ERDC TR-16-4

Impact of Incremental Sampling
Methodology (ISM) on Metals Bioavailability

Engineer Research and
Development Center

Jay Clausen, Brandon Swope, Anthony Bednar, Laura Levitt,
Timothy Cary, Thomas Georgian, Marienne Colvin, Kara
Sorensen, Nancy Parker, Sam Beal, Dale Rosado, Michael Catt,
Kristie Armstrong, and Charolett Hayes

Approved for public release; distribution is unlimited.

May 2016


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ERDC TR-16-4
May 2016

Impact of Incremental Sampling
Methodology (ISM) on Metals Bioavailability

Jay Clausen, Laura Levitt, Timothy Cary, Nancy Parker, and Sam Beal
U.S. Army Engineer Research and Development Center (ERDC)
Cold Regions Research and Engineering Laboratory (CRREL)
72 Lyme Road
Hanover, NH 03755-1290

Anthony Bednar, Dale Rosado, Michael Catt, Kristie Armstrong, and Charolett Hayes
U.S. Army Engineer Research and Development Center
Environmental Laboratory (EL)
3909 Halls Ferry Road
Vicksburg, MS 39180-6199

Brandon Swope, Marienne Colvin, and Kara Sorensen
Space and Naval Warfare Systems Command (SPAWAR) Systems Center Pacific
Pacific Bioassay Laboratory
53475 Strothe Road, San Diego, CA 92152

Thomas Georgian
U.S. Army Corps of Engineers, Environmental and Munitions Center of Expertise
1616 Capital Avenue
Omaha, NE 68102-9200

Final Report
Approved for public release; distribution is unlimited.

Prepared for

Under

U.S. Army Environmental Command

2450 Connell Road, Building 2264
Fort Sam Houston, TX 78234
Project 404632, “Metal Bioavailability Assessment”


ERDC TR-16-4

Abstract
This study assessed the impact of the incremental sampling methodology
(ISM) on metals bioavailability through a series of digestion and in vivo
experiments. These tests used Eisenia fetida and Lolium rigidum in both
milled and unmilled loam and sand soil containing antimony, copper,
lead, and zinc obtained from Donnelly Training Area, Alaska. No significant differences in metal levels were evident between milled and unmilled
soil for E. fetida, and uptake of lead by L. rigidum in sand yielded lead recoveries comparable with Method 3050 analysis of soil. In contrast, L.
rigidum grown in loam had much lower recoverable lead. Milling of the
soil as part of the ISM process had no significant impact on the lead species distribution. In comparison with Method 3050, the alternative digestion tests involving the use of glycine; oxalate; ethylenediaminetetraacetic
acid (EDTA); or alternative digestion procedures, such as the synthetic
precipitation leaching procedure (SPLP) and the toxicity characteristic
leaching procedure (TCLP), yielded lower recoveries of lead for all soil particle sizes and soil types. Diffusive gradient in thin films experiments
yielded metal concentrations positively correlated with E. fetida concentrations. The physiologically based extraction technique (PBET) positively
correlated with bulk soil concentrations and E. fetida tissue concentrations for all soils evaluated.

DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products.
All product names and trademarks cited are the property of their respective owners. The findings of this report are not to
be construed as an official Department of the Army position unless so designated by other authorized documents.
DESTROY THIS REPORT WHEN NO LONGER NEEDED. DO NOT RETURN IT TO THE ORIGINATOR.

ii



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iii

Contents
Abstract................................................................................................................................................... ii
Illustrations.............................................................................................................................................. v
Preface .................................................................................................................................................. viii
Acronyms and Abbreviations ................................................................................................................ix
1

Introduction..................................................................................................................................... 1
1.1 Background ..................................................................................................................... 1
1.2 Objectives ........................................................................................................................ 3
1.3 Approach ......................................................................................................................... 3

2

Incremental Sampling Methodology............................................................................................ 5

3

Methods........................................................................................................................................... 8
3.1
3.2
3.3
3.4

Field sampling ................................................................................................................. 9
Laboratory sample preparation ................................................................................... 10

