RESEARCH ARTICLE

Soil quality indicators to assess functionality

of restored soils in degraded semiarid ecosystems
Miriam Muñoz-Rojas1,2,3,4 , Todd E. Erickson1,2 , Kingsley W. Dixon1,2,3 , David J. Merritt1,2

A thorough knowledge of soil functionality is critical to successful restoration of disturbed ecosystems, and its evaluation
involves the assessment of soil properties and processes as a component of a healthy ecosystem. Here, we propose a set of soil
quality indicators to assess the soil status in restored soils (topsoil and waste material) and test new methods that are easy to
apply, interpret, and cost-effective for the analysis of soil biological indicators in restored ecosystems. We show that in addition
to organic carbon and C:N ratio, biological indicators (microbial diversity and activity in particular) are the most sensitive
indicators to detect differences among reconstructed soils and analogue undisturbed soils in semiarid areas. The use of the
1-day CO2 test is proven to be an alternative cost- and time-effective method to measure microbial activity and assess soil
functionality of restored soils. Our results show a positive effect of vegetation on reconstructed soils and a recovery of soil
functionality in waste material to levels similar to topsoil once vegetation is established. However, soil functionality in both
restored waste materials and topsoils is still far from that in undisturbed native soils. We conclude that soil functionality is
critical in the restoration process, particularly in semiarid areas, and the methods used here could be effectively applied in a
broad range of restoration projects in arid and semiarid environments.
Key words: 1-day CO2 test, ecosystem services, microbial activity, mine restoration, Pilbara, soil health

degraded ecosystems should aim to not only recover the capac-

Implications for Practice
• Vegetation has a positive effect on restored soils, improv-
ing soil functions and processes through increasing micro-
bial activity and diversity, and levels of organic C and the
C:N ratio (connected to nutrient cycling processes).
• Waste materials used in extractive operations may be a
suitable alternative to topsoil to support vegetation estab-
lishment in restored areas. However, the use of organic
amendments is recommended to provide additional inputs
of organic matter.
• Microbial diversity and microbial activity are sensitive
and robust indicators to assess functionality of restored
soils in degraded arid and semi arid ecosystems.
• The 1-day CO2 test to measure soil microbial activity is
a rapid assessment tool for determining soil quality and
functionality in restoration projects.
Introduction
Soil functionality is commonly defined as the capability of a
specific soil to provide key functions in terrestrial ecosystems
such as biological productivity, nutrient cycling, or physical sta-
bility and support for plant growth (Brussaard 1997; Fitter et al.
2005). Approximately 80% of ecosystem services can be con-
nected to soil functions (Lal 2001), but these services can be
critically altered in disturbed ecosystems such as those resulting
from extractive activities (Oliveira et al. 2011). Thus, restora-
tion and/or rehabilitation (hereafter referred to as restoration) of
ity of soil to support vegetation but also to restore ecosystem
functions and services (Costantini et al. 2015; Lamb et al. 2015;
Perring et al. 2015).
At the same time, the effect of restored vegetation commu-
nities can be critical to regenerate soil functionality (Harris
2009; van der Putten et al. 2013). The feedbacks between the
soil–plant systems can function through pathways that involve
soil physicochemical and biological properties (Ehrenfeld
et al. 2005). An adequate vegetation cover not only provides
physical protection from erosion and increases organic matter
that aid water holding capacity (Cerdá 1997; Brevik et al.
2015) but also activates cycling of nutrients and microbial
processes, for example, through root exudates that serve as
substrates for the soil microbial communities (Garcia et al.
2005). These processes are particularly important in arid and
Author contributions: MMR conceived and wrote the manuscript, performed the
experimental research and analyzed the data; TE, KD, DM conceived and edited the
manuscript.
1 School of Plant Biology, The University of Western Australia, Crawley, 6009 WA,
Australia
2 Kings Park and Botanic Garden, Perth, 6005 WA, Australia
3 Department of Environment and Agriculture, Curtin University, Perth, 6845 WA,
Australia
4 Address correspondence to M. Muñoz-Rojas, email
miriam.munoz-rojas@uwa.edu.au
© 2016 Society for Ecological Restoration
doi: 10.1111/rec.12368
Supporting information at:
http://onlinelibrary.wiley.com/doi/10.1111/rec.12368/suppinfo

