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Arabian Journal for Science and Engineering

https://doi.org/10.1007/s13369-018-3641-0

RESEARCH ARTICLE - CIVIL ENGINEERING

Assessing the Effect of Density and Water Level on the Degree of


Compaction of Sand Using Dynamic Cone Penetration Test
Abdulrahman M. Hamid1 · Omar S. Baghabra Al-Amoudi2 · Saad A. Aiban2

Received: 30 June 2018 / Accepted: 18 November 2018


© King Fahd University of Petroleum & Minerals 2018

Abstract
Saudi Arabia has witnessed unparalleled development in all types of construction, especially in eastern Saudi Arabia where
the major oil and petrochemical industries are located. The major projects are mostly located on sandy soil, and it is difficult
to assess the physical properties of this soil using conventional methods. Consequently, laboratory and field studies were
conducted to evaluate the density of sandy soil utilizing dynamic cone penetration test (DCPT). The laboratory study was
conducted on sand specimens prepared with different relative densities (40%, 60% and 90%). Additionally, the effect of the
level of water table on DCPT values was investigated. The density of soil at two field sites was assessed by using DCPT and
nuclear gauge. Test results indicated that an increase in the dry density and shear strength was associated with a decrease in
the dynamic cone penetration index. Further, the variation in the water table level had a significant impact on DCPT results
due to the changes in the effective stress. The DCP resistance for fully drained sand increased significantly by as much as
170% compared with the fully submerged or dry sand, due to the development of negative pore water pressure. Further, the
results of this investigation have shown that DCPT is an effective and reliable tool to assess the degree of compaction of
sandy soils. Reliable correlations between density, relative density and degree of compaction of sandy soils have also been
developed.

Keywords Dynamic cone penetration test · Nuclear gauge · Sandy soil · Relative density · Water table · Field and laboratory
tests

1 Introduction electrical resistivity. However, the nuclear gauge is the most


widely used method to measure the in situ density of soil
Density, which is the ratio of mass to volume, is one of as it provides rapid and accurate results with minimal effect
the essential engineering properties of soil that is used in of human error [1]. However, there are some restrictions for
the design and construction of foundations, dams, retaining using a device with a nuclear source due to its negative effects
walls, embankments and other structures. The in situ den- on human health [1]. Furthermore, the nuclear gauge tech-
sity of soil is determined using various techniques, such as nique can be used to a depth of about 300 mm only.
sand replacement (sand cone), balloon, nuclear gauge and Sand is the most predominant type of soil in the Eastern
Province of Saudi Arabia, which is confined by gigantic sand
B Abdulrahman M. Hamid deserts from three directions (the Empty Quarter, Ad-Dahna
A7hamid@uwaterloo.ca and Al-Nafud). Because of the lack of cohesion in sandy soil,
Omar S. Baghabra Al-Amoudi it is not possible to obtain undisturbed samples. Since the
amoudi@kfupm.edu.sa dynamic cone penetration test (DCPT) has been widely used
Saad A. Aiban for field exploration and quality assessment of subsoil layers,
saiban@kfupm.edu.sa this technique may be useful to assess the density of sandy
1 Department of Civil and Environmental Engineering, soils. In addition, the DCP technique can be used for the
University of Waterloo, 200 University Ave W., Waterloo, characterization of subgrade and base materials. Perhaps, the
ON, Canada most important advantage of the DCP technique is its ability
2 Department of Civil and Environmental Engineering, King to provide a continuous record of the relative soil strength
Fahd University of Petroleum and Minerals, Dhahran 31261, with depth [2]. Moreover, the DCP device is distinguished
Saudi Arabia