Soil characterization ..................................................................................................... 11
In vitro experiments ...................................................................................................... 12

3.4.1

Organism procurement and handling ................................................................................... 12

3.4.2

Test material........................................................................................................................... 12

3.4.3

Earthworm survival, growth, and bioaccumulation test ....................................................... 14

3.4.4

Diffusive gradients in thin films (DGT) .................................................................................. 18

3.4.5

Physiologically based extraction technique (PBET) .............................................................. 19

3.4.6

Metals analysis ....................................................................................................................... 19

3.5 Vegetation experiments ................................................................................................ 21
3.6 Analytical methods ....................................................................................................... 25
4


Results ........................................................................................................................................... 26
4.1

Soil properties ............................................................................................................... 26

4.1.1

Lead speciation ...................................................................................................................... 29

4.1.2

Other digestion approaches .................................................................................................. 30

4.2 Earthworm bioaccumulation experiments .................................................................. 31
4.2.1

Phase I—Particle size impacts ............................................................................................... 31

4.2.2

Phase II—Soil toxicity .............................................................................................................. 32

4.2.3

Worm tissue metals bioaccumulation................................................................................... 37

4.2.4

Soil metal concentrations ...................................................................................................... 42


4.2.5

Diffusive gradients in thin films (DGT) bioavailability assessment...................................... 44

4.2.6

Physiologically based extraction technique (PBET) metal bioaccessibility ......................... 46

4.3 Vegetation bioaccumulation......................................................................................... 48
5

Discussion ..................................................................................................................................... 51
5.1 Bioavailability assessment ........................................................................................... 51
5.2 Incremental sampling methodology impact on metal bioavailability......................... 56


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5.3 Oversize fraction disposition ........................................................................................ 58
6

Conclusion..................................................................................................................................... 61

References............................................................................................................................................ 62
Report Documentation Page

iv



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v

Illustrations
Figures
1

Comparison of prior digestion results for tungsten ................................................................. 6

2

Collection of field samples from the small-arms range berm at the Texas Range
on the Donnelly Training Area, AK ............................................................................................. 9

3

Study design sample processing hierarchy ............................................................................ 10

4

Earthworm experimental layout .............................................................................................. 14

5

Earthworms used in the study ................................................................................................. 16

6

Vegetation .................................................................................................................................. 23


7

Vegetation uptake experiment holders................................................................................... 24

8

Image of scanned leaf and root sample for Test 12 contaminated loam (CL-1AUa)
in <250 µm to >2 mm soil ....................................................................................................... 24

9

Particle size distribution and general chemical properties for the loam and sand
used in this study ...................................................................................................................... 26

10

Lead soil concentrations for background and contaminated study materials ................... 29

11

Lead speciation for study soils ................................................................................................ 29

12

Various lead soil concentrations by digestion method compared with Method
3050B ........................................................................................................................................ 30

13


Mean percent earthworm survival (±SD) from spiking studies............................................ 31

14

Earthworm 14-day mean survival (±SD) in all samples ........................................................ 32

15

Earthworm 14-day mean survival (±SD) in sand ................................................................... 33

16

Earthworm 14-day mean wet weight (±SD) in sand .............................................................. 34

17

Earthworm 14-day mean survival (±SD) in loam ................................................................... 35

18

Earthworm 28-day mean survival (±SD) in loam ................................................................... 36

19

Earthworm 28-day mean wet weight (±SD) in loam.............................................................. 37

20

Earthworm 14-day copper bioaccumulation (mg/kg) in sand.............................................. 38


21

Earthworm 14-day zinc bioaccumulation (mg/kg) in sand ................................................... 38

22

Earthworm 14-day lead bioaccumulation (mg/kg) in sand .................................................. 39

23

Earthworm 14-day antimony bioaccumulation (mg/kg) in sand.......................................... 39

24

Earthworm 28-day copper bioaccumulation (mg/kg) in loam ............................................. 40

25

Earthworm 28-day zinc bioaccumulation (mg/kg) in loam................................................... 40

26

Earthworm 28-day lead bioaccumulation (mg/kg) in loam .................................................. 41

