Ecotoxicology and Environmental Safety 214 (2021) 112070

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Review

Sewage sludge in agriculture – the effects of selected chemical pollutants

and emerging genetic resistance determinants on the quality of soil and
crops – a review
Martyna Buta , Jakub Hubeny, Wiktor Zieliński, Monika Harnisz , Ewa Korzeniewska *
Department of Water Protection Engineering and Environmental Microbiology, The Faculty of Geoengineering, University of Warmia and Mazury in Olsztyn,
Prawocheńskiego 1 Str., 10-720 Olsztyn, Poland

A R T I C L E I N F O A B S T R A C T

Edited by Professor Bing Yan In line with sustainable development principles and in order to combat climate change, which contributes to
progressive soil depletion, various solutions are being sought to use treated sewage sludge as a soil amendment to
Keywords: improve soil quality and enrich arable soils with adequate amounts of biogenic compounds. This review article
Sewage sludge focuses on the effects of the agricultural use of biosolids on the environment. The article reviews the existing
Antibiotics
knowledge on selected emerging contaminants in treated sewage sludge and describes the impact of these
Heavy metals
pollutants on the environment and living organisms based on 183 publications selected from over 16,000 papers
Antibiotic resistance genes
Co-resistance on related topics published over the last ten years. This study deals not only with chemical contaminants but also
genetic determinants of resistance to these compounds. Current research has questioned the agricultural use of
biosolids due to the presence of mutual interactions between antibiotics, heavy metals, the genetic determinants
of resistance (antibiotic resistance genes - ARGs and heavy metal resistance genes - HMRGs) and non-steroidal
anti-inflammatory drugs as well as the risks associated with their transfer to the environment. This study em­
phasizes the need for more extensive legal regulations that account for other pollutants of environmental concern
(PEC), particularly in countries where sewage sludge is applied in agriculture most extensively. Future research
should focus on more effective methods of eliminating PEC from sewage sludge, especially from the sludge that is
used to fertilize agricultural land, because even small amounts of these micropollutants can have serious im­
plications for the health and life of humans and animals.

1. Introduction
Despite the fact that WWTPs play a major role in reducing the
amount of inflowing pollutants, significant quantities of contaminating
substances are released into water bodies (Ben et al., 2017; Korze­
niewska and Harnisz, 2018; Krzeminski et al., 2019; Martín-Pozo et al.,
2019). The above applies not only to organic compounds but also to
micropollutants such as microorganisms, including pathogenic and
opportunistic microorganisms that are resistant to popular antimicrobial
drugs (Osińska et al., 2017). The group of environmental concern pol­
lutants includes a wide range of personal care products (PCPs) such as
anti-inflammatory drugs and antimicrobials or heavy metals. In the
wastewater can also occur residues of PCPs as an active or inactive form
of these chemical compounds (Ahmed et al., 2017). Heavy metals that
reach WWTPs with wastewater from industrial plants and livestock
farms also pose a massive risk. These highly toxic compounds are
characterized by stability and a long half-life in the environment (Möller
and Schultheiß, 2015; Kang et al., 2018; Rensing et al., 2018), which
complicates their removal.
Wastewater treatment plants generate massive quantities of sewage
sludge as an indirect product of wastewater treatment (Fijalkowski
et al., 2017; Lindholm-Lehto et al., 2017). The quantity and the quality
of the produced sewage sludge are closely linked to the applied tech­
nological system and the degree to which wastewater is processed by a
WWTP. According to estimates, sewage sludge accounts for up to 3% of
the wastewater flowing through a WWTP (Statistics Poland Spatial and
Environmental Surveys Department, 2019). This amount could appear
insignificant, but the total quantity of raw wastewater reaching a WWTP
and the plant’s processing capacity have to be taken into consideration
to estimate the total amount of sewage sludge generated by a WWTP.

* Corresponding author.
E-mail addresses: ewa.korzeniewska@uwm.edu.pl, ewakmikr@uwm.edu.pl (E. Korzeniewska).

https://doi.org/10.1016/j.ecoenv.2021.112070
Received 18 November 2020; Received in revised form 13 February 2021; Accepted 15 February 2021
Available online 2 March 2021
0147-6513/© 2021 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Buta et al.