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by its economy and simplicity to operate and its superiority 2 Laboratory Study
to provide repeatable results and rapid property assessment.
Further, DCPT has the main features of both cone penetration 2.1 Material
test (CPT) and standard penetration test (SPT) [3].
Some applications of the DCPT include correlations with The sand used in this investigation was brought from the
resilient modulus, California bearing ratio (CBR), uncon- dunes located in eastern Saudi Arabia. It was light golden in
fined compressive strength, shear strength, as well as its use color and had a uniform gradation, as shown by the grain-
in quality control of compaction of fills and performance size distribution in Fig. 1. The soil can be classified as poorly
evaluation of pavement layers [4–9]. Consequently, several graded sand (SP) according to the Unified Soil Classification
DCP–CBR correlations have been developed for different System and as A-3 according to the American Association
materials, for both laboratory and field conditions [10–20]. of State Highway and Transportation Officials (AASHTO)
Undoubtedly, DCPT has proven to be an efficient technique classification system.
in assessing the properties of soil and evaluating the degree of The maximum dry density (ASTM D 4253) was
compaction of embankments and other soil structures. How- 1.84 g/cm3 , and the minimum dry density (ASTM D 4254)
ever, due to the difficulties in using CPT, correlations between was 1.63 g/cm3 . The other properties of the selected sandy
DCPT and CPT data have been used, thereby increasing the soil are summarized in Table 1.
applicability of DCPT [21–23].
Abu-Farsakh et al. [24] reported, based on laboratory and
field studies using DCP, plate load test (PLT), falling weight
deflectometer (FWD), CBR and dynamic cone penetration 100

tests can be used to evaluate the density of subgrade and


80
pavement layers. Moreover, they developed empirical corre- Percent Passing
lations between the DCP data with elastic modulus, resilient
60
modulus and CBR. They also indicated that the DCP test
was an efficient tool to control the degree of compaction in 40
earthen structures.
Mohammadi et al. [25] developed relationships between 20
the dynamic cone penetration index, relative density, modu-
lus of elasticity, shear modulus, modulus of subgrade reaction 0
0.0 0.1 1.0 10.0
and the friction angle of soil with a high coefficient of deter-
mination (more than 90%). They used a mold measuring Grain size (mm)
700 mm in diameter and 700 mm in height and conducted
Fig. 1 Grain-size distribution curve for the natural sand used in the
DCPT and PLT. laboratory study
Kim et al. [26] conducted DCPT and Clegg impact ham-
mer tests to develop criteria for assessing the degree of
soil compaction. Based on their field and laboratory results,
minimum-required dynamic cone penetration blow counts Table 1 Properties of the dune sand used in the laboratory study
were proposed for various types of soils. Property Value
Since sandy soils are widely prevalent in eastern Saudi
Arabia and many parts of the world, there is a genuine need to Particle size range, mm 0.1–0.9
investigate the possibility of using DCPT to assess the in situ Specific gravity 2.68
density of such soils. In addition, the influence of water table D10 , mm 0.146
level on the DCPT values needs also to be studied [27,28]. D30 , mm 0.2
Consequently, the reported study also evaluated the effect of D60 , mm 0.29
the water level on the penetration resistance of a sandy soil Cu 1.98
using the DCPT. Hence, the main objective of the reported Cc 0.94
study was to assess the effect of density and level of water AASHTO Classification A-3
table on the DCPT values and to develop reliable correlations USCS Classification SP
between the density of sand and its dynamic cone penetration γd (max), g/cm3 1.84
index (DCPI). Both laboratory and field tests were conducted γd (min), g/cm3 1.63
to achieve the objectives of the study. Clay (%) 0
Silt (%) 1