Restoration Ecology 1
Indicators to assess soil functionality
semiarid ecosystems, where vegetation patchiness is a common
characteristic of the environment and the landscape is shaped by
mosaics that include well-defined vegetated and nonvegetated
spaces (Cerdá 1997; Maestre & Cortina 2002). In these areas,
soil fertility and improved environmental conditions are usually
found in the plant-covered patches under the plant canopy, also
known as fertility islands (Goberna et al. 2007).
A common practice in open-cut and strip mining operations
involves an initial high-impact disturbance of removing the top
layer of soil prior to starting the extractive operations and sub-
sequently respreading of this stockpiled topsoil in areas tar-
geted for restoration (Keipert et al. 2002; Rivera et al. 2014;
Muñoz-Rojas et al. 2015). This topsoil layer is a source of seeds,
plant propagules, nutrients, and microorganisms (Jasper 2007;
Golos et al. 2016), and consequently is a key element in land
restoration programs (Lamb et al. 2015). However, topsoil is a
scarce resource and its deficit has been identified as an impor-
tant issue for the success of these restoration efforts. Hence,
alternative soil substrates such as waste materials produced in
mining operations are being used as growth media in restoration
(Machado et al. 2013). The use of these soil substrates can incor-
porate coarser materials that help to overcome slope instabil-
ity, hydraulic discontinuity, and prevent erosion processes (Kew
et al. 2007). These materials can also have pH or salinity levels
adequate for plant growth (Machado et al. 2013); however, these
substrates are often depleted in organic materials and nutrients,
making the establishment of plant species extremely difficult
(Shrestha & Lal 2006). In dry environments, these altered soil
conditions are also compounded by unpredictable precipitation
and long periods of drought, limiting plant establishment suc-
cess (Audet et al. 2013; James et al. 2013; James & Carrick
2016).
A thorough knowledge of soil functionality is critical to suc-
cessful restoration of disturbed ecosystems and its evaluation
involves the assessment of soil properties and processes as they
relate to the ability of soil to function effectively as a compo-
nent of a healthy ecosystem (Schimann et al. 2012). The use of
soil quality indicators may be an effective approach to assess
functionality of topsoil and novel substrates used in restora-
tion (Costantini et al. 2015). However, identifying indicators
that are sufficiently simple and robust yet provide enough infor-
mation to address the state of the soil and its trajectory of
change has proved to be a complex task for scientists and land
managers (Gonzales-Quiñones et al. 2011). The soil parame-
ters commonly used to estimate soil quality or functionality in
restoration include individual determinations of basic physical
and chemical factors (Schoenholtz et al. 2000; Sheoran et al.
2010); however, these fail to account for biological processes
(Knoepp et al. 2000; Izquierdo et al. 2005; Haney et al. 2008a).
Soil indicators need to integrate soil physical, chemical, and
importantly, biological properties such as microbial biomass,
microbial activity (MA), and microbial community composition
(Schimann et al. 2012; Mukhopadhyay et al. 2014).
Soil physical indicators such as bulk density (BD) or soil tex-
ture and structure are highly related to water holding capacity
and root development which are essential factors to sustain plant
communities through prolonged drought (Sheoran et al. 2010).
2 Restoration Ecology
Soil chemical properties, e.g. pH, electrical conductivity (EC),
organic carbon (OC), nitrogen, and other nutrients, are associ-
ated with soil fertility, and suitable levels of these parameters
are crucial for the establishment and survival of plant commu-
nities (Garcia et al. 2005; Hueso-González et al. 2014; Brevik
et al. 2015). However, microbial indicators are more sensitive
than physical and chemical properties to disturbances (Bastida
et al. 2008) and need to be routinely incorporated in soil assess-
ment studies in restoration programs (Haney et al. 2008a). For
example, soil MA and diversity play key roles in the sustain-
ability of vegetation communities by maintaining vital functions
in the soil ecosystem, involving carbon and nutrient cycling
(Harris 2009; Brevik et al. 2015; Orwin et al. 2015). In general,
biological indicators for assessing soil functionality can include
soil microbial biomass and number (Muscolo et al. 2014), soil
MA (Haney et al. 2008a), and community structure (Nannipieri
et al. 2003; Dimitriu et al. 2010). Other indicators commonly
used to assess soil functionality are the microbial indices and
quotients (Jangid et al. 2010), and the ratio between fungal and
bacterial communities (Bárcenas-Moreno et al. 2011).
There exist broad possibilities for methods and techniques
to assess soil indicators; nevertheless, these need to fulfill a
number of requirements to be practical quantitative tools for
soil functionality assessment in restoration (Bastida et al. 2008;
Mukhopadhyay et al. 2014; Costantini et al. 2015). Different
criteria have been proposed to select appropriate indicators, such
as those suggested by Doran and Zeiss (2000), who consid-
ered that they should be (1) sensitive to variations in manage-
ment, (2) strongly correlated with soil functions, (3) useful for
describing ecosystem processes, (4) easy to use and understood
by land managers, and (5) simple and cost-effective. Currently,
one of the available methods to measure soil respiration is the
1-day CO2 analysis or Solvita test (Haney et al. 2008b) that is
based on the evolution of CO2 during 24 h of a dried soil that
has been rewetted. This method has proved to be simple and
easy to interpret and can be a valuable tool for soil function-
ality assessment in ecosystem restoration (Haney et al. 2008a;
Muñoz-Rojas et al. 2016).
Through the research presented in this study, we propose a set
of soil quality indicators to assess the soil status in reconstructed
soils and we test new and inexpensive methods that are easy to
apply and interpret for the analysis of soil biological indicators
and the trajectory of restored ecosystems. Our main objectives
were to: (1) analyze soil physicochemical and biological quality
indicators in natural and restored soils; (2) test a new method
(1-day CO2 analysis) for analysis of soil MA as a biological
indicator of soil functionality in restored ecosystems; (3) ana-
lyze the relationships between soil physicochemical and biolog-
ical quality indicators to be used in restoration; and (4) assess the
effects of vegetation cover on soil functionality in restored areas.
Materials and Methods
Site Description
This study was conducted in the Pilbara interim biogeographical
region (north Western Australia, 22∘ 03′ S, 118∘ 07′ E–23∘ 19′ S,