The quantity of raw wastewater that is received by a WWTP is deter­
mined by the local population and the degree of urban and industrial
development in a given region (Eid et al., 2019). Wastewater and sewage
sludge production in selected countries is presented in Table S1. At
present, the global production of sewage sludge is estimated at 45
million megagrams (Mg) of dry matter (DM) per year (Zhang et al.,
2017). Europe, East Asia, and North America are considered to be the
main producers of sewage sludge in the world (Shaddel et al., 2019).
According to the Final Report for the European Commission (Milieu Ltd,
2010), sewage sludge production in the EU countries alone increased by
1.5 million Mg of DM in the last 10 years, from 11.5 million Mg in 2010
to 13 million Mg of DM in 2020. Based on the data for 2016, around 30
million Mg of wet sludge with a moisture content of up to 80% are
produced each year in China alone (K. Wang et al., 2017). As the amount
of sewage sludge generated in that country is expected to double in the
coming years (Q. Wang et al., 2017), China has implemented a project
aiming at increasing the number of WWTPs. The growing production of
sewage sludge also leads to a worrying increase in the ratio of total
generated sludge to managed sludge.
Many industrialized and developing countries are considering
alternative measures for disposing sewage sludge produced by WWTPs.
As a result, sewage sludge is no longer regarded as a waste product
(Rorat et al., 2019). Over the years, various measures had been applied
to manage sewage sludge, including thermal processing, composting,
agricultural use, and landfilling (Leblanc et al., 2006; Lamastra et al.,
2018). Such practices are still in use today, but their proportions
continue to change, not only in many European countries but also
globally. The percentage share of different sewage sludge management
methods in selected countries is presented in Fig. 1. For practical and
legal reasons, sewage sludge is increasingly reused rather than land­
filled. This approach is consistent with the zero waste strategy which
Fig. 1. Sewage sludge disposal from urban WWTPs by treatment method, reporting year 2017. (1) Data for 2016. (2) Data for 2003. (3) Data for 2019 (Leblanc et al.,
2006; USEPA, 2019; EUROSTAT, 2020).
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Ecotoxicology and Environmental Safety 214 (2021) 112070
aims to minimize the amount of the generated waste (Song et al., 2015)
and to promote the development of the bioeconomy which provides
intelligent waste management solutions that deliver tangible environ­
mental benefits (Duan et al., 2020). In Western members of the Euro­
pean Union, land application constitutes a major way of sewage sludge
management, but this method is currently banned in some of the
countries, e.g. Germany or Netherlands. South America’s sewage sludge
management is underdeveloped when compared to the United States.
The differences result from legal regulations and various infrastructure
levels (Shaddel et al., 2019). The methods of handling sewage sludge
also differ across Asia. Especially South-East Asia has many contrasting
regions in terms of the economic situation and population size, so the
priority and management of sediments also differ significantly (Spinosa,
2015). Discrepancies in the approach to sludge management are also
noticeable between South Korea and Japan. Main sewage sludge
disposal routes in Japan are incineration (about 70%) and landfill
application, whereas South Korea’s sludge management were based on
dumped sewages into the sea until 2012. Nowadays, the sewage sludge
generated there is sent mainly to landfills (Shaddel et al., 2019).
The article reviews selected emerging pollutants in treated sewage
sludge and describes the impact of these contaminants on the environ­
ment and living organisms based on 183 publications selected from over
16.000 papers over the last ten years (PRISMA Diagram, Supplementary
Materials). This research aimed to review the quality of agricultural used
sewage sludge in view of legally selected regulated and unregulated
pollutants of environmental concern (PEC), such as heavy metals, anti­
biotics, non-steroidal anti-inflammatory drugs, and genetic de­
terminants of resistance to antibiotics and heavy metals. This review
also discusses the mutual relations/interactions and the impact of pol­
lutants on sludge-amended soil and on the crops growing on sludge
amended-soil, which plays an important role in consumer risk
M. Buta et al.
assessments.
2. Composition and disposal methods of sewage sludge
The parameters of sewage sludge are determined by its moisture
content, the content of organic and mineral compounds, and its tech­
nological properties, including dewatering abilities and calorific value.
