10 ppm BaP

10 ppm BaP*

10 ppm BaP*

10 ppm BaP

10 ppm BaP

b

BaP Soot

1, 10, 100 ppm

10 ppm BaP

BaP Fuel Oil

1, 10, 100 ppm

10 ppm BaP

BaP a

Baseline synthetic soil, 70% sand, 20% clay, 10% peat

b

Baseline synthetic soil with 2% charcoal added

c

Baseline synthetic soil with peat reduced to 1%

d

Baseline synthetic soil with clay reduced to 2%

* Not carried through the study because of evidence for BaP degradation during weathering



Page 24 of 29

Table 2. Benzo(a)pyrene Concentrations in Test Soils After Weathering Post-weathering concentrations (% pre-weathering concentration) a 3 3 Soil Source Target H content following H content following Concentration Method 3550C acid digestion (ppm) extraction BSS Solvent 1 36 105 10 28 78 100 60 101 Soot 1 50 98 10 66 90 100 43 87 Fuel oil 1 38 93 10 56 103 100 52 139 BSS + charcoal Solvent 10 56 96 Soot 10 36 93 Fuel oil 10 64 100 BSS, reduced peat Solvent 10 46 67 Fuel oil 10 85 82 BSS, reduced clay Solvent 10 41 114 Fuel oil 10 41 118 BSS, montmorillonite Solvent 10 28 64 BSS, humus Solvent 10 60 76 a BSS = Baseline synthetic soil; “+ charcoal” = 2% charcoal added; “reduced peat” = peat content reduced to 1%; “reduced clay” = clay content reduced to 1%; “montmorillonite” = montmorillonite instead of kaolinite; “humus” = humus instead of peat. b BaP concentration determined based upon radioactivity measured and BaP specific activity.


24

Page 25 of 29


Table 3. Relative Oral Bioavailability of BaP from Soils Matrix

Baseline Synthetic Soil

BaP, ppm (µg dose)

Source Material Solvent Soot a AUC RBA AUC RBA a

Fuel Oil AUC RBA a

1 (0.5)

455 ± 30

0.558 ± 0.082

442 ± 27

0.542 ± 0.074

529 ±3

0.648 ± 0.008

10 (5)

413 ±8

0.505 ± 0.021

550 ± 26

0.673 ± 0.072

785 ± 97

0.961 ± 0.267

100 (50)

426 ± 65

0.552 ± 0.078

192 ± 13

0.235 ± 0.035

869 ± 119

1.064 ± 0.325

Baseline Synthetic Soil + Charcoal

10 (5)

103 ± 16

0.126 ± 0.020

171 ±4

0.210 ± 0.013

249 ± 24

0.305 ± 0.066

Baseline Synthetic Soil – Reduced Clay

10 (5)

335 ± 49

0.410 ± 0.060

NA

b

NA

b

698 ± 40

0.854 ± 0.111

Baseline Synthetic Soil – Reduced Peat Food

10 (5)

—

NA

b

NA

b

621 ± 70

0.761 ± 0.191

d

c

—

c

817 ± 28

a

nCi-h/ml; mean ± SD, N=5 NA = not determined per experimental design; see Table 1 c Not evaluated due to evidence of BaP degradation during weathering d Average of AUCs obtained from animals receiving BaP in food in concentrations of 1, 10, and 100 ppm, corresponding to doses of 0.5, 5, and 50 µg total BaP b


25


Page 26 of 29

Figure Legends Figure 1. Linear Pharmacokinetics of BaP/3H-BaP in Blood Following Doses Ranging from 0.1 to 100 µg. Rats were administered a single dose of BaP ( 0.1, 1, 10, or 100 µg) in food by gavage. Each dose contained 25 µCi 3H-BaP. Serial blood samples were taken from 2 to 144 hours after the dose and the concentration of radiolabel determined (mean ± SD, N=5). Blood concentration versus time profiles were similar for all BaP doses and the AUCs derived from these profiles were not significantly different (see Results). The absence of a change in pharmacokinetics indicates linear pharmacokinetics over the dose range examined. Figure 2. Blood Concentrations of 3H Following Oral Administration of BaP/3H-BaP in Food and Weathered Soils. Rats were administered a single dose of either food with BaP (100 ppm; 25 µCi 3H-BaP) or weathered soils (baseline soil and baseline soil with charcoal) BaP (10 ppm; 25 µCi 3H-BaP). The solvent was spiked with BaP/ 3H-BaP and was added to the soil prior to weathering. The bioavailability of BaP from weathered soils was calculated relative to bioavailability of BaP from food. The shaded area represents the food-reference AUC used to calculate the soil RBAs


Page 27 of 29


Figure 1


27


Page 28 of 29

Figure 2


28

Page 29 of 29


TOC Art


29

Effects of Source and Concentration on Relative Oral Bioavailability of

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