27

Earthworm 28-day antimony bioaccumulation (mg/kg) in loam ......................................... 41

28


Soil to earthworm-tissue concentration comparisons for copper ........................................ 43

29

Soil to earthworm-tissue concentration comparisons for zinc ............................................. 43

30

Soil to earthworm-tissue concentration comparisons for lead............................................. 44

31

Soil to earthworm-tissue concentration comparisons for antimony .................................... 44

32

Diffusive gradients in thin films for copper flux ..................................................................... 45

33

Diffusive gradients in thin films for zinc flux........................................................................... 45


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vi

34

Diffusive gradients in thin films for lead flux .......................................................................... 46


35

Diffusive gradients in thin films for antimony flux ................................................................. 46

36

Physiologically based extraction technique copper bioaccessibility .................................... 47

37

Physiologically based extraction technique zinc bioaccessibility ......................................... 47

38

Physiologically based extraction technique lead bioaccessibility......................................... 48

39

Physiologically based extraction technique antimony bioaccessibility ................................ 48

40

Lead uptake (mg/kg) into the leaves (green) and roots (brown) of rye grass in
contaminated loam................................................................................................................... 49

41

Lead uptake (mg/kg) into the leaves (green) and roots (brown) of rye grass in
contaminated sand................................................................................................................... 49


42

Average lead uptake (mg/kg) in the leaves (green) and roots (brown) of rye grass
in contaminated loam and sand ............................................................................................. 50

43

Average lead uptake (mg/kg) in earthworms versus soil concentration by
digestion method ...................................................................................................................... 52

44

Average copper uptake (mg/kg) in earthworms versus soil concentration by
digestion method ...................................................................................................................... 53

45

Average lead uptake (mg/kg) in ryegrass leaf tissue versus soil lead by digestion
method....................................................................................................................................... 55

46

Average lead uptake (mg/kg) in ryegrass root tissue versus soil lead by digestion
method....................................................................................................................................... 55

47

Milled versus unmilled lead (mg/kg) tissue levels ................................................................ 58


Tables
1

Artificial soil mixtures and treatments .................................................................................... 13

2

Field-collected soils................................................................................................................... 13

3

Earthworm toxicity and bioaccumulation test specifications ............................................... 15

4

Initial quality parameters for field-collected soils samples ................................................... 17

5

Experimental design for the vegetation study........................................................................ 21

6

Initial soil concentration measurements ................................................................................ 27

7

Initial metal soil concentration (mg/kg) measurements ...................................................... 28

8


Earthworm 14-day survival in sand......................................................................................... 33

9

Earthworm 14-day mean Individual wet weight (± SD) in sand ........................................... 34

10

Earthworm 14-day survival in loam......................................................................................... 35

11

Earthworm 28-day survival in loam ........................................................................................ 36

12

Earthworm 28-day mean individual wet weight (±SD) in loam ............................................ 37

13

Earthworm 14-day tissue metal concentrations (mg/kg) wet weight (±SD) in
sand ........................................................................................................................................... 42

14

Earthworm 28-day tissue metal concentrations (mg/kg) wet weight (±SD) in
loam ........................................................................................................................................... 42

15


Summary of metal concentrations (mg/kg) in sand ............................................................. 42

16

Summary of metal concentrations (mg/kg) in loam ............................................................. 43

17

Lead (mg/kg) worm tissue versus soil concentration ........................................................... 52


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vii

18

Copper (mg/kg) worm tissue versus soil concentration ....................................................... 53

19

Lead (mg/kg) ryegrass leaf tissue versus soil concentration ............................................... 54

20

Lead (mg/kg) ryegrass root tissue versus soil concentration .............................................. 54

21


Lead concentration by soil type and processing method ..................................................... 57

22

Computed metal mass by soil particle size ............................................................................ 60