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2.2 Density Control Procedure shear mold, tamping by a small square wooden plate with a
dimension of 60 × 60 mm was used. All the direct shear tests
Several trials were conducted to determine the most appro- were carried out in dry condition, as per ASTM D 5321.
priate and accurate way to obtain the required density. The
trials were conducted in a relatively small mold (750 mm 2.3 Control of the Water Table Level
diameter and 450 mm height) [27]. Then, the required den-
sity was achieved in a cylindrical glass reinforced plastic pipe Two drain holes (25 mm diameter), which were connected
(GRP pipe) measuring 1600 mm in diameter and 1500 mm in by two valves (see Fig. 2), were drilled in the chamber to
height and, thereafter, the DCPT was determined. It is to be facilitate the drainage of the water from the chamber during
noted that the soil chamber is a well-established technique the tests. Furthermore, in order to control the water level in
for the standardization of in situ testing devices, where the the chamber during the test, the valves were connected to
soil sample is prepared in the chamber and tested under con- transparent pipes to monitor the water level. The inlets of
trolled boundary conditions [29]. The GRP pipe was fixed the valves were covered with a filter to prevent the discharge
on a stiff steel plate (width = 1700 mm, length = 1700 mm, of soil particles during drainage, as schematically shown in
thickness = 300 mm) using epoxy. The schematic diagram of Fig. 2. The following four cases were investigated to study
the soil chamber is shown in Fig. 2. the effect of the water table level on the DCP resistance: (1)
Two techniques were utilized to achieve the required den- dry condition, (2) when the water table was at a distance of
sity: pluviation and vibration. In the pluviation technique, a 466 mm above the base (equating to one-third of the layer
large funnel was used to prepare loose sand sample in the thickness of the soil), (3) fully submerged sand and (4) the
chamber with the desired density. The relative density of effect of negative pore water pressure (full drainage, whereby
the loose sand deposited in the chamber was controlled by the water was removed from the sample) to study the effect
two factors: (1) size of the nozzle at the bottom of the fun- of the water table level on the DCP resistance.
nel (25 mm maximum diameter) and (2) sand drop height
(1000 mm), as shown in Fig. 3a. However, the medium 2.4 Test Procedure
and dense sand samples were prepared using the vibration
technique, whereby the sample in the testing mold were com- The DCP tests under laboratory conditions were conducted in
pacted in five 300-mm-thick lifts. The compaction effort was accordance with ASTM D 6951, using an 8 kg hammer. Nine
applied using a 300-mm-long vibrating rod with a circular DCP tests were performed to assess the density of sand for a
plate of 300 mm diameter and 17 kg weight, whereby the total penetration of 1500 mm, three DCP tests for each rela-
plate contained a 50 mm opening in the middle to allow the tive density (40%, 60% and 90%). The DCPI value for each
rod to enter in and out of the sand sample. The relative den- test was obtained by dividing the total penetration through
sity of medium and dense sand deposited in the chamber was the sample (in mm) by the number of blows [30]. It is to
controlled by the time of vibration. Medium density sand be noted that the readings for the top 300 mm were always
samples were vibrated for 1 s while the dense density sand neglected due to the loose nature of soil at that location.
samples were vibrated for 10 s, by inserting the rod in the Two DCPTs were conducted on medium dense sand for
sand sample, as depicted in Fig. 3b. each case of the following water table levels: (1) at the base of
To prepare the soil sample for the direct shear test, a square the chamber (zero level or dry condition), (2) at one-third of
shear box with a 60 × 60 mm internal dimension and 25 mm the soil thickness, (3) at the top of the soil (full saturation) and
height was used. To achieve a uniform compaction in the (4) at full drainage (suction). The DCP test was performed
for a total penetration depth of 1400 mm.

Transparent GRP Pipe


pipes
Chamber
(25 mm Thick) 3 Field Study
1600 mm diameter and 3.1 Location Description
1500 mm height

Pipe (25 mm) The field study was performed at two construction areas
Valve
along the Arabian Gulf. The purpose of the field study was to
Filter fabric
assess the in situ soil density using the DCPT and the nuclear
gauge techniques. DCP was used to assess the variation of
Stiff Steel Plate the density of the backfill at depths ranging from 1.2 to 2.4 m,
while the nuclear gauge was used to assess the dry density at
Fig. 2 Schematic diagram of the soil chamber depths ranging from 1.2 to 1.6 m.

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Fig. 3 Density control


procedure a pluviation
technique, b vibration technique

The DCPT and nuclear gauge tests were performed at ran- three tests were conducted for each density set. Figure 5
domly selected locations, distributed over a large area within presents the laboratory results of the nine series of tests for
each of the two construction sites. The backfill material con- three different relative densities (40%, 60% and 90%). The
sisted mainly of poorly graded dune sand, and the backfilling data for each relative density were clustered together, thereby
thickness was variable depending on the level of the original indicating the consistency and repeatability of the test results.
ground surface. Additionally, it should be noted that the DCP blow counts
increased with depth, particularly in the case of dense sand,
3.2 Test Procedure whereby the increase was noted to be significant. For exam-
ple, at a depth of 1400 mm, the average number of blows was
The DCP tests were conducted in accordance with ASTM 23, 39 and 118 for loose (40%), medium (60%) and dense
D 6951, using an 8 kg hammer. The variation in the density (90%) sand, respectively. This may be attributed to the fact
of the dry soil with depth was determined using the nuclear that during the compaction process, the lower layers get com-
gauge at random locations of the selected sites. The readings pacted further. The increased resistance to the impact load
at the referenced locations were considered as the control val- could also be partly attributed to the tendency of dense sands
ues. Figure 4 shows the locations at which DCP and nuclear to dilate upon penetration of the cone [27]. On the contrary,
gauge tests were conducted. in the case of the loose samples, the DCP resistance did not
exhibit a significant change with the depth.
4 Results and Discussions
4.1 Laboratory Study 4.1.2 DCPI Correlations