119∘ 43′ E) at an iron ore mine site that was restored in 2011.
Climate in this region is semiarid with mean annual rain-
fall ranging between 250 and 400 mm (considering the
1971–2000 period), mostly concentrated in the summer
months (December–March), that register approximately 72%
of the total annual rainfall. These precipitations are mainly a
result of occasional thunderstorms and tropical cyclones. Mean
annual temperatures vary between 19.4 and 33.2∘ C with average
maximums over 40∘ C in the summer (Bureau of Meteorology
2015). Geology is complex with very ancient rock deposits (e.g.
Phanerozoic, Proterozoic, and Archaean sedimentary, granite,
volcanic rocks, and Devonian limestone) and soils comprise red
shallow stony soils on hills and ranges and sands on plains with
predominance of Red Kandosols, Red Ferrosols, and Leptic
Rudosols (Isbell 2002). Vegetation is predominantly composed
of hummock grasslands, tussock grasslands, sclerophyll shrub-
lands, and shrublands and woodlands with a tussock grass
understorey.
Experimental Design and Soil Sampling
The restored area covered 7.9 ha and included two subareas
with different soil materials used as growth medium: topsoil
retrieved from nearby stockpiles that were set aside for use in
restoration programs, which covered an area of 3 ha, and a lat-
eritic waste material utilized for its erosive stability and physical
competency, which covered an area of 4.9 ha. Following the
respread of these soil materials in 2011, a seed mix of native
legumes, grasses, and trees collected from the Pilbara region
was broadcast at the same dose of approximately 7 kg/ha. An
undisturbed natural shrub–grassland ecosystem dominated by
Triodia spp. and Acacia spp. representative of the restored area
was selected as the analogue reference site. Soil sampling was
carried out in March 2015 (at the end of the wet season). In
all the studied areas (restored and undisturbed), the dominant
plant species were similar but the vegetation cover differed in
both sites: 30% in the site restored with waste material, 55% in
the area with respread topsoil, and more than 70% in the nat-
ural reference site (plant cover estimates were obtained from
unpublished field observations and monitoring data). All sites
had similar conditions of elevation (between 200 and 300 m)
and slope (ranging from 14∘ to 16∘ ). Six plots (2 × 2 m) were
established at each of the restored subareas and the natural site
covering vegetated patches (n = 3) and nonvegetated patches or
bare soil (n = 3). Distance between plots was at least 4 m. From
each plot, a bulked soil sample composited from three individ-
ual samples was collected from the top 5 cm of the soil surface.
Samples were air-dried, sieved (2 mm mesh), and divided in two
subsamples, one was used for physical and chemical analysis,
and the other was stored at 4∘ C for 2 weeks before microbial
analysis.
Soil Physicochemical Analyses
Soil particle size (soil texture) was determined by laser diffrac-
tion using a Mastersizer 2000 (Malvern Instruments, Malvern,
U.K.) and BD and plant available water capacity (AWC) were
Restoration Ecology
Indicators to assess soil functionality
determined according to the methods proposed by Rawls (1983)
and Saxton and Rawls (2006) which use pedotransfer functions
based on soil texture and organic matter. Soil water repellence
(SWR) was measured using the water drop penetration time
(WDPT) test (Wessel 1988) and classified according to Bis-
dom et al. (1993). Soil pH and EC were measured in deion-
ized water (1:2.5 and 1:5 w/v, respectively), using a AD8000
microprocessor-based pH, conductivity, and temperature bench
meter (Adwa Instruments, Szegen, Hungary). OC was ana-
lyzed by dichromate oxidation following the Walkey and Black
method and total N with the Kjeldahl method. Available P was
determined by Mehlich Extraction (Mehlich 1984) followed
by inductively coupled plasma atomic emission spectroscopic
(ICP-AES) analysis.
Soil Microbiological Analyses
Soil MA was measured with the 1-day CO2 test, a cost-effective
and rapid method to determine soil microbial respiration rate
based on the measurement of the CO2 burst produced after
moistening dry soil (Haney et al. 2008a, 2008b). The method
is described in detail in Muñoz-Rojas et al. (2016).
Soil microbial abundance of specific groups was measured
by phospholipid fatty acid analysis (PLFA) as explained in
Muñoz-Rojas et al. (2016). The fungi to bacteria ratio (F:B)
was calculated from the amounts of PLFAs specific to these
particular groups (Frostegård & Bååth 1996). Soil microbial
biomass C (MBC) was determined based on the conversion
for bacterial and fungal biomass proposed by Frostegård and
Bååth (1996) and Klamer and Bååth (2004). Microbial diversity
(MD) was calculated by the total number of PLFAs and using
the exponential of Shannon’s Diversity index (Shannon 1948)
as follows: N = exp [−Σpi (ln pi)], where pi is the ratio of
the activity of each substrate to the sum of the activities on
all substrates. Species are represented by individual fatty acid
relative to the concentration of all fatty acids. The exponential
form of the Shannon index determines the effective number
of species or true diversity (Hill 1973). The respiratory or
metabolic quotient (qCO2 ) was determined as the microbial
respiration or CO2 -C evolution per unit of MBC (measured
with the 1-day CO2 test) and time (Anderson & Domsch 1993),
and the microbial quotient (Cmic: Corg) as the MBC to total
OC ratio (Anderson & Domsch 1990). The metabolic quotient
indicates loss (positive values) and enrichment (negative) of
MBC in the ecosystem. It declines with succession following
recovery after disturbance since microbial communities become
more efficient at conserving C under “equilibrium” conditions
(Muñoz-Rojas et al. 2016).
Statistical Analyses
Soil indicators and indexes were tested for normality and vari-
ance homogeneity using the Shapiro–Wilk and Levene tests,
and data were log transformed as necessary (presented data are
nontransformed). Differences in soil indicators among soil types
and vegetation cover type (vegetated and nonvegetated patches)
were tested using one-way analysis of variance (ANOVA) and
3
Indicators to assess soil functionality