Untreated sewage sludge usually contains 2–8% of DM (with a pre­
dominance of volatile solids), protein, fat, mineral compounds, organic
acids, cellulose and others (Rorat et al., 2019). The composition of raw
sewage sludge varies considerably both between WWTPs and within the
same plant, mainly due to the applied processing technology, and the
quantity and the origin of raw wastewater. Dehydrated sewage sludge
contains around 20–45% of DM, and its suitability for agricultural uti­
lization is determined mainly by a high content of organic matter (up to
70%) and biogenic elements, including carbon (approx. 4%), nitrogen
(approx. 4%) and phosphorus (approx. 2.5%) (Cheng et al., 2014;
WWAP, 2017), which increase soil fertility and are essential substrates
for the healthy growth and the development of plants and soil micro­
biota (Hei et al., 2016; Samara et al., 2017; Tyrrell et al., 2019). Stabi­
lized sewage sludge contains around 45–55% of organic compounds
(Drechsel et al., 2015; WWAP, 2017; Rorat et al., 2019). However, apart
from the above advantages, the available literature indicates that
sewage sludge also contains dangerous or potentially toxic biological
and chemical compounds that undermine its use in agriculture. These
include heavy metals, persistent organic pollutants (POPs), and micro­
organisms that are pathogenic for both humans and animals (Bondarc­
zuk et al., 2016; Iglesias et al., 2018).
Various technological processes, mostly compaction, stabilization,
conditioning, and dewatering, are deployed to process and to neutralize
sewage sludge before it is applied as an agricultural fertilizer (Fig. S1).
Aerobic and anaerobic digestion is one of the most popular methods of
stabilizing sewage sludge and mineralizing organic compounds (Keles­
sidis and Stasinakis, 2012; Youngquist et al., 2014). Digestion methods
reduce the counts of pathogenic microorganisms, eliminate odor and
prevent the decomposition of sewage sludge over time (Rorat et al.,
2019). Chemical stabilization methods (including liming) are less
widely used (Kelessidis and Stasinakis, 2012). Microorganisms are
highly effectively removed through thermal conditioning during which
sewage sludge is subjected to a temperature of 393.15–423.15 K
(120–150 ◦ C) and the pressure of 0.5–2 MPa for 0.5 h-1. Other condi­
tioning methods, including chemical and physical processing, rinsing
and freezing, are also used to remove pollutants (including pathogenic
microorganisms) with varied efficacy (Verlicchi and Zambello, 2015;
Rorat et al., 2019). Incineration (thermal degradation) is one of the key
methods for the final disposal of sewage sludge, aimed at its further
purification or use as an alternative source of energy (Drechsel et al.,
2015). Sewage sludge intended for the remediation of industrial land­
scapes and agricultural soils (Christodoulou and Stamatelatou, 2016;
Statistics Poland Spatial and Environmental Surveys Department, 2019)
is frequently composted (Haiba et al., 2017). Most of the discussed
treatment methods completely remove or minimize microbial loads in
sewage sludge, but there are no equally effective methods for elimi­
nating heavy metals which are highly stable in the environment (Kang
et al., 2018).
To address the problem of heavy metal toxicity for humans, animals,
and plants, in 1986, the European Community adopted the European
Council Directive 86/278/EEC on the protection of the environment and
of the soil, particularly when sewage sludge is used in agriculture. The
directive lays down mandatory limit values for toxic elements in soils.