4.1.1 Effect of Soil Density on DCPT Based on the laboratory results, correlations were established
between the DCPI and relative density of sand. The relative
The repeatability of the DCP test results is an important con- density of sand was calculated using the following equa-
sideration that was evaluated for all the test sets. Therefore, tion [31]:

Fig. 4 Field test arrangement


Nuclear gauge

Dynamic cone penetration test 1000 mm

300 mm

1000 mm 300 mm
A A
Section (A-A)
Top View

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0
Table 2 Statistical data for the developed equations
DCP1-Loose Sand
Relation Correlation equation Coefficient of
DCP2-Loose Sand
DCP3-Loose Sand correlation (R 2 )
DCP1-Medium Sand
DCP2-Medium Sand γd –DCPI γd = 2.02/(DCPI)0.04 0.99
DCP3-Medium Sand
Penetration Depth (mm)

500 DCP1-Dense Sand Dr –DCPI Dr = 320.38/(DCPI)0.5 0.99


DCP2-Dense Sand
DCP3-Dense Sand e–DCPI e = 0.457 + 0.002 ∗ (DCPI) 0.94
φpeak –DCPI φpeak = 51.58/(DCPI)0.03 0.99
φpeak –Dr φpeak = 42.5 + 0.06 ∗ (Dr ) 0.99

1000

in the relative density resulted in an increase in the friction


angle of sand similar to that noted in the reported study.

1500 4.1.3 Effect of Water Table

0 25 50 75 100 125 150


Figure 8 presents the variation of penetration resistance with
No. of Blows
depth for the two DCP tests on the dry medium dense sand
Fig. 5 Variation of number of blows with depth for natural dry sand at conducted at two different locations. The DCP resistance
different densities value was around 3 blows/100 mm. It should be noted that
there was a vivid increase in the penetration resistance for
   the medium dense sand layer at a depth between 1300 and
γd − γmin γmax
Dr = (1) 1400 mm from the surface due to an increase in the density
γmax − γmin γd
as a result of the vertical confining pressure.
Figure 9 depicts the effect of the water table level on the
where Dr is the relative density, γd is the dry density, γmax DCP when the water level was at one-third of the thickness of
is the maximum dry density and γmin is the minimum dry the soil layer (at 466 mm from the base). The DCP resistance
density of sand. increased from 2 blows/100 mm within the top third layer of
The data were statistically analyzed to determine the best the stratum (similar to that depicted in Fig. 8) to a value of
correlation between DCPI and the relative density, as shown about 5 blows/100 mm above the water table level. The high-
in Table 2. It could be noted that an increase in the relative est DCP resistance was noted at a depth between 634 mm
density of sand tends to decrease the value of DCPI, as shown and 934 mm from the surface of the sand sample (466 mm
in Fig. 6a, b. On the contrary, an increase in the void ratio of and 766 mm from the base). This relatively high value (5
sand resulted in an increase in the DCPI, as shown in Fig. 6c. blows/100 mm) occurred at the level under suction (capil-
The void ratio was calculated using the following equation lary zone) above the water table. The suction zone is known
[31]: to induce negative pore water pressure, thereby increasing
the effective stress and shear strength of the soil leading to
Gs the stiffening of the soil deformation response and increas-
e= −1 (2)
γd ing the DCP resistance [33,34]. Further, the DCP resistance
decreased to 4 blows/100 mm below the groundwater table
where e is the void ratio, G s is the specific gravity and γd is since the pore water pressure became positive.
the dry density of sand. In the case of fully submerged sand, the DCP resistance
The correlation between the relative density and DCPI was between 2 and 3 blows/100 mm throughout the full sand
with the peak friction angle of sand is illustrated in Fig. 7 thickness, as shown in Fig. 10, whereby the sand pore voids
and Table 2. An increase in the relative density from 40 to were fully filled with water and the soil could be assumed
90% resulted in an increase in the peak friction angle from to be saturated. However, the hydrostatic pressure resulting
45 to 48 degrees. Furthermore, it was also noted that an from the weight of water and the uplift force, due to buoy-
increase in the friction angle resulted in a decrease in the ancy from the effective weight of water-filled sand, might
DCPI. It is to be reported that several correlations between have induced small skeletal forces for the submerged sand
the relative density and friction angle have been proposed by compared to dry sand [35]. Therefore, the effective stress of
different researchers including Meyerhof [32] and Moham- sand has decreased due to the development of positive pore
madi et al. [25]. These researchers indicated that an increase water pressure, as indicated in the following equation:

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Fig. 6 Correlation between 1.9 100


DCPI with: a dry density, b
relative density and c void ratio d = 2.02*(DCPI)
-0.04
Dr = 320.38*(DCPI)
-0.5

R2=0.99 R2=0.99

Relative Density (%)


Density (ton/m3)
75
1.8

50

1.7

25

0 20 40 60 80 0 20 40 60 80
DCPI (mm/blow) DCPI (mm/blow)
(a) (b)

e = 0.002(DCPI)+0.457
0.8 R2=0.94
Void Ratio

0.6

0.4

0.2

0
0 20 40 60 80

DCPI (mm/blow)

(C)

Fig. 7 Correlation between 60 60


friction angle with: a relative
density and b DCPI ɸpeak = 42.5 + 0.06*(Dr) ɸpeak = 51.58/(DCPI)0.03
55 55
R2 = 0.99 R2 = 0.99
Friction Angle (deg)
Friction Angle (deg)

50 50

45 45

40 40

35 35

30 30

40 60 80 100 0 20 40 60 80

Relative Density (Dr) DCPI (mm/blow)

(a) (b)

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0 0
DCP1
DCP2
DCP1
DCP2
DCP1

Penetration Depth (mm)


Penetration Depth (mm)

500 500

DCP1
DCP2

DCP2

1000 1000

1500 1500

0 5 10 15 20 0 5 10 15 20
Blow Count/100mm (N/100mm)
Blow Count/100mm (N/100mm)

Fig. 8 DCP resistance for dry medium dense sand Fig. 10 Effect of full submergence (WT level = 1400 mm) on DCP
resistance of medium dense sand

0
DCP1 significant negative influence on the stiffness and resistance
DCP2 of sand to DCP penetration.
In the case of full drainage (water was removed from
the sample), the DCP resistance value increased to about 8
DCP1 blows/100 mm at a depth of 700 mm, as depicted in Fig. 11,
Penetration Depth (mm)

500
DCP2
compared with 3 blows/100 mm for submerged or dry sand.
The high resistance in the full drainage situation may be
ascribed to the development of negative pore water pressure
that increases the effective stress of the sand sample, whereby
1000
the effective stress is given by the following equation:

σ́ = σ − (−u o ) (4)

where σ́ is the effective stress, σ is the total stress of soil and


1500 u o is the pore water pressure. It is to be noted that the increase
in the DCP resistance is about 170% compared with that for
0 5 10 15 20 the submerged or dry sands, as schematically summarized in
Blow Count/100mm (N/100mm) Fig. 12.

Fig. 9 Effect of water table level (WT level = 466 mm) on DCP resis-
tance of medium dense sand 4.2 Field Results

Figures 13 and 14 present typical field data on the varia-


σ́ = σ − (+u o ) (3) tion of DCP blow count and dry density with depth at the
selected construction sites along the Arabian Gulf. The data
in Fig. 13 indicate that the soil is very densely compacted at
where σ́ is the effective stress, σ is the total stress of soil and a depth between 300 and 700 mm with a dry density of about
u o is the pore water pressure. Such a decrease in the effective 1.85 g/cm3 and 1.75 g/cm3 (more than 5 blows/100 mm).
stress for the case of submerged sand explains the reason for Furthermore, at depth from 700 to 1400 mm, the penetration
the decrease in the penetration resistance (in other words, resistance decreased as verified by a very loose material (with
when the pore pressure was positive), thereby indicating a a dry density of about 1.65 g/cm3 and 1.55 g/cm3 ) because of