Table 1. Soil physical and chemical indicators (SQ Indicator) across soil types (mean ± SE, n = 3, total n = 18). Different lower case letters indicate significant
differences within soil types across the three sites with vegetation cover or within the three sites with bare soil, and upper case (capital) letters indicate significant
differences between vegetated and nonvegetated patches for each soil type (LSD test, p < 0.05). NS, natural soil; TS, topsoil; WS, waste; AWC, available water
capacity; BD, bulk density; EC, electrical conductivity; OC, soil organic C; N, total N; C/N, organic C/N; P, total available P.

Vegetation Cover Bare Soil

SQ Indicator NS TS WS NS TS WS

AWC (%) 5.8 ± 0.2Aa 5.6 ± 0.1Aa 5.5 ± 0.3Aa 5.6 ± 0.1Aa 5.9 ± 0.1Aa 4.71 ± 0.1Ab
Sand (%) 82.2 ± 1.7Aa 83.8 ± 1.2Aa 83.9 ± 2.5Aa 83.4 ± 1.3Aa 82.0 ± 0.9Aa 88.6 ± 0.5Ab
Silt (%) 13.7 ± 1.2Aa 13.7 ± 1.1Aa 13.4 ± 2.0Aa 12.5 ± 0.8Aa 15.1 ± 0.8Ab 9.5 ± 0.4Ac
Clay (%) 4.1 ± 0.5Aa 2.5 ± 0.2Ab 2.7 ± 0.5Ab 3.8 ± 0.2Aa 2.8 ± 0.2Ab 1.9 ± 0.1Ac
BD (g/cm3 ) 1.5 ± 0.0Aa 1.5 ± 0.0Aa 1.6 ± 0.0Aa 1.6 ± 0.0Aa 1.6 ± 0.0Aa 1.5 ± 0.0Aa
EC (dS/m) 32.0 ± 4.6Aa 30.3 ± 3.5Aa 48.5 ± 4.0Aa 23.5 ± 3.6Aa 22.7 ± 3.9Aa 29.7 ± 7.4Aa
pH 6.4 ± 0.1Aa 6.4 ± 0.2Aa 6.8 ± 0.1Aa 6.2 ± 0.1Aa 6.4 ± 0.1Ab 6.9 ± 0.1Ab
OC (%) 0.6 ± 0.1Aa 0.5 ± 0.1Aa 0.2 ± 0.1Ab 0.4 ± 0.1Ba 0.2 ± 0.1Ba 0.1 ± 0.0Bb
N (%) 0.02 ± 0.0Aa 0.01 ± 0.0Ab 0.0 ± 0.0Ac 0.04 ± 0.0Aa 0.02 ± 0.0Aab 0.01 ± 0.0Ab
C/N 10.1 ± 0.3Aa 11.8 ± 0.4Aa 6.03 ± 0.3Ab 14.1 ± 0.6Bb 15.2 ± 0.4Bb 12.4 ± 0.6Bc
P (mg/kg) 2.3 ± 0.3Aa 1.3 ± 0.3Aa 1.7 ± 0.7Aa 2.3 ± 0.3Aa 1.7 ± 0.3Aa 1.5 ± 0.4Aa

Table 2. Soil microbial PLFAs across soil types (mean ± SE, n = 3, total n = 18). Different lower case letters indicate significant differences within soil types
across the three sites with vegetation cover or within the three sites with bare soil, and upper case (capital) letters indicate significant differences between
vegetated and nonvegetated patches for each soil type (LSD test, p < 0.05). NS, natural soil; TS, topsoil; WS, waste; TM, total microbial; TB, total bacteria;
TF, total fungi; PS, pseudomonas; AC, actinomycetes; GP, Gram (+) bacteria; GN, Gram (−) bacteria; MF, mycorrhizal fungi.