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Preface
This study was conducted for the U.S. Army Environmental Command
(AEC) under Project 404632, “Metal Bioavailability Assessment.” The
technical monitors were Drs. Doris Anders and Robert Kirgan with AEC.
This report was prepared by Dr. Jay Clausen, Laura Levitt, Timothy Cary,
Nancy Parker, and Dr. Sam Beal (Biogeochemical Sciences Branch, Dr.
Justin Berman, Chief), U.S. Army Engineer Research and Development
Center (ERDC), Cold Regions Research and Engineering Laboratory
(CRREL); Dr. Anthony Bednar, Dr. Dale Rosado, Michael Catt, Dr. Kristie
Armstrong, and Charolett Hayes, ERDC Environmental Laboratory (EL);
Dr. Brandon Swope, Marienne Colvin, and Dr. Kara Sorensen, Space and
Naval Warfare Systems Command (SPAWAR) Systems Center Pacific, Pacific Bioassay Laboratory; and Dr. Thomas Georgian, U.S. Army Corps of
Engineers, Environmental and Munitions Center of Expertise. At the time
of publication, Dr. Loren Wehmeyer was Chief of the Research and Engineering Division. The Deputy Director of ERDC-CRREL was Dr. Lance
Hansen, and the Director was Dr. Robert Davis.
COL Bryan S. Green was the Commander of ERDC, and Dr. Jeffery P. Holland was the Director.

viii


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ix

Acronyms and Abbreviations
ABA

Absolute Bioavailability

AEC

U.S. Army Environmental Command

AFO

Oral Absorption Fraction

Al

Aluminum

As

Arsenic

ASTM

American Society of Testing Methods

Ba


Barium

Ca

Calcium

CaCO3

Calcium Carbonate

Cr

Chromium

CRREL

Cold Regions Research and Engineering Laboratory

Cu

Copper

DGT

Diffusive Gradients in Thin Films

DI

Deionized


DOD

U.S. Department of Defense

DU

Decision Units

EC50s

Half Maximal Effective Concentration

EDTA

Ethylenediaminetetraacetic Acid

EL

Environmental Laboratory

ERDC

Engineer Research and Development Center

ESTCP

Environmental Security and Technology Certification Program

Fe


Iron


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x

HCl

Hydrochloric Acid

HDPE

High-Density Polyethylene

HNO3

Nitric Acid

ICP-MS

Inductively Coupled Plasma–Mass Spectrometry

ICP-OES

Inductively Coupled Plasma–Optical Emission Spectroscopy

ISM

Incremental Sampling Methodology


ITRC

Interstate Technology Regulatory Council

K

Potassium

LC50s

Half Maximal Lethal Concentration

Mg

Magnesium

Mn

Manganese

MS

Mass Spectroscopy

Na

Sodium

Ni


Nickel

NIST

National Institute of Standards and Tests

NRC

National Resource Council

NT

Not Tested

OLS

Ordinary Least Squares

P

Phosphorus

Pb

Lead

Pb(II)

Exchangeable Lead


Pb2+

Residual Lead

PBET

Physiologically Based Extraction Technique

PbC

Organic Lead


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xi

PbCO3

Lead Carbonate

PbO

Lead Oxide

PbS

Lead Sulfide


PbSol

Soluble Lead

RBA

Relative Bioavailability

Sb

Antimony

SD

Standard Deviation

Si

Silicon

SPAWAR

Space and Naval Warfare Systems Command

SPLP

Synthetic Precipitation Leaching Procedure

SRM


Standard Reference Material

TCLP

Toxicity Characteristic Leaching Procedure

TMG

Trace-Metal Grade

USACE

U.S. Army Corps of Engineers

USEPA

U.S. Environmental Protection Agency

V

Vanadium

WDOE

Washington State Department of Ecology

Zn

Zinc



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xii


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1

1

Introduction

1.1

Background
The U.S. Environmental Protection Agency (USEPA) has adopted the incremental sampling methodology (ISM) as the accepted method (Method
8330B, 8330C) for sample collection and processing of soils containing
energetic residues on U.S. Department of Defense (DOD) training and
testing ranges (USEPA 2014, 2006a; Hewitt et al. 2009; Walsh et al. 2005;
Jenkins et al. 2004, 2005; and Pitard 1993). In addition to energetics, incremental sampling and associated processing procedures are increasingly
being adopted for other constituents introduced in particulate form, such
as metals (Hewitt et al. 2011, 2009; ITRC 2012; Alaska 2009; Hawaii
2008). ISM is a
structured composite sampling and processing protocol
that reduces data variability and provides a reasonably unbiased estimate of mean contaminant concentrations in a
volume of soil targeted for sampling. ISM provides representative samples of specific soil volumes defined as decision units (DUs) by collecting numerous increments of soil
(typically 30–100 increments) that are combined, processed, and sub-sampled according to specific protocols
(ITRC 2012).