Before it is used in agriculture, sewage sludge has to be treated with the
use of biological, chemical, or thermal methods, long-term storage, or
any other appropriate process to significantly reduce its fermentability
and the health hazards resulting from its agricultural use (European
Council, 1986). The sewage sludge that has undergone biological sta­
bilization, digestion, or composting contains nutrients for soil
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Ecotoxicology and Environmental Safety 214 (2021) 112070
conditioning and energy generation. This sludge are referred to as bio­
solids, and it is applied to soil to enhance crop growth or is further
processed as compost or fertilizer (Leblanc et al., 2006; Drechsel et al.,
2015). Based on the provisions of the discussed directive, the EU
countries have developed internal documents regulating the use of
municipal sewage sludge on agricultural land, including their chemical
quality (heavy metals), biological quality (pathogenic microorganisms,
parasites, and parasite eggs) as well as reference methods for analyzing
sewage sludge and soils where biosolids are applied (Minister of Envi­
ronment, 2015). Similar regulations have been introduced by the United
States Environmental Protection Agency in 40 Code of Federal Regula­
tions (CFR), Part 503 (USEPA, 2018). These guidelines regulate the use
and stabilization of sewage sludge, and they set limit values for the
contaminants present in sewage sludge. In addition to heavy metals,
sewage sludge often contains other micropollutants whose concentra­
tions are not legally controlled, such as pharmaceuticals and their
degradation products, pesticides, genetic material containing antibiotic
resistance genes (ARGs), and genes encoding other microbial virulence
factors (Rorat et al., 2019). Wastewater effluents, which have been
identified as hotspots for the dissemination of ARGs and
antibiotic-resistant bacteria (ARB) (Rizzo et al., 2013), as well as heavy
metal resistance genes (HMRGs), are the source of micropollutants in
sewage sludge (Di Cesare et al., 2016b). Sewage sludge is obtained from
dehydrated and processed municipal and industrial wastewater, and it is
characterized by very high concentrations of dangerous compounds
(Halecki et al., 2016). Sewage sludge accumulates significant quantities
of micropollutants because it acts as an environmental “sponge” that
temporarily binds chemical compounds such as metals and antibiotics
on its surface. The previous study presents also the risk of bio­
accumulation of POPs and the impact on MGEs, which play a significant
role in spreading ARGs (Islam et al., 2018). Sewage sludge contains DM
that is abundant in organic matter and offers a supportive environment
for the adhesion and the growth of microorganisms (Andreoilo et al.,
2017).
3. Antibiotics and ARGs in sewage sludge - sources, causes,
persistence
Rapid industrial and agricultural development, as well as the
excessive, inadequate use of fertilizers, promote the transfer of signifi­
cant amounts of pollutants to the soil environment (Drechsel et al.,
2015). Despite its suitability for agricultural use, raw or digested sewage
sludge is heavily contaminated with pharmaceuticals. According to the
literature, around 100,000 to 200,000 Mg of antibiotics are consumed
each year in the world (Jeong et al., 2010). In the United States, annual
antibiotic consumption is estimated at 16,000 Mg, but up to 70% of
antibiotics are not used for therapeutic purposes (Ezzariai et al., 2018).
The consumption of antibiotics in human medicine in selected European
countries is presented in Fig. 2. Greece, Cyprus, and Spain are leading
antibiotic consumers in the EU (ECDC, 2020a). Beta-lactams, tetracy­
clines, and macrolide-lincosamide-streptogramin (MLS) antibiotics are
most widely prescribed in human medicine (ECDC, 2020b), whereas
tetracyclines, beta-lactams, and sulfonamides (SAs) are most widely
used in veterinary medicine (Tasho and Cho, 2016; Spielmeyer, 2018).
The entry route of antimicrobials into the environment begins with
human and animal patients, because antibiotics are not fully metabo­
lized in their bodies (Korzeniewska and Harnisz, 2018). Antibiotic res­
idues in unmodified form and their degradation products are evacuated
with feces, and they reach the environment directly - with manure
applied to the soil, or indirectly - with treated wastewater. Wastewater
processed in WWTPs is discharged to water bodies, and the accumulated
biosolids reach the soil environment (McEneff et al., 2014; Bondarczuk
et al., 2016). Beta-lactam antibiotics such as amoxicillin and ampicillin,
as well as macrolide antibiotics are readily hydrolyzed (Pikkemaat et al.,
2016). Raw sewage is supplied to WWTPs in liquid form, which creates a
supportive environment for hydrolysis. Nevertheless, nearly all groups
M. Buta et al.
Fig. 2. Consumption of antibacterials for systemic use in the community and the hospital sector in Europe, reporting year 2018. *Country provided only total care
data (ECDC, 2020a).
of antibiotics have been identified in semi-liquid and solid sewage
sludge, within a concentration range of ng to mg kg-1 DM (Jelić et al.,
2012; Li et al., 2013). The above can be attributed to the fact that large
amounts of pharmaceuticals are adsorbed on the surface of solid parti­
cles in sewage sludge, and adsorption is the main mechanism of anti­
biotic removal from wastewater (Xu et al., 2015). As a result, around
70% of non-degraded antibiotics are transferred to sewage sludge (Sun
et al., 2019). Numerous researchers have reported on the presence of
tetracycline, quinolone, macrolide, and sulfonamide antibiotics in
sewage sludge (Li et al., 2013; Cheng et al., 2014; Ashfaq et al., 2016).
