Abstract
The aim of the study was to determine the effect of stand species
composition on PAHs accumulation. The study covered the soils of the
stands of the Rybnik Forest District, which are under the influence of
one of the highest deposition of industrial emissions in Europe. Pine,
pine-oak and oak stands growing in the same soil conditions were
selected for the study. Samples for further analyses were collected from
the organic horizon, from the humus mineral horizon and from the mineral
horizon. Organic C content, N content, pH, alkaline cation content, as
well as microbiological biomass of C, N, extracellular and intracellular
enzyme activity were determined in the soil samples. PAH content was
determined in the soil samples. Additionally, the soil organic matter
fractions were determined: free light fraction (fLF), occluded light
fraction (oLF) and mineral associated fraction (MAF). Pine stand soils
were characterized by the highest average PAHs content. The oak stand
soils were characterized by the lowest PAHs accumulation and high
enzymatic activity. The study confirms the important role of the stand
species composition in shaping the quality and quantity of SOM and soil
acidification, which in turn is reflected in microbial activity and PAHs
accumulation in forest soils. PAHs accumulation in forest soils is
related to the fractional composition of SOM, which is the effect of the
influence of species composition through the supplied aboveground and
belowground biomass. A strong correlation between the PAH content and C
content of the light soil fraction of OM was noted.
Keywords: enzyme activity; organic contamination; soil organic
matter; PAHs; forest soils degradation
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are organic compounds composed
of two or more interconnected benzene rings causing soil environment
contamination (Sun et al., 2012; Ghosal et al., 2016; Cao et al., 2020).
The prevalence of PAHs in the soil environment is mostly of
anthropogenic origin (Zhou et al., 2005), due to the increasing use of
energy from the combustion of fossil fuels, oil and internal combustion
engines (Tiwari et al., 2011; Lui et al., 2017). As a result of the
sorption capacity of soils, nearly 90% of total PAHs emissions present
in the ecosystem are accumulated (Eom et al., 2007). In forest soils,
organic pollutants are stored, captured and delivered to the litter by
tree crowns (Matzner, 1984). The accumulation of PAHs in soils depends
in particular on the amount and quality of soil organic matter and soil
pH (Chaineau et al., 2005; Wu et al., 2008; Lladó et al., 2009; Aichner
et al., 2015). As a result of the impact of woody vegetation, forest
soils are characterized by a high content of organic carbon, which is
reflected in a higher accumulation of PAHs compared to agricultural
soils. In addition to soil organic matter, pH and fine fraction content
is important in the accumulation of PAHs in the soil environment (Luo et
al., 2008). The pH value is an important parameter in the PAH
biodegradation process as it is a key factor for the availability of
nutrients and thus for the development of microorganisms involved in
PAHs degradation. The amount and quality of organic matter of forest
soils is determined by the species composition of the stand (De Deyn et
al., 2008; Błońska et al., 2016; Błońska et al., 2017). The stand,
through litter fall and root systems, shapes soil pH and soil chemical
properties (Reich et al., 2005; Mareschal et al., 2010). Coniferous
stands soils have a lower pH compared to mixed or deciduous stands soils
(Jandl et al., 2004). Strong soil acidification results in a reduction
of sorption capacity, causing reduced biological activity and disruption
of C, N and P cycle in the forest ecosystem (Mueller et al., 2012). The
fall of litter in deciduous stands has a positive effect on soil pH and
provides macroelements to improve biological activity of forest soils
and faster circulation of organic matter (Hobbie et al., 2007; Błońska
et al., 2016).
The soil organic matter, due to its heterogeneous structure and
composition, shows strong sorption properties for most of the persistent
organic pollutants, in particular PAHs (Ehlers & Loibner, 2006;
Ukalska-Jaruga et al., 2015). Three fractions of soil organic matter are
distinguished: free light fraction (fLF), aggregate-occluded light
fraction (oLF) and mineral associated fraction (MAF) (Wambsganss et al.,
2017). The fractional composition of soil organic matter determines its
sorption capacity (Six et al., 2002). Labile C can remain in the ground
from a week to years and recalcitrant C can persist for decades or even
centuries. SOM associated with minerals can be stabilized due to its
protection from mineralization, whereas the light fraction is more
susceptible to changes to soil use and altered litter inputs (Grüneberg
et al. 2013; Li et al. 2019a). Identification of SOM fraction can serve
as an indicator of changes in store carbon (Błońska et al. 2017).