Vegetation Cover Bare Soil

PLFAs (mg/kg) NS TS WS NS TS WS

TM 34.5 ± 9.1Aa 13.6 ± 0.5Ab 12.3 ± 0.9Ab 19.3 ± 1.9Aa 7.4 ± 2.2Ab 3.3 ± 0.5Bb
TB 10.2 ± 3.2Aa 3.9 ± 0.2ba 3.2 ± 0.2Ab 4.8 ± 0.5Aa 2.3 ± 0.5Bb 1.2 ± 0.2Bc
TF 23.4 ± 8.1Aa 9.8 ± 0.3Ab 8.6 ± 0.7Bb 14.2 ± 1.3Aa 5.1 ± 1.8Ab 2.0 ± 0.3Bc
PS 0.6 ± 0.0Aa 0.3 ± 0.0Ab 0.5 ± 0.1Ab 0.3 ± 0.0Aa 0.1 ± 0.0Bb 0.2 ± 0.0Bb
AC 2.5 ± 0.6Aa 1.0 ± 0.1Ab 0.8 ± 0.1Ab 1.4 ± 0.2Aa 0.8 ± 0.1Bb 0.5 ± 0.0Bb
GP 1.8 ± 1.1Aa 1.6 ± 0.1Aa 1.2 ± 0.1Aa 0.4 ± 0.1Aa 0.9 ± 0.2Bb 0.5 ± 0.1Bb
GN 2.3 ± 0.9Aa 2.3 ± 0.2Aa 2.1 ± 0.2Aa 1.1 ± 0.1Aa 1.4 ± 1.3Ba 0.8 ± 0.1Ba
MF 2.7 ± 1.1Aa 1.7 ± 0.0Aa 1.3 ± 0.2Aa 1.5 ± 0.2Aa 0.1 ± 0.1Bb 0.0 ± 0.0Bb

comparisons between means were performed with the Tukey’s
HSD (honestly significant difference) test (p < 0.05). Principal
component analysis (PCA) was used to assess differences and
clusters in soil variables between soil materials. Pearson’s corre-
lations were used to analyze further relationships between mea-
sured soil indicators. The relationships between MA measured
with the 1-day CO2 test and OC, MBC, MD, and PLFAs were
analyzed using linear and nonlinear regressions. All analyses
were performed with R statistical software version 3.1.2 (R Core
Team 2014). The R package FactomineR, which auto scales and
centers the variables, was used for the PCA analysis.
Results
Soil physical, chemical, and microbiological indicators were
generally significantly (p < 0.05) different across growth media
types, but the extent of these variations depended on the vege-
tation cover (Tables 1 and 2). Soil characteristics from restored
areas, in particular from those with returned topsoil, tended to
be similar to those of natural sites in areas covered by vegetation
for both substrates (topsoil and waste).
There were significant differences in AWC (p < 0.05) and soil
texture (sand and silt contents) across soil types, but these differ-
ences were only significant under bare soil (Table 1). Neverthe-
less, although statistically significant, differences in AWC were
rather small and would not make a real difference of soil condi-
tions in the field. All the soil samples analyzed were hydrophilic,
or nonwater repellent (<5 seconds). Values of OC and N were
significantly (p < 0.05) higher in natural soils and topsoil com-
pared to the waste for both the vegetated sites and bare soil sites
(Table 1). Although soil pH was higher in the waste material
of vegetated and bare sites, similar values were found for bare
soil and areas covered by vegetation for natural soils and top-
soil. Under bare soil, there were not significant differences in
EC between soil materials, but under vegetation, this chemical
indicator was significantly (p < 0.05) lower in the natural soils
and topsoil, compared to the waste. Between vegetated and non-
vegetated patches, only OC and C:N were significantly different
(p < 0.05); higher values of both indicators were observed in
areas covered with vegetation.
Microbial abundance of both bacteria and fungi groups dif-
fered significantly (p < 0.05) between soil types under bare soil
(natural soil > topsoil > waste), but under vegetated areas, the

4 Restoration Ecology
 Indicators to assess soil functionality

(A) (B)

25 2.5
NS TS WS
Aa

MA (ppm CO2–C h–1)

20 2 Aa
MBC (mg kg–1)

15 1.5
Aa
10 1 Aa Aa
Ab Ab Aa
Ba
5 Ab 0.5
Bc Bb

0 0
Vegetation cover Bare soil Vegetation cover Bare soil

(C) (D)
4
120 Aa
Aa Ba
3 Aa
90 Aa
Bb Ab
MD

F:B
2 Bc
60
Ab Ab
Ab
30 1
Bc

0 0
Vegetation cover Bare soil Vegetation cover Bare soil

(E) (F)
4
0.25
Aa Aa
Aa
0.2 3
Aa
Cmic:Corg

Aa Aab
qCO2

0.15
Aa 2
Aa Aa
Aa Aa
0.1 Ab
1
0.05

0 0
Vegetation cover Bare soil Vegetation cover Bare soil

Figure 1. Soil microbial indicators: (A) microbial biomass C (MBC), (B) microbial activity (MA), (C) microbial diversity (MD), (D) F:B ratio, (E) metabolic
quotient (qCO2), and (F) microbial quotient (Cmic:Corg) across soil types (mean ± SE, n = 3, total n = 18). Different lower case letters indicate significant
differences among soil types within the same vegetation cover and upper case (capital) letters indicate significant differences between vegetated and
nonvegetated (bare) patches for each soil type (LSD test, p < 0.05).

differences between the reconstructed soils (topsoil and waste)
were not significant (Table 2). Under bare soil, mycorrhizal
fungi (MF) was absent in the waste, and very low in the topsoil,
compared to soils from the analogue sites (e.g. on average 40%
less in topsoil than in natural soils). However, although in both
reconstructed soils MF differed significantly (p < 0.05) with nat-
ural soils in these spaces between plants, similar levels of MF
were found for all soil types under the canopy of plants (Table 2).
PLFAs were more abundant in the vegetated areas compared to
bare soil, but differences were only significant (p < 0.05) for the
reconstructed soils (both topsoil and waste).
Both MBC and MA showed significant (p < 0.05) differ-
ences between soil types under bare soil (Fig 1A & 1B). In
these nonvegetated bare patches, MBC of areas restored using
topsoil was on average 55% less than in natural soils and these
values were much lower (88%) in areas restored with waste
materials. However, due to the large variation of both MBC
and MA found in natural soils, together with higher values of
both microbial indicators under vegetated areas, differences
between restored soils in vegetated areas were not significant
(p < 0.05). Similar trends were found in MD (Fig. 1C) where
differences between soil types were significant under bare soil