Ashfaq et al. (2016) identified five antibiotics in samples of sewage
sludge obtained from hospital waste, where moxifloxacin was charac­
terized by the highest concentration (219 µg kg-1). The cited authors
were also the first researchers to detect gemifloxacin in the analyzed
sewage sludge samples (Ashfaq et al., 2016). Cheng et al. (2014) iden­
tified four fluoroquinolone antibiotics (FQs), eight sulfonamide antibi­
otics (SAs), and four tetracycline antibiotics (TCs) in sewage sludge. In
their study, SAs were detected in far smaller concentrations (mean of
55.4 μg kg-1 DM) than TCs (mean of 8326 μg kg-1 DM) and FQs (mean of
8274 μg kg-1 DM) (Cheng et al., 2014). Research has also demonstrated
that FQs, SAs, and TCs are susceptible to photodegradation (Łukasze­
wicz et al., 2016) whose effectiveness is determined by the chemical
structure of antimicrobials and the applied matrix (Zessel et al., 2014).
Fluoroquinolones undergo photolysis more rapidly in aquatic than in
soil environments. According to research, FQs are not completely
degraded under exposure to light in soil and sewage sludge, and these
compounds can persist in soil for up to several years (Jechalke et al.,
2014). In aquatic environments, the photodegradation products of FQs
can be restored to their active form within a pH range of 2–6 (Solliec
et al., 2016). The environmental pH and the chemical structure of the
drug also affect the photodegradation of SAs. Zessel et al. (2014)
demonstrated that in contrast to SAs containing a six-membered het­
erocyclic ring, the photolysis of SAs that contain five-membered het­
erocyclic rings is influenced by the environmental pH.
There is considerable research evidence to indicate that the man­
agement of sewage sludge affects its quality (Table S2) (Verlicchi and
Zambello, 2015; Ju et al., 2016). The concentrations of quinolone and
tetracycline antibiotics are even twice higher in raw sludge than in
primary sludge. According to the critical review conducted by Verlicchi
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Ecotoxicology and Environmental Safety 214 (2021) 112070
and Zambello (2015), secondary sludge may contain significant quan­
tities of antibiotics such as sulfamethoxazole (68,000 µg kg-1 DM),
clarithromycin (67,000 µg kg-1 DM), azithromycin (64,000 µg kg-1 DM),
and ofloxacin (21,000 µg kg-1 DM).
A comparison of tetracycline and sulfonamide concentrations in
biosolids and animal manure, i.e. in environments where these anti­
biotic groups are most ubiquitous, revealed that biosolids can contain
36.6–743.6 µg kg-1 of TCs and 4.8–668.9 µg kg-1 of SAs. In samples of
animal manure, the concentrations of TCs and SAs were determined in
the range of 4.02–136,000 µg kg-1 and 6.26–91,000 µg kg-1, respectively
(Łukaszewicz et al., 2016). The comparison made by Cycoń et al. (2019)
also confirms that biosolids include much lower concentrations of an­
tibiotics than animal manure. These findings suggest that in terms of
antibiotic contamination, sewage sludge is a more suitable soil amend­
ment than animal manure. Therefore, biosolids could become a more
popular alternative fertilizer in the following years.
The existing wastewater treatment methods differ in their ability to
decrease antibiotic concentrations in wastewater. However, due to the
significant amounts of highly biodiverse microorganisms in wastewater
and sewage sludge (Karkman et al., 2018), even small, sublethal con­
centrations of antimicrobials (Giebułtowicz et al., 2018) can contribute
to the rapid emergence of antibiotic resistance (Rizzo et al., 2013).