The processes of soil organic matter decomposition depend on the
diversity and activity of soil microorganisms. The primary decomposers
(bacteria and fungi) release extracellular hydrolytic enzymes into their
immediate environment and these catalyze organic matter decomposition
(Berg & McClaugherty, 2008). Enzymatic activity informs about the
condition of the soil environment and the activity of soil
microorganisms (Gil-Sotres et al., 2005; Li et al., 2019b). Soil
microorganisms and enzymes secreted by them are responsible for the
circulation of basic nutrients such as C, N and P (Makoi & Ndakidemi,
2008). Thanks to microorganisms and their enzymes, toxic organic
compounds such as PAHs are removed from the soil environment (Haritash
& Kaushik, 2009). Biodegradation of these pollutants depends primarily
on the moisture, pH, soil temperature and bioavailability of organic
matter contained in the soil (Johnsen et al., 2005). The biological
breakdown of persistent, organic pollutants by microorganisms is one of
the most important and effective methods of removing PAHs from the
environment. The rate of biodegradation changes is influenced by the
composition and activity of the microorganisms, the properties and age
of the contamination, the presence of other compounds and the physical
and chemical properties of the environment (Lors et al. 2012).
The aim of the study was to determine the effect of stand species
composition on PAH accumulation in forest soils. Pine, pine-oak and oak
stands growing on the same soil conditions were selected for the study.
The following hypotheses were tested during the study: 1) stand species
composition through the amount and quality of soil organic matter and
soil acidification influences the PAHs accumulation; 2) deciduous
species positively influence the soil enzymatic activity as a result
increasing the PAHs degradation; 3) PAHs accumulation in forest soils is
related to the fractional composition of soil organic matter.
Materials and methods
Study area and soil sampling
The study was conducted in the Rybnik Forest District of southeastern
Poland (50°05′55″N; 18°32′42″E) (Fig. 1). The average annual temperature
in this area was 8.4°C and the average annual rainfall was 705 mm. The
field sites were located in an area where the soils derived from glacial
sand; they are dominated by Brunic Arenosols (WRB 2015). The Rybnik
Forest District was located in one of the most exposed areas of
industrial emissions in Europe (Fig. 2) (EEA 2020). The average annual
concentrations of benzo(a)pyrene [BaP] for the Rybnik Forest
District for the years 2017-2026, exceeded the admissible standards
according to the Directive 2004/107/EC (1
ng.m3) reaching the result in the
range of 5-12 ng.m3 (Environmental
Impact Assessment Report, 2016). Average annual PM10 concentration of 53
µg.m3 recorded in Rybnik Forest
District exceeded the permissible standard (20
µg.m3) and as same as average annual
concentrations of PM2.5, reaching the result of 26
µg.m3 exceeded the permissible standard (10
µg.m3) (WHO, 2005).
The study areas were selected during field observations. The research
covered stands of similar age, one-story structure and the same canopy
density. The age of the stands is 80 years. Three coniferous stands, the
main species of which was Scots pine (Pinus sylvestris L.), three
mixed stands, formed by Scots pine (Pinus sylvestris L.) and
Pedunculate oak (Quercus robur L.), and three deciduous stands,
formed by Pedunculate oak (Quercus robur L.), were selected for
the study. The stands grew on soils with a similar grain size, the
average sand content was 81%, silt content 16% and clay content 3%.
The samples were taken from the organic horizons (Of, Oh, Ofh), humus
mineral horizons (AEes, A) and mineral horizons (B) (Fig. 3). In order
to determine enzyme activity, microbial biomass and PAH content, fresh
samples of natural moisture were taken, sifted through a sieve (ø 2 mm)
and stored at 4°C in the dark before analysis.
Laboratory analysis
The particle size distribution was determined using laser diffraction
(Analysette 22, Fritsch, Idar-Oberstein, Germany). The soil pH was
determined in H2O and KCl using the potentiometric method. C and N were
measured using an elemental analyser (LECO CNS TrueMac Analyzer Leco,
St. Joseph, MI, USA). The concentration of calcium, potassium, magnesium
and sodium was determined by an ICP (ICP-OES Thermo iCAP 6500 DUO,
ThermoFisher Scientific, Cambridge, U.K.). Available phosphorus was
measured using a Bray–Kurtz method.