Restoration Ecology 5
Indicators to assess soil functionality

(A) (B)
1 20
y = 9.564x0.9816
y = 0.5332x1.0245
r2 = 0.8124
0.8 r2 = 0.8096
15

MBC (mg kg–1)

Soil OC (%)

0.6
10
0.4

5
0.2

0 0
0 0.4 0.8 1.2 0 0.4 0.8 1.2
(C) (D)
40 120
y = 14.176e1.9735x
y = 20.594x0.9826
PLFA microbial (mg kg–1)

r2 = 0.6421
r2 = 0.8123
30 90

20 60

MD
10 30

0 0
0 0.4 0.8 1.2 0 0.4 0.8 1.2
Microbial activity (ppm CO2–C h–1) Microbial activity (ppm CO2–C h–1)

Figure 2. Relationships between microbial activity and (A) soil organic C; (B) microbial biomass C; (C) total abundance of PLFA microbial; and
(D) microbial diversity.

(p < 0.05), but vegetated areas showed similar values between
reconstructed soils. Under bare soil, the F:B index was lower
in the rehabilitated soils compared to the analogues, but an
opposite trend was found under vegetated patches where both
topsoil and waste showed a higher index than the topsoil
(Fig. 1D). The qCO2 was slightly higher in topsoil compared
to natural soils and waste (Fig. 1E), but these differences were
not significant (p < 0.05). The Cmic:Corg revealed an opposite
trend with lowest values in the topsoil in both vegetated and
nonvegetated patches (Fig. 1F). However, these differences
were only significant under bare soil (p < 0.05). Among these
soil microbial indicators, MA and MD showed the largest
differences between the vegetated areas and bare soil.
The correlation matrix (Table S1, Supporting Information)
displayed correlation coefficients for the combination of linear
relationships among all the studied soil variables, showing the
direction and the strength of these relationships. Soil pH was
significantly (p < 0.05) and negatively correlated with % clay
(r = 0.52), N (r = −0.50), and MD (r = −0.60). The abundance
of all microbial groups measured with PLFA (Table 2) was sig-
nificantly correlated with texture, e.g. negative with % sand (r
ranging between −0.51 and −0.55) and positive with % clay (r
ranging between 0.71 and 0.80). PLFAs also correlated signif-
icantly with OC and N (r = 0.62–0.80), MD (r = 0.70–0.73),
and particularly with MA (r = 0.94–0.95). MA was also
significantly (p < 0.05) and positively correlated with OC and
N (r = 0.78 and r = 0.62, respectively), and MD (r = 0.60).
The power model was the best fit to describe the relationship
between MA and OC (r2 = 0.8096), MBC (r2 = 0.8124), total
PLFAs (r2 = 0.8123), and MD (r2 = 0.6421) (Fig. 2). All these
models were significant at the p < 0.001 level. The PCA (Fig. 3)
indicated that the first two axes could explain 66.5% of the total
variation of the soil samples: 54.5% by axis 1 and 12% by axis
2. All the soil microbial parameters, OC, and N, accounted for
a large amount of the variation in the distribution of samples
along axis 1, which discriminated natural soils and topsoil from
waste. In particular, samples from waste under bare soil showed
a clear clustering.
Discussion
In this study, we assessed a number of soil quality indicators
in areas restored using different substrates as reconstructed
growth media (topsoil and waste material) and compared these
to natural analogue sites. Soil samples were taken 4 years
after restoration under the canopy of reinstated vegetation,
and in spaces without vegetation cover (bare soil). Measuring
soil characteristics in these nonvegetated patches allowed us
to have a reference of soil conditions similar to those before
vegetation establishment. Our results showed that most of the