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0
tested locations for the thickness of the backfills (less than 5
DCP1
DCP2
blows/100 mm).
In general, the typical field results depicted in Fig. 13
indicate that there is significant variation in the degree of
DCP1
compaction within the same location. Such variability was
Penetration Depth (mm)

500 clear both laterally and with depth. The DCP blow counts
per unit depth decreased significantly as the depth increased
for both tests. This is an indication of inconsistent degree
DCP2 of compaction (the contractor might have compacted the top
lifts of the stratum very well but did not do the same effort
1000 with the deeper layers). It is known that when sand exists
at low densities, it can settle easily upon wetting or due to
loading or vibration from heavy structures. This will certainly
have a negative effect on the future settlement of the buildings
and utility pipes supported on or buried within such soils. It is
1500 therefore recommended to have a rectification methodology
that takes into account the deficiency in compaction. Such a
0 5 10 15 20
rectification methodology should consider the impacts on the
Blow Count/100mm (N/100mm) installed underground utilities and other installations as well.
Fig. 11 Effect of full drainage on DCP resistance of medium dense
Comparison of the field results (Figs. 13 and 14) indicate
sand that the soil compaction was very good in one location com-
pared to the other. The top layers (within 1000 mm) exhibited
little cementation between the sand particles, as reflected
by high penetration resistance values at most of the tested
locations. The penetration resistance at a depth between 500
and 1000 mm of the top layer decreased but it still indicated
a very dense material as the DCP blow count per 100 mm
was more than 5 [27]. This observation is confirmed by the
nuclear gauge density readings, as depicted in Fig. 14. It was
observed that an increase in the dry density resulted in an
increase in the number of blows. Based on the field inves-

Dry Density, g/cm3


2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2
0

DCP 1
DCP 2
400 Dry Density
Penetration Depth (mm)

800

Fig. 12 Effect of water level for four cases of medium dense sand

1200

the low DCP blow count (less than 5 blows/100 mm) for the
sand in the field, which indicates the presence of a very loose 1600
material [27]. This finding is confirmed by the nuclear gauge
density readings, as shown in Fig. 13. It can be observed
0 5 10 15 20 25 30 35 40 45
that the density of the sand backfill was reflected by the
Blow Count per 100 mm (N/100mm)
DCP results which indicate good correlation between the
two techniques. The soil compactness, as measured by the Fig. 13 Typical variation of DCP data and dry density data with depth
blow count, was less than the acceptable range for most of the at Al-Jubail area

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Arabian Journal for Science and Engineering

Dry Density, g/cm3 5. In the case of submerged sand, the effective stress
2.2 2.1 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 of sand decreased due to the development of positive
0
pore water pressure, thereby leading to a negative
influence on the strength of sand and a decrease in
400 the DCP resistance.
DCP 1 6. In the full drainage situation, the DCP resistance
Penetration Depth (mm)

DCP 2 increased by as much as 170% compared to that for


800 Dry Density the submerged or dry condition due to the develop-
ment of negative pore water pressure thereby leading
1200
to a significant increase in the shear strength and stiff-
ness of sand.
7. The DCPT technique was found to be useful in assess-
1600 ing the effect of water level in sand and has the
potential to be used as a quality control and quality
assurance tool.
2000
0 5 10 15 20 25 30 35 40 45 • Field study:
Blow Count per 100 mm (N/100mm)
1. There was a good correlation between the nuclear
Fig. 14 Typical variation of DCP and dry density data with depth at gauge and the DCP techniques in predicting the den-
Ras Al-Khair area sity of sand.
2. The field data have clearly indicated the non-
tigation, it appears that the soil stratum at one of the sites homogeneity in the compaction effort in the two
(Fig. 14) was consistent and consisted of very dense sandy selected sites. Variations in the soil density with depth
soil. and between different locations were noted.
3. The DCPT data have indicated that this technique is
5 Conclusions an effective tool for the assessment of in situ strength
of sand backfills.
Laboratory and field tests were conducted to assess the relia-
Acknowledgements The authors express their gratitude to Hadhramout
bility of the DCPT in evaluating the properties of sandy soil Establishment for Human Development and King Fahd University of
under different conditions. The following conclusions could Petroleum and Minerals (KFUPM) for the opportunity and support
be drawn based on the field and laboratory tests: to accomplish this work. Special thanks are to Sheikh Abdullah A.
Bugshan for his continuous support.

• Laboratory study:
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