The PAHs were determined in 10 g of each soil sample, extracted using 70
ml of 2-propanol. The samples were centrifuged (4500 rpm, 5 min) and the
supernatant collected. The supernatants were extracted to the solid
phase (5 ml/min) using solid-phase extraction (CHROMABOND® CN/SiOH). The
residue was dissolved in acetonitrile and analysed using high-pressure
liquid chromatography (HPLC) with a Dionex UltiMate 3000 HPLC system,
equipped with a fluorescence detector and a Dionex UltiMate 3000 Column
Compartment C18 5 μm with a 4.6x100-mm HPLC column. The mobile phases
were water (A) and acetonitrile (B) at a flow rate of 1 ml/min. Based on
the standard PAH Calibration Mix (CRM 47940) at a concentration of 10
μg/ml, calibration solutions at different concentrations (i.e. 0.1, 0.2,
0.5, 1 and 2 μg/ml) were prepared. Each prepared solution was placed
into the chromatography column, the chromatograms obtained being used to
produce a calibration curve. The soil samples were then analysed in
triplicate. After every nineth analysis, a control sample (a calibration
solution with a concentration of 0.1 μg/ml) was injected. Naphthalane
[Nft], Acenapthene [Ace], fluoren [Flu], phenanthrene
[Phe], antracen [Ant], fluoranthene [Flt], pyrene [Pyr],
benzo(a)anthracene [BaA], chrysene [Chr], benzo(k)fluoranthene
[BkF], benzo(b)fluoranthene [BbF], benzo(a)pyrene [BaP],
dibenzo(ah)anthracene [DBahAnt] indeno(1,2,3-c,d)pyren [IcdP],
and benzo(g,h,i)perylene [BghiP] were determined. The activity of
extracellular enzymes (β-D-cellobiosidase [CB], β-glucosidase
[BG], xylanase [XYL], N-acetyl-β-D-glucosaminidase [NAG],
phosphatase [PH] was determined using fluorogenically-labeled
substrates (Pritsch et al., 2004; Turner, 2010; Sannaullah et al.,
2016).
The fluorescence was measured on a multidetection plate reader
(SpectroMax), with excitation at a wavelength of 355 nm and emission at
460 nm. The dehydrogenase activity (DH) was determined using Lenhard’s
method according to the Casida procedure (Alef & Nannipieri, 1995). For
the determination of microbial biomass C, microbial biomass N and
microbial biomass P, the fumigation and extraction method was used
(Jenkinson & Powlson, 1976; Vance et al., 1987).
Physical separation of soil organic matter fractions was performed using
the method described by Sohi et al. (2001). A sample of soil (15 g) was
placed in a 200-ml centrifuge tube and 90 ml of NaI (1.7 g cm−3) was
added. Each tube was gently shaken for 1 min and centrifuged for 30 min.
The free light fraction (fLF) was removed using the pipette and
collected on a glass fiber filter. The soil remaining at the bottom of
the centrifuge tubes was mixed with another portion of 90 mL of NaI and
subjected to sonication (60 watts for 200 s) to destroy aggregates.
After centrifugation, the matter released from aggregates occluded light
fraction (oLF) was collected on glass fiber filter. The remaining
fraction was assumed to consist of mineral associated fraction (MAF) of
SOM. After drying (40 °C), the subsamples of different fraction were
weighted and analyzed for CfLF, CoLF, and CMAF, respectively using an
LECO CNS True Mac Analyzer (Leco, St. Joseph, MI, USA).
Statistical anaylsis
The Pearson correlation coefficients for the soil characteristics were
calculated. Principal component analysis (PCA) was used to evaluate the
relationships between the soil properties and PAH content. Analysis of
variance was employed to assess the differences between the average
values of the soil properties and PAH content. Properties of different
humus types were compared using a parametric honestly significant
difference (HSD) test. The multiple regression method was used to
develop models describing the relationship between the PAH content and
soil properties. All the statistical analyses were performed using
statistical package R (R Core Team 2020) and R Studio (RStudio Team
2020).
Results
Physicochemical properties
Soil pH ranged from 3.57 to 3.98 and increased with soil depth (Tab. 1).