Antibiotic resistance is not a new problem, but its spread and severity
continue to increase, mainly due to the absence of legal regulations on
limit values for antibiotics that are released into the environment with
treated wastewater and sewage sludge (Korzeniewska and Harnisz,
2018). Over the years, genes encoding resistance to nearly all known
antibiotic groups have been identified in raw wastewater reaching
WWTPs; therefore, many resistance genes can be transferred to sewage
sludge (Guo et al., 2017). The presence of ARGs in treated wastewater,
including ampC (encoding resistance to most beta-lactams), ermB
(encoding resistance to erythromycin), blaNDM-1 (encoding resistance to
many beta-lactams and carbapenem antibiotics regarded as a last-resort
treatment for bacterial infections), tem1 (encoding resistance to peni­
cillins) and vanA (encoding resistance to vancomycin), is of particular
environmental concern (Luo et al., 2013; Munck et al., 2015). The
presence of ARGs and the discussed antibiotics in sewage sludge is
determined by the applied treatment methods. Research into the effec­
tiveness of anaerobic digestion methods in removing ARGs from sewage
M. Buta et al. Ecotoxicology and Environmental Safety 214 (2021) 112070

Table 1
Resistance to antibiotics detected in various types of sludge and amended soil.
ARGs type Source References

Aminoglycoside (aac(3)IV, aadA, strA, strB) Anaerobic digested (Yang et al., 2014; Bengtsson-Palme et al., 2016)
sludge
Dried sludge
Biosolids (Karkman et al., 2016)
Biosolid amended soil (Rahube et al., 2014)
(Ross and Topp, 2015)
Bacitracin (bacA, UppP) Anaerobic digested (Yang et al., 2014)
sludge
Beta-lactam (CARB-8, NPS-1, OXA-1, OXA-2, OXA-10, ROB-1, TEM, VEB-1, GIM-1, Anaerobic digested (Jechalke et al., 2014; Sittig et al., 2014; Łukaszewicz et al.,
IMP, IMP-1, NDM-1, SIM-1, SPM-1, VIM, VIM-2) sludge 2016; Felden and Cattoira, 2018)
Dried sludge
Biosolid (Karkman et al., 2016)
Biosolid amended soil (Rahube et al., 2014)
Crops from biosolid (Rahube et al., 2014; Ross and Topp, 2015)
amended soil
(Rahube et al., 2014)
Chloramphenicol (catA2, catB3, catD, cmlA9, cmxA) Anaerobic digested (Zessel et al., 2014; Ashfaq et al., 2016; Pan and Chu, 2016)
sludge
Dried sludge
Biosolid amended soil (Karkman et al., 2016)
Crops from biosolid (Rahube et al., 2016)
amended soil
(Rahube et al., 2016)
MLS (ereA, ereB, ermA, ermB, ermC, ermE, ermF, ermG, macB, mefA, mefE, vatB, vga, vgb) Anaerobic digested (Sittig et al., 2014; Zessel et al., 2014; Ashfaq et al., 2016; Pan
sludge and Chu, 2016)
Dried sludge
Biosolid (Karkman et al., 2016)
Biosolid amended soil (Rahube et al., 2014)
Crops from biosolid (Sittig et al., 2014; Barra Caracciolo et al., 2015; Pan and Chu,
amended soil 2016)
(Barra Caracciolo et al., 2015; Pan and Chu, 2016)
Multidrug (acrA, acrB, emrD, mdtF, mdtG, mdtH, mdtL, mdtN, mexB, mexF, norM, sdeY) Anaerobic digested (Sittig et al., 2014; Łukaszewicz et al., 2016)
sludge
Dried sludge (Karkman et al., 2016)
Oxazolidinone (linezolid) Anaerobic digested (Bengtsson-Palme et al., 2016)
sludge
Polymyxin (arnA) Anaerobic digested (Sittig et al., 2014; Łukaszewicz et al., 2016)
sludge
Quinolone (qnrA, qnrB, qnrS) Anaerobic digested (Jechalke et al., 2014; Sittig et al., 2014)
sludge
Biosolid (Rahube et al., 2014)
Biosolid amended soil (Sittig et al., 2014; Pan and Chu, 2016)
Crops from biosolid (Rahube et al., 2016)
amended soil
Sulfonamide (sul1, sul2) Anaerobic digested (Sittig et al., 2014; Zessel et al., 2014; Ashfaq et al., 2016; Pan
sludge and Chu, 2016)
Dried sludge
Biosolid (Karkman et al., 2016)
Biosolid amended soil (Munir and Xagoraraki, 2011; Rahube et al., 2014)
Crops from biosolid (Ross and Topp, 2015; Rahube et al., 2016)
amended soil
(Rahube et al., 2014)

Tetracycline (tet32, tet33, tetA, tetC, tetG, tetL, tetM, tetO, tetPA, tetPB, tetQ, tetS, tetT, Anaerobic digested (Jechalke et al., 2014; Sittig et al., 2014; Łukaszewicz et al.,