Initially, ISM is focused on correct field sampling, then various manipulations of the samples are performed to create a single homogenized sample
that is analyzed for the constituents of interest, providing a more representative average concentration of the selected study area.
The Environmental Security and Technology Certification Program
(ESTCP) funded ER-0918 project, which developed new sampling and
sample preparation procedures falling under the ISM umbrella for soils
containing metal particulates (Clausen et al. 2012, 2013a, 2013b, 2013c).
USEPA Method 3050C will be introduced in the Method VI update to SW846 in 2016 (USEPA, forthcoming). However, the impact on sample processing, principally machining of the sample to reduce particle size, and its
effect on metal bioavailability and ultimately human and ecological risk is


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2

unknown (Clausen 2015). The ISM protocols may introduce a positive bias
in extraction efficiencies and bioavailability; the multi-increment sampling
methodology dictates that samples be ground to a particle size of 75 µm to
achieve a fundamental error of less than 15% (Hewitt et al. 2009). The act
of milling to such a fine particle size may increase the exposure and bioavailability of contaminants to test organisms used in toxicological bioassays.
The DOD has established directives mandating that all DOD facilities implement procedures to assess environmental impacts of munitions on
training and testing ranges (DOD 2004, 2005). Presently, many DOD installations are being directed to implement changes to their sample and
sample processing of soil and sediment samples for metals (Alaska 2009;
Hawaii 2008) in the absence of data showing that these changes are appropriate for assessing human and ecological risk and for establishing soil
cleanup levels. However, a common approach for calculating risk associated with soil exposure is by collecting and analyzing soil by using USEPA
Method 3050B (USEPA 1996) for digestion followed by Methods 6010 and
6020 for analysis (USEPA 2006b, 2006c). Method 3050B states
This method is not a total digestion technique for most samples. It is a very strong acid digestion that will dissolve almost all elements that could become “environmentally
available.”
Unfortunately, Method 3050B (USEPA 1996) does not define what is
meant by “environmentally available” and whether this is equivalent to bioavailability. Within the environmental industry, there is a lack of consensus on the proper sample preparation and analysis methods for soils containing metallic residues at military ranges. Studies with different physiologically based bioavailable extraction tests yield different results. The

document Bioavailability of Contaminants in Soils and Sediments: Processes, Tools, and Applications (NRC 2003) states the following:
Replacing default values with site-specific information
should be encouraged. . . . There is no clear regulatory
guidance or scientific consensus about the level and lines of
evidence needed for comprehensive bioavailability process
assessment.


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Therefore, the U.S. Army Environmental Command (AEC) funded this
project to address a U.S. Army concern on whether the widespread adoption of ISM as part of USEPA Method 3050B update would lead to a bias
in metals bioavailability results. Specifically, the concern relates to the
milling step of the ISM process and the assumption that this activity would
result in elevated metals levels as compared to the conventional sample
processing approach.

1.2

Objectives
The three objectives for this study were (1) to determine whether incorporating sample processing changes similar to those in Method 8330B into
Method 3050B yield soil or sediment metal concentrations appropriate for
human and ecological risk assessment; (2) to identify the appropriate bioavailability tests for various metals, depending on the different soil and
sediment types for ranges and to establish the relationship with Method
3050C; and (3) to determine whether the oversize fraction, >2 mm in size,
can be ignored as USEPA does not consider this material to be soil. Further, this study proposes providing context for the modified USEPA
method for metals in relation to bioavailability assessment approaches.
Our hypothesis is that milling (sometimes referred to as grinding) of soil
will change the estimated bioavailability of a particular metal; that metal
bioavailability is dependent on soil type, which has bearing on the appropriateness of a given bioavailability test; and that the oversize fraction contains a significant metal mass that should not be ignored.