Soils with pine stands were characterized by the lowest pH. Pine-oak
stands were characterized by a higher pH, while oak stands were
characterized by the highest pH. The content of carbon, nitrogen and
phosphorus was statistically higher in the organic horizons of the
tested soils (Tab. 1). In the mineral horizons, there was a clear
decrease in C, N and P content. The highest content of organic carbon,
i.e., 33.32%, was recorded in the organic horizons of pine stand soils.
Mixed pine-oak stands were characterized by a lower organic carbon
content of 23.23% and the lowest C content, i.e., 20.18% was found in
oak stands. The highest nitrogen content was recorded in organic
horizons of pine stand soils (1.42% in the Of horizon and 1.06% in the
Oh horizon). Lower nitrogen content was recorded in the soils of
pine-oak stand (1.04% in the Ofh horizon) and the lowest nitrogen
content was recorded in the organic horizons of oak stand soils
(1.00%). The C/N ratio showed the highest values in pine stand soils,
lower in pine-oak stand soils and the lowest in oak stand soils. The sum
of alkaline cations reached statistically higher values in the organic
horizons of the tested soils (Tab. 1).
Biochemical parameters
In all stand variants, organic soil horizons were characterized by
significantly higher enzymatic activity (Tab. 2). The activity of
enzymes decreased significantly with the depth of soil profile. In
general, the highest activity of the studied extracellular enzymes in
terms of percentage of organic carbon was found in organic horizons,
especially organic horizons of pine stands. In the case of DH and NAG
activity, the highest values were recorded in the organic horizons of
oak stand soils. In the case of mineral humus horizons, the highest
activity of DH and PH was recorded in the oak stand soils (Tab. 2). As
in the case of enzymatic activity, significantly higher microbiological
biomass of C and N was recorded in surface soil horizons (Tab. 2). The
highest MBC was recorded in the top horizon of pine stands (2414.77
µg·kg-1), while the second one was recorded in the top
horizons of oak stands (2296.67 µg·kg-1), and the
lowest MBC z was noted in the soils of pine-oak stands (2097.24
µg·kg-1). The highest MBN was recorded in topsoil
levels of oak stands (461.25 µg·kg-1), lower in
pine-oak stand soils (421.62 µg·kg-1), and the lowest
in pine stand soils (335.18 µg·kg-1) (Tab. 2). In
mineral humus horizons, the highest MBC and MBN was recorded in oak
stand soils and the lowest in pine stand soils (Tab. 2).
Soil organic matter fraction parameters
Statistically significant highest average C and N contents in the free
light fraction were found in the top horizons of the soils studied (Tab.
3). The highest C and N content in the free light fraction was recorded
in the organic horizon of pine stand soils (266.30 and 7.99
g·kg-1, respectively), the surface horizons of
pine-oak stands were characterized by lower values (134.19
g·kg-1 and 5.10 g·kg-1,
respectively) and surface horizons of oak stands by the lowest ones
(105.22 g·kg-1 and 5.05 g·kg-1,
respectively). Significantly higher mean C and N contents in the
aggregate-occluded light fraction were recorded in the humus mineral
horizons of the soils studied (Tab. 3). The highest content of
CoLF and NoLF was found in the Oh
horizon of pine stand soil (13.87 and 0.58 g·kg-1,
respectively). In the case of C and N of the mineral associated
fraction, the highest values were determined in the humus horizon of
mineral soils of oak and pine-oak stands (Tab. 3).
Polycyclic aromatic hydrocarbons content
The highest PAHs content was recorded in organic soil horizons of all
stand variants (Tab. 3, Fig. 4, Fig. 5). The PAHs content decreased with
soil depth, regardless of the stand variant. The highest amount of PAHs
in organic horizons was recorded in pine stand soils (4816.13
µg·kg-1 in Of and 2793.60 µg·kg-1 in
Oh horizon). In the Ofh horizon of pine-oak stand soils, the average PAH
content was 3087.09 µg·kg-1 and in oak stand soils it
was 2936.20 µg·kg-1 (Tab. 3). In the surface horizons
of pine and pine-oak stands soils, a clear domination of 3- and 4-ring
PAHs was noted (Fig. 4). In the case of oak stand soils, 4-ring PAHs
dominated, followed by 3 and 5-ring PAHs. The highest mean content of
3-ring PAHs in the organic soils levels was noted in case of fluorine
and phenanthrene (Fig. 5). In turn, fluoranthene, pyrene and chrysene
showed the highest values among 4-ring PAHs. For 5- and 6-ring PAHs, the
highest values were reached by benzo(b)fluoranthene and
ideno(c,d)pyrene, respectively.