tetV, tetW, tetX) sludge 2016; Felden and Cattoira, 2018)
Dried sludge
(Karkman et al., 2016)

Trimethoprim (dfrA) Anaerobic digested (Jechalke et al., 2014; Sittig et al., 2014)
sludge
Vancomycin (vanA, vanB, vanC1, vanC2/C3, vanR, mrsA, mrsB) Anaerobic digested (Jechalke et al., 2014; Łukaszewicz et al., 2016; Sittig et al.,
sludge 2014)
Dried sludge
Biosolid (Karkman et al., 2016)
Biosolid amended soil (Rahube et al., 2014)
(Rahube et al., 2014)
Fosfomycin (fosA) Biosolid (Rahube et al., 2014)
Biosolid amended soil (Rahube et al., 2014)

5
M. Buta et al.
sludge has produced contradictory results. The efficacy of anaerobic
digestion in reducing the concentrations of ARGs in post-digestion
municipal thickened primary sludge (TPS), thickened secondary acti­
vated sludge (TSAS) and combined sludge was evaluated in the litera­
ture to reduce the abundance of ARGs in sewage sludge. The results
indicate that anaerobic digestion is not a highly effective method of ARG
removal (estimated removal efficiency of 20–50%), and it is somewhat
more effective in eliminating genes that confer resistance to selected
antibiotics, mostly TCs (Yang et al., 2014; Ju et al., 2016). According to
some reports, anaerobic digestion can also increase the concentrations
of ARGs. Yang et al. (2014) demonstrated that only 20.7% of ARGs in
sewage sludge were removed during anaerobic digestion. The evaluated
treatment method was effective in eliminating only selected ARGs,
whereas the concentrations of genes encoding resistance to MLS, poly­
myxin, tetracycline, and vancomycin increased after anaerobic diges­
tion. Genes encoding resistance to tetracyclines, polymyxin,
vancomycin, and macrolide-lincosamide-streptogramin B (MLSB) anti­
biotics were also identified in digested sewage sludge (Table 1) (Yang
et al., 2014; Karkman et al., 2016). The abundance of the analyzed ARGs
was considerably reduced by prolonged hydraulic retention time (HRT)
during mesophilic anaerobic digestion (Sun et al., 2019). Sun et al.
(2019) analyzed the effectiveness of anaerobic digestion conducted
under various conditions on the content of antibiotics, ARGs, and mobile
genetic elements (MGEs) in sewage sludge. They identified 245 ARGs
and 12 MGEs in the studied samples. Sewage sludge harbored genes
encoding resistance to nearly all antibiotic groups, and genes encoding
resistance to aminoglycosides, MLSB, SAs, tetracyclines, and multidrug
resistance genes were most abundant. Total gene concentrations ranged
from 8.39 × 105 to 2.46 × 107 copies μL-1, and the content of ARGs and
MGEs was lowest in the samples that had undergone thermal hydrolysis
pretreatment. Bengtsson-Palme et al. (2016) identified genes encoding
resistance to linezolid, the first antibiotic of the oxazolidinone group
that targets multidrug-resistant bacteria (including
vancomycin-resistant enterococci, VRE), in samples of anaerobically
digested sludge (Gawryszewska et al., 2017). Other studies revealed that
sludge stabilization methods such as anaerobic digestion or the addition
of lime effectively eliminate ARGs from sewage sludge (Munir et al.,
2011). However, some ARGs, including genes encoding resistance to
SAs, beta-lactams, tetracyclines, and vancomycin, are still present in
digested and composted sewage sludge intended for application to
agricultural land (Bengtsson-Palme et al., 2016). Su et al. (2015) iden­
tified 156 ARGs and MGEs in composted sewage sludge, with the pre­
dominance of genes encoding resistance to tetracyclines, MLSB,
aminoglycosides and multidrug resistance genes. The number of ARGs
increased significantly in the final stage of composting relative to the
initial stage of the process (Su et al., 2015). The concentrations of ARGs
are generally reduced with a treatment-induced decrease in the pop­
ulations of ARB that carry these genes in wastewater and sewage sludge.