1.3

Approach
Our study approach involves standard soil toxicity tests and novel techniques conducted in several phases. The in vitro studies focused first on
the development of toxicity metrics (e.g., half maximal effective concentrations [EC50s] and half maximal lethal concentrations [LC50s]) for the
common lumbriculid worm, Eisenia fetida, in soils spiked with copper using standardized protocols (ASTM 1997; WDOE 1996). Second, our study
tested both uncontaminated and contaminated soils having undergone
grain size partitioning prior to testing (as part of the ISM protocol). The
study used the earthworm (Eisenia fetida) for toxicology and bioaccumulation bioassays (ASTM 2009) and the ryegrass (Lolium rigidum) for a
seed germination bioassay (ASTM 2004). Various digestion experiments
(Clausen et al. 2010; USEPA 2007; Rodriguez et al. 1999; Ruby et al. 1996,

3


ERDC TR-16-4

1999) were conducted to assess the relative bioavailability (RBA) of metals
in soil or soil-like samples by measuring the rate and extent of metal solubilization in an extraction solvent that resembles gastrointestinal fluids.
The fraction of metal that solubilizes in an in vitro system is referred to as
in vitro bioaccessibility. This method may provide a faster and less costly
alternative for estimating RBA of metals than in vivo methods.

4


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2


Incremental Sampling Methodology
Multi-increment sampling has been established as the proper methodology for evaluating particulate deposition of energetic residues (Hewitt et
al. 2009; Ramsey and Hewitt 2005; Walsh et al. 2005; Jenkins et al. 2005,
2004; and Pitard 1993) and has been adopted as a new USEPA Method
8330B (USEPA 2006a) and 3050C (USEPA, forthcoming). There has been
increasing push to adopt the multi-increment sampling methodology for
other analytes, including metals (Hewitt et al. 2011; ITRC 2012; Alaska
2009; Hawaii 2008). One sample processing step of the multi-increment
sampling methodology involves machining (or grinding) of the sample to
increase the number of particulate contaminants of interest present in the
sample. ISM dictates that the samples be ground to a particle size less than
75 µm to achieve a fundamental error of less than 15% (Hewitt et al.
2009).
The act of grinding to such a fine particle size may increase the exposure
and bioavailability of contaminants to test organisms used in toxicological
bioassays; a topic explored in this study. Bioavailability studies are commonly used to assess the toxicity and bioavailability of a particular metal
to human and ecological receptors.
Another issue relates to the standard USEPA Method 3050B used for metals digestion (USEPA 1996), which according to the method yields the environmental fraction that a human or ecological receptor may encounter.
However, there is no documentation in the literature that establishes the
relationship between this environmental available fraction and accepted
bioavailability tests. Consequently, it is not possible to place the Method
3050B value in the proper context in regards to bioavailability. Our earlier
work evaluating different digestion procedures for tungsten in soil
(Clausen et al. 2010) indicated Method 3050B recovered considerably less
tungsten than did some commonly used European Union digestion methods (Figure 1). In addition, a modified Method 3050B involving milling
and some other changes to the sample preparation methods (Clausen et al.
2012) yielded results closer to presumed total digestion methods. Yet, the
results from using Method 3050B are typically used by risk assessors to
compute the human and ecological risk or to compare against soil reme-


5


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6

dial action levels. The present study will identify the appropriate bioavailability test for metals and will establish the relationship between Method
3050C and Method 3050B.
Figure 1. Comparison of prior digestion results for tungsten.