6 Restoration Ecology

Figure 3. Biplot of the first and second axes obtained from the principal
component analysis showing dependencies between soil quality indicators.
Points represent soil samples (n = 18). BS, bare soil; VC, vegetation cover;
Abbreviations of soil variables as in Tables 1 and 2 and Figure 1.
soil physicochemical parameters measured and proposed as
quality indicators differed among soil types (reconstructed and
natural soils) in the bare spaces between plants, but these
indicators tended to be similar in areas covered by vegetation.
The biological indicators also captured differences between
vegetated and nonvegetated patches and showed that microbial
abundance, activity, and diversity largely diverged from natural
sites in both reconstructed soil types.
Soil quality indicators can be effectively used as direct
measurements of soil properties, or as indirect measures of soil
functions (Izquierdo et al. 2005; Costantini et al. 2015). Conse-
quently, they are being increasingly used in the assessment of
soil quality and health, and importantly, in monitoring and eval-
uating their direction of change with time or after disturbances
(Zornoza et al. 2007; Bastida et al. 2008; Muñoz-Rojas et al.
2016). The choice of suitable indicators is challenging because
of the difficulty in standardizing methods and the varying spatial
scales in which they can be applied (Conant & Paustian 2002;
Bastida et al. 2008). And, although some indicators are more
suitable than others to capture differences among different soil
types, the use of individual soil properties (in particular phys-
ical and chemical) can have serious limitations (Doran & Zeiss
2000; Zornoza et al. 2007). Our results showed that some of the
soil physicochemical indicators are, in general, able to detect
differences between soil materials. However, except for OC and
C:N ratio, these parameters are rather similar between vege-
tated and nonvegetated patches, reflecting the poor influence of
established plants on these soil properties. Soil physicochem-
ical characteristics have been extensively used as soil quality
indicators, particularly in agriculture (Mukherjee & Lal 2014)
but also in the assessment and monitoring of restoration of
degraded ecosystems (Schoenholtz et al. 2000; Mukhopadhyay
Restoration Ecology
Indicators to assess soil functionality
et al. 2014). A key soil chemical indicator is soil OC that has
been widely used as a key attribute of soil quality because of
the many functions that it provides and supports (Muñoz-Rojas
et al. 2012; Willaarts et al. 2015). Low contents of soil OC are
generally associated with decreased values of soil fertility and
biodiversity and a loss of soil structure, which as a consequence
can diminish water holding capacity, and increase BD and hence
soil compaction (Lal 2004; Shrestha & Lal 2006). Although
lower values of soil OC were found in the area restored with
topsoil, compared to the analogue site, differences were not sig-
nificant, and values of OC in the topsoil were almost double in
the vegetated patches than in the nonvegetated patches. There-
fore, even though the removal of these topsoil layers occurs prior
to extractive operations, there might have been a short-term neg-
ative effect on soil fertility, such as topsoil stockpiling that can
have a detrimental effect on soil fertility (Keipert et al. 2002).
However, processes associated with soil OC such as nutrient
cycling seem to have recovered after 4 years postrestoration pro-
vided those areas have had successful vegetation establishment.
The C:N ratio, a property that is also related to soil productivity,
was lower in the area restored with waste in both vegetated and
nonvegetated patches, which is in agreement with Ganjegunte
et al. (2009) and Banning et al. (2011a). In their study of a
postmining forest rehabilitation chronosequence, Banning et al.
(2011a) found that C:N ratios of the older restoration sites
were lower than nonmined forest. Ganjegunte et al. (2009) also
obtained significantly lower C:N ratios in mined soils compared
to undisturbed and attributed this difference to altered rates of
mineralization. In our study, these ratios are significantly lower
under the canopy of restored plants, which might be explained
by additional N inputs from the legumes (Acacia shrubs) used
in the restored operation (Banning et al. 2011a).
Soil biological (microbiological) indicators have proved to be
the most sensitive for detecting differences not only among the
soil types but also between vegetated and nonvegetated patches.
Both the diversity and abundance of soil microorganisms are
related to functions such as decomposition of C and N, and plant
and root development (Garcia et al. 2005; Goberna et al. 2007).
Assuming that soil conditions in both bare soil and vegetated
patches are similar prior to mining, these microbial indicators
provide valuable surrogates of the trajectory of pedogenesis
following restoration. Soil microbial biomass has been exten-
sively used in soil quality assessments (Bastida et al. 2008) as it
comprises the living part of the soil organic matter (e.g. fungi,
bacteria, protozoa, algae). Usually, an increase in the microbial
biomass is considered beneficial to the soil system, whereas a
decline is considered negative; but a comparison of a degraded
soil with a reference undisturbed soil can provide a higher level
of interpretation (Gonzales-Quiñones et al. 2011). Our results
show a considerably larger abundance of microbial biomass in
the analogue or reference areas compared to the restored soils
in both vegetated and nonvegetated patches. Higher contents of
PLFAs were found under the canopy of plants particularly in
the waste soil, with four times the amount of microbial biomass
in the vegetated areas compared to the bare soil. This significant
increase reflects the recovery of the microbial community in
the waste substrate to levels similar to those found in topsoil,
7
Indicators to assess soil functionality
yet these contents are 30% less than the values of analogue
sites.
The metabolic (qCO2) and the microbial (Cmic: Corg) quo-
tients represent the energy optimization as the ecosystems
recover and the proportion of MBC over the total OC, respec-
tively (Anderson & Domsch 1990, 1993). Differences in both
indices among soil types were unclear, a similar finding to pre-
vious studies (Garcia et al. 2005; Hedo et al. 2015). Although
these microbial indicators have been successfully used to assess
substrate quality and ecosystem response to disturbances across
different soil types and environments (Jangid et al. 2010; Ban-
ning et al. 2011b; Muñoz-Rojas et al. 2016), they have also been
subjected to criticism by a number of authors for the lack of
sensitivity in distinguishing between the effects of disturbance