Corelations
In the soils studied, the activity of enzymes strongly correlated with
carbon and nitrogen content, in the case of CB, BG, XYL and PH it was a
positive relationship. In the case of most of enzymes studied,
statistically significant correlations with PAH content were noted,
apart from DH and CB activity (Tab. 4). Also a positive relationship
between the amount of accumulated PAHs and CfLF,
NfLF and CMAF was found in the soils
studied (Tab. 4). A significant positive correlation between PAH content
and the amount of organic carbon was clearly shown (Fig. 5), especially
in pine stands soils, where PAHs increase exponentially with the amount
of organic carbon. (Fig. 6). Multiple regression analysis confirmed
relationships between PAHs content and the C of light and heavy fraction
of soil organic matter. Multiple regression models explained 84% of the
variance in PAH content (Tab. 5).
Factors 1 and 2, distinguished in the PCA analysis, explain a total of
66.8% of the variance of properties of the tested soils (Fig. 7). The
PCA analysis confirms the negative correlation between PAH content and
pH of the tested soils. The PAHs content positively correlated with
CfLF and NfLF (Fig. 7). The PCA analysis
confirms a higher PAHs content in the organic horizons of the soils
studied, where a lower pH and a higher C content were observed. Mineral
horizons are associated with a higher content of C and N of the
aggregate-occluded light fraction and mineral associated fraction (Fig.
7).
Discussion
The obtained results confirm the hypothesis of significant impact of
stand species composition on the amount and quality of soil organic
matter supplied to the soil and on acidification, and consequently on
PAHs accumulation in forest soils. In all cases of the investigated
soils, the amount of PAHs accumulated in organic horizons indicated the
third, moderate degree of soil contamination (1000 - 5000
µg·kg-1) according to the classification proposed by
Maliszewska-Kordybach (1996). The highest average accumulation of PAHs
in organic horizons was recorded in soils of pine stands, lower in soils
of mixed pine-oak stands and the lowest in soils of oak stands. At the
same time, the soils with the highest PAHs accumulation were
characterized by strong acidification and less decomposed organic
matter. The quality and quantity of soil organic matter is an effect of
the stand species composition, which has already been described by other
authors (Jandl et al., 2007; Schulp et al., 2008; Vesterdal et al.,
2008; Zhang et al., 2008). The share of deciduous species in the stand
improves the biogeochemical cycles of the matter by providing nutrients
(Augusto et al., 2015; Ammer, 2019). Coniferous species, especially
pine, provide organic fall to the soil, which acidifies the top soil
horizons. As a result of the influence of coniferous species, the
processes of microbiological decomposition of the fallen organic matter
on the soil surface are generally slower (Schulp et al., 2008; Eisalou
et al., 2013; Magh et al., 2018). Deciduous species provide the soil
with carbon substrates and nutrients used by microorganisms in the
decomposition processes through the fall of litter and root systems
(Błońska, 2015; Błońska et al. 2021). The beneficial effect of deciduous
species on the topsoil was reflected in higher pH value and higher
content of alkaline cations. It was also observed that the share of
deciduous species had a significant positive effect on the biochemical
properties of soils expressed by enzymatic and microbiological activity
of biomass, which was reflected in organic matter decomposition
processes and PAHs biodegradation potential. An increased share of
deciduous tree species that co-create the stand leads to the relocation
of carbon to ground biomass, resulting in a beneficial increase in
nutrient utilization efficiency compared to coniferous monocultures
(Epron et al., 2013). An increased PAHs accumulation capacity in organic
horizons of coniferous stand soils is also due to the effect of the
assimilation of pollutants in the atmosphere through the presence of
assimilation apparatus throughout the growing season (Hill et al., 2002;
Peng et al., 2012). An increased PAHs accumulation in coniferous forest
soils can also be explained by the presence of wax and lipids on the
needle surface, which are able to absorb and accumulate lipophilic PAHs
present in the contaminated atmosphere (Li et al., 2017).