1400

Tungsten (mg/kg)

1200
1000
800
600

Unground (HNO3)
Unground (HNO3+H3PO4)
Unground (HF+HNO3+H3PO4)
Ground M (HNO3+H3PO4)
Ground C (HNO3+H3PO4)

400
200
0

0

200

400

600

800

1000

1200

1400

Method 3050B Tungsten (mg/kg)

The U.S. Army questioned whether the subsequent reduction in soil and
contaminant particle size through milling to control subsampling analytical errors might alter the relationship between the concentration of metals
reported and their actual bioavailability as compared to the unground or
conventionally prepared soil or sediment sample. Such an effect would
have a significant impact on inferences of human and ecological risk when
using Method 3050. Because metals in soils are found in a variety of mineral associations and chemical combinations of varying stability or solubility, the total metal content of a soil or sediment based on Method 3050B
often does not correlate well with toxicity or bioavailability measures due
to differences in digestion efficiencies (Rodriguez et al. 1999; Ruby et al.
1999, 1996). The bioavailable metal is typically only a fraction of the total
metal content that is truly available and capable of producing a toxic response. Despite this fact, risk assessors often use Method 3050 digestion
procedure to determine human or ecological risk or to set soil remedial action levels. If Method 3050 is to be used as an index of that risk, the relationship between toxicity/bioavailability and the analytical concentrations
reported by the modified 3050 method must be understood.



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7

A term often used when discussing bioavailability is absolute bioavailability (ABA), which is the ratio of the amount of metal absorbed compared to
the amount ingested, also referred to the oral absorption fraction (AFO):
ABA = AFO / Ingested Dose

(1)

RBA = (|ABA| × test material) / (|ABA| × reference material)

(2)

For example, if 100 micrograms (μg) of lead dissolved in drinking water
were ingested and a total of 50 μg entered the body, the ABA would be
50/100 or 0.50 (50%). Likewise, if 100 μg of lead contained in soil were ingested and 30 μg entered the body, the ABA for soil would be 30/100 or
0.30 (30%). If the lead dissolved in water were used as the frame of reference for describing the relative amount of lead absorbed from soil, the
RBA would be 0.30/0.50 or 0.60 (60%).

RBA is the ratio of the absolute bioavailability of a metal present in some
test material compared to the absolute bioavailability of the metal in some
appropriate reference material.


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3


Methods
Our study used a variety of methods to determine the amount of metal
present in the two soils tested and included the following:
•

•
•
•
•
•
•
•

Digestion methods using nitric acid (HNO3) (Method 3050C and B),
oxalate, glycine, ethylenediaminetetraacetic acid (EDTA), synthetic
precipitation leaching procedure (SPLP), toxicity characteristic leaching procedure (TCLP), and sequential digestion (Tessier et al. 1979)
In vitro bioaccessibility (Drexler and Brattin 2007)
Varying particle sizes (sieving and grinding)
In vivo survival and bioaccumulation studies over 14 and 28 days in the
earthworm (Eisenia fetida)
In vivo survival and bioaccumulation studies over 8 months in the
ryegrass (Lolium rigidum)
Physiological based extraction technique (PBET)
Diffusive gradients in thin films (DGT)
Analysis with inductively coupled plasma–optical emission spectroscopy (ICP-OES) and ICP–mass spectroscopy (MS) (Methods
6010/6020)

The earthworm (E. fetida) was used for toxicology and bioaccumulation
bioassays (ASTM 2009), and ryegrass (L. rigidum) was used for a seed

germination bioassay (ASTM 2004). The study evaluated in vitro bioaccessibility by using the method of Drexler and Brattin (2007), which the
USEPA has approved for lead. Initial particle size testing looked at any effects the milling process alone had on both plant and invertebrate bioassays. The smaller particle size itself may be toxic and influence the results
of the bioassays without any related contaminant toxicity. Clean artificial
control soil was made based on the formula outlined in American Society
of Testing Methods (ASTM) Methods E1676-04 and E1963-09 for the
earthworm and ryegrass, respectively (ASTM 2009, 2004). Our study performed a series of toxicity tests on a control (unmilled) soil and on a series
of processed soil milled to different particle sizes (e.g., <2 mm to 250 µm
and <250 µm). A split of these same samples was analyzed using the
USEPA Method 3050B. The results of this set of experiments guided the

8


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9

particle sizes used for the site soil testing and allowed a point of comparison between the total metal content of the soil and the environmentally
available metal as ascertained by Method 3050C.