and stress (Bastida et al. 2008). The F:B ratio showed that fungi
levels were similar in natural soils and topsoil regardless of veg-
etation cover. However, a pronounced difference was observed
in the waste between vegetated and nonvegetated patches which
indicates a successful recolonization of the fungal microbial
communities in this soil substrate. Fungal communities can
effectively recover within the first 4 years following natural dis-
turbances in semiarid areas (Muñoz-Rojas et al. 2016). Other
studies have shown a complete recovery of microbial fungi after
5 years since mine reclamation in cold environments (Dangi
et al. 2012). However, this process can require a longer period
of time in semiarid environments (Frost et al. 2001) although it
might be accelerated by nutrient inputs of established vegetation
(Martínez-García et al. 2011). Both MA and MD have an impor-
tant role in sustaining essential soil functions that predominantly
involve carbon and nutrient cycling (Orwin et al. 2015). In our
study, these indicators, in particular, have shown sensitivity to
detect differences among soil types and vegetation cover.
An extensive number of techniques are currently available to
determine soil microbial parameters and many have been used
to assess restoration capability. These methods can be biogeo-
chemical and physiological, e.g. chloroform fumigation extrac-
tion, and substrate induced respiration (Gonzales-Quiñones
et al. 2011), metabolic, e.g. enzymatic activities (Izquierdo et al.
2005; Dimitriu et al. 2010), and more recently, molecular, such
as the analysis of soil extracted nucleic acid sequences (DNA,
RNA) (Banning et al. 2011a). An alternative to molecular meth-
ods is the determination of PLFAs a biogeochemical approach
that provides information on both microbial taxonomic and
functional diversity (Dimitriu et al. 2010; Muñoz-Rojas et al.
2016). However, here we have shown that the 1-day CO2 test
to measure MA can be effective in discriminating between soil
substrates and natural soils, even in arid environments such
as our study area that are characterized by lower amounts of
soil C and MA (Conant et al. 2004; Wang et al. 2014). Previ-
ous studies showed the strong correlation of the MA measured
with this method with MBC or N mineralization (Haney et al.
2008a, 2008b), but our results confirmed also a positive and sig-
nificant correlation with soil organic C, microbial abundance,
and MD. Therefore, the use of this test provides an alternative
to other expensive and time consuming specialized techniques
for an effective assessment of soil functionality in restored
soils.
8 Restoration Ecology
In general, our results reflect the effective recovery of soil
functions of the waste material to levels similar to the topsoil
after 4 years following restoration once vegetation is estab-
lished, which means that native vegetation can increase the lev-
els of fertility of these poor soils relatively quickly. Therefore, in
the absence of topsoil as a growth media, the use of certain types
of waste materials can be a promising alternative in restoration
of degraded areas. However, the assessment of soil quality indi-
cators, particularly those related to critical soil functions such
as OC, microbial abundance, or MD, reflect that restored soils
(both topsoil and waste) are still far from reaching the quality
levels of natural analogue sites. Moreover, in the restored areas,
the established vegetation covers approximately 35 and 55% of
the area for waste and topsoil, respectively, which is dissim-
ilar to the vegetation cover of reference areas (>70%). It has
been reported that the patchy distribution of vegetation in arid
and semiarid systems creates locally improved conditions of soil
fertility under the plant canopy that consequently support micro-
bial mediated ecosystem processes and favors C mineralization
and nitrification processes (Cerdá 1997; Goberna et al. 2007;
Martínez-García et al. 2011). Thus, reinstating a higher vegeta-
tion cover density in the restored areas, similar to undisturbed
ecosystems, could promote a faster recovery of soil functions
and more importantly, a long-term sustainability of the restored
ecosystems.
The positive effect of vegetation on the restored soils is evi-
dent in this study, but additional inputs of organic matter would
be beneficial to increase the vegetation cover and optimize the
restoration success. Thus, the use of soil amendments at appro-
priate doses could be an effective strategy to increase soil OC
and optimize soil functions (water holding capacity, nutrient
cycling processes), particularly during the initial plant growth
stages of seedling emergence and early establishment when the
positive effects of vegetation may not be evident (Curtis &
Claassen 2009; Jordán et al. 2011; Hueso-González et al. 2014).
These plant stages are particularly critical in restoration of arid
and semiarid areas where very low rates of success are attained
(James et al. 2013) and degraded soils could be sustained with
external sources of OC.
In conclusion, our results show the recovery of soil function-
ality of the waste material to levels similar to the topsoil once
vegetation is established, but both topsoil and waste do not reach
the levels of OC, MA, or MD of undisturbed soils, at least within
4 years postrestoration. Here, we show that the role of soil func-
tionality is critical in the restoration process, particularly in arid
and semiarid areas, and the methods used in this research could
be easily applied in a broad range of restoration projects.
Acknowledgments
We thank Brad Stokes and Cameron Mounsey for their help
with the collection of soil samples. This research was supported
by a BHP Billiton Iron Ore Community Development Project
(contract no. 8600048550) under the auspices of the Restoration
Seedbank Initiative, a partnership between BHP Billiton Iron
Ore, The University of Western Australia, and the Botanic
Gardens and Parks Authority.

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Supporting Information
The following information may be found in the online version of this article:
Table S1. Pearson’s correlation with relationships between main soil indicators (n =
18). Correlation coefficients (r) with boldface are significant at p < 0.05. BD, bulk
density; EC, electrical conductivity; OC, soil organic C; N, total N; C/N, carbon to
nitrogen ratio; P, total available P; MA, soil microbial activity (basal respiration); TM,
total microbial abundance; TB, total bacteria abundance; TF, total fungi abundance;
MBC, microbial biomass C; qCO2 , MA to MBC; Cmic:Corg, MBC to OC ratio; F:B,
fungi to bacteria ratio; MD, microbial diversity.
Received: 4 January, 2016; First decision: 4 February, 2016; Revised: 15 March,
2016; Accepted: 15 March, 2016
Restoration Ecology