Our research confirmed the importance of the fraction of soil organic
matter in the accumulation of PAHs in forest soils. In our study, the
PAHs content was closely related to the amount of organic carbon and
nitrogen of free light fraction of soil organic matter. According to
Cachada et al., (2018), the amount and quality of soil organic matter
and its fractions are the factor controlling the accumulation of PAHs in
soils. Soils of pine stands are characterized by a higher proportion of
light fraction of soil organic matter, which is due to the slowdown of
decomposition processes. Additionally, high value of C to N ratio of
light fraction in soils of pine stands confirms weaker decomposition of
organic residues. In the case of soils of oak stands, a significantly
lower C/N ratio of light fraction of soil organic matter was recorded.
The light fraction present in the soil is formed by partially decomposed
plant residues, animal remains in the intermediate stage of
decomposition between stabilized carbon (Błońska, 2015). According to Li
et al., (2014), the organic fraction has stronger sorption properties
compared to the mineral fraction. The biodegradation efficiency of PAHs
by microorganisms is related to the bioavailability of compounds
dissolved in the soil solution (Mackay et al., 2001). According to
Jastrow et al. (2007), the biological activity of soils is regulated by
the structures of organic matter, and in particular is determined by the
size of soil aggregates. The relationship between C and N of free light
fraction of organic matter and PAHs content and enzymatic activity in
soils has been reported. Strong sorption of PAH compounds within the
organic matter structures reduces bioavailability as well as desorption
and diffusion of pollutants by microorganisms (Ukalska-Jaruga et al.,
2015).
According to Oleszczuk et al. (2007), strong sorption of PAH compounds
occurs under low pH conditions, which is characteristic for soils with
coniferous stands in our study. The low soil pH may result from a
significant amount of humic and fulvic acids (Maliszewska-Kordybach et
al., 2010), which significantly reduce the bioavailability of organic
matter and PAHs in soil solution (Haynes, 2005). Strongly absorbed,
soil-accumulated PAH compounds inaccessible to microorganisms are
subject to aging process (Zhang et al., 2011). Ni et al. (2008) claim
that an effective method of bioremediation of soils contaminated with
PAHs is to reduce the content of free light fraction of organic matter.
Wilson et al. (1993) claim that the bioremediation method by reducing
the free light fraction of organic matter is only effective with respect
to low molecular weight PAHs. However, our research also confirms the
effect of light fraction organic matter reduction on high molecular
weight PAHs due to the beneficial effect of nutrients supplied to the
soil by oak stands. The contribution of oak forming a stand can be an
effective factor in the process of natural soil bioremediation and
elimination of toxic soil contamination levels especially for stands
exposed to intensive industrial emissions. However, more research is
needed on the impact of other deciduous species forming the stands on
the reduction of PAHs in the soil through beneficial effects on the
amount and quality of soil organic matter and the amount of nutrients.
In our study we noted a strong positive relationship of enzymatic
activity with organic carbon and PAHs content in soils. The lack of
limitation of enzyme activity in soil samples with high PAHs content can
be explained by the masking effect of soil organic matter. Strong
affinity of soil organic matter to PAHs and at the same time high
enzymatic activity in samples with high content of organic carbon
explains the obtained relationships. Similar results were obtained in
the case of relations between enzyme activity and heavy metal content
with simultaneous high accumulation of organic carbon (Lasota et al.
2020).
Conclusions
Our research confirms the importance of stand species composition in
PAHs accumulation in forest soils. The stand species composition
influences the amount and quality of soil organic matter and
acidification, which in turn is reflected in soil biochemical activity
and decomposition of organic pollutants. A significant influence of
deciduous species on the surface properties of soil levels and
consequently on the PAHs content was found. The soils of oak stands were
characterized by the lowest accumulation of PAHs with high biochemical
activity expressed by enzyme activity and microbiological biomass. Pine
stand soils are strongly acidified, the decomposition of soil organic
matter is slowed down, resulting in a high accumulation of PAHs. Our
results confirmed the importance of fractional composition of soil
organic matter in PAHs accumulation. The free light fraction of soil
organic matter and the mineral associated fraction are of greatest
importance in PAHs accumulation.