3.1

Field sampling
Soil samples were collected on 2 October 2013 from the Texas small-arms
range berms (Figure 2) at the Donnelly Test Area, AK, where 200 rounds
of 7.62 mm ammunition were fired with an M-16 rifle. A total of 50 increments were collected from each berm following ISM (Clausen et al. 2013b,
2012; ITRC 2012). Soil contamination consisted of the metals antimony,
copper, lead, and zinc. The contaminated berms sampled were constructed
of loam and sand. Uncontaminated control berms of each material were
also sampled for a total of four site samples. Samples were collected using

the multi-increment sampling methodology sampling guidelines, Method
3050C, with a minimum of 50 increments used to create one single sample. To accurately address variability, each of the four berms was sampled
in triplicate, resulting in three replicate samples (each consisting of 50 increments).
Figure 2. Collection of field samples from the small-arms range berm at the
Texas Range on the Donnelly Training Area, AK.


ERDC TR-16-4

3.2

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Laboratory sample preparation
Once the field samples were collected, they were shipped back to the
ERDC Cold Regions Research and Engineering Laboratory (CRREL) in
Hanover, NH, for laboratory preparation. The samples were air dried and
sieved with a No. 10 mesh sieve to remove the >2 mm fraction, which is
commonly discarded (Figure 3). The USEPA does not consider >2 mm to
be soil even though this fraction can be a sizeable portion of the total metal
mass. The <2 mm fraction was then split in half with a Lab Tech Essa sectorial rotary splitter (Model RSD 5/8, Belmont, Australia) operated at 100
rpm. The weight for both splits was recorded. One of the <2 mm splits was
used for the unmilled experiments and the other for the milled experiments.
Figure 3. Study design sample processing hierarchy.

The ground fraction was created using the ISM techniques, which involved
using a Lab Tech Essa chrome steel ring mill grinder (Model LM2, Belmont, Australia) for five 60 sec intervals with 60 sec of cooling between
each interval. This length of grinding typically yields a material size less
than 150 µm (Hewitt et al. 2009).
The unground <2 mm sample was sieved with a no. 60 sieve, yielding

>250 µm and <250 µm fractions. The <250 µm fraction can stick to the


ERDC TR-16-4

hand due to electrostatic forces. Therefore, some risk assessors require
that analysis of this material yields a conservative risk calculation.
Each soil sample yielded 7 subsamples with 2 contaminated soils (loam
and sand) and two controls (loam and sand) for a total of 28 subsamples.
This material was then digested using a variety of extractants and methods.

3.3

Soil characterization
Solid samples were digested according to USEPA Method 3050B using nitric acid and hydrogen peroxide. Hydrochloric acid was not used to reduce
matrix interferences from chloride ions in the subsequent ICP-MS analyses. In certain cases, such as with plant tissues, additional hydrogen peroxide was used above the 10 mL described in the method if required to destroy residual organic matter prior to filtration, dilution, and analysis.
A series of sequential extractions was also performed to determine the speciation for lead (Baumann and Fisher 2011; Tessier et al. 1979). The most
bioavailable metals fraction is the labile fraction, which is loosely associated with soil particles. This labile fraction is easily extractable with magnesium chloride and sodium acetate at pH 5. Magnesium chloride yields
what is referred to as the exchangeable lead. Sodium acetate recovers lead
species associated with carbon, and the soluble fraction of lead is obtained
using deionized (DI) water. Hydroxylamine hydrochloride is used to recover lead oxides; and a mixture of hydrogen peroxide, nitric acid, and hydrochloric acid is used to recover lead species associated with organic matter and sulfides. Any remaining lead after the sequential digestion is referred to as the residual lead, which tends to be the insoluble solid lead
species. The sequential metal extraction process allows for a better discrimination of the influence of milling on the bioavailable fractions of the
metals versus total metal concentrations alone.
Glycine was used as an extractant following the procedures in USEPA
(2007). The glycine procedure is supposed to simulate a synthetic gastric
juice and has been previously validated using in vivo juvenile swine tests
(Drexler and Brattin 2007). An extraction using disodium EDTA at pH 7.0
was performed following the method of Quevauviller et al. (1997). This reagent sequesters metal ions associated with calcium (Ca2+) and iron (Fe3+),

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