However, some studies have demonstrated that the applied treatment
processes are much more effective in eliminating ARB than ARGs from
wastewater and sewage sludge (Bondarczuk et al., 2016; Korzeniewska
and Harnisz, 2018). Moreover, some advanced treatment methods
promote the initiation of the SOS response in bacteria, which accelerates
mutations and horizontal gene transfer (HGT) (Qin et al., 2015).
4. Transfer and bioavailability of antibiotics and ARGs in soil
and neighbouring environments
In Europe, more than 50% of sewage sludge is used in agriculture
(Kelessidis and Stasinakis, 2012; Malmborg and Magnér, 2015; Ezzariai
et al., 2018). In view of the information presented in the previous sec­
tion, the risks associated with potential antibiotic transfer from sewage
sludge to the environment should be carefully considered. The transfer
of antibiotics from biosolids in the soil environment to groundwater and
plants begins with the key process of antimicrobial adsorption on the
surface of solid particles. Hydrophobic and electrostatic interactions in
6
Ecotoxicology and Environmental Safety 214 (2021) 112070
adsorption directly influence the transport of chemical compounds
(González-Pérez et al., 2017), which become immobilized on soil par­
ticles and biosolids (Łukaszewicz et al., 2016). However, the effective­
ness of the adsorption mechanism varies across antibiotic groups.
Research has demonstrated that anaerobic digestion affects the
adsorption of antibiotics to sludge particles by enhancing the accumu­
lation of these compounds in sewage sludge, delaying biotic and abiotic
degradation processes, and potentially decreasing the bioavailability of
antimicrobials (Łukaszewicz et al., 2016; Urra et al., 2019). The
persistence of antimicrobials in sewage sludge, in particular biosolids,
has not been studied extensively, but more is known about their
behaviour in the soil environment. According to the literature, antibi­
otics released into the soil with biosolids undergo various processes,
such as transport, fixation, degradation, and inactivation, and the rate
and efficiency of these processes are significantly determined by the
structure and biochemical properties of soil, the soil microbiome, and
weather conditions (Barra Caracciolo et al., 2015; Verlicchi and Zam­
bello, 2015). Biodegradation plays a key role in the elimination of xe­
nobiotics, including antimicrobials. During this process, xenobiotics are
degraded or mineralized with the involvement of soil-dwelling micro­
organisms (Barra Caracciolo et al., 2015). The effectiveness of biodeg­
radation is largely dependent on the activity and quantitative and
qualitative composition of native soil bacteria and allochthonous mi­
croorganisms that reach soil with sewage sludge, as well as the physi­
cochemical properties of pharmaceuticals, which determine their
susceptibility to biodegradation mechanisms (Pan and Chu, 2016). Ac­
cording to the literature, the widely used FQs, SAs, and trimethoprim are
most resistant to microbial degradation (Sittig et al., 2014). The
persistence of antibiotics in the environment is considerably influenced
by local conditions, including oxygen concentration, pH, insolation,
temperature, and seasonal variations in these parameters (Barra Car­
acciolo et al., 2015). Sitting et al. (2014) found that the transformation
of sulfadiazine in soil was inhibited with an increase in depth. They
attributed their findings to a decrease in microbial activity resulting
from the limited availability of carbon (Sittig et al., 2014), as well as the
kinetics and non-linear adsorption of sulfadiazine to soil material (Sittig
et al., 2012). Sulfamethoxazole has a half-life of 24.8–57.8 days in soil,
and it is lower under aerobic conditions (Pan and Chu, 2016). The rate at
which SAs are degraded in the environment is very difficult to deter­
mine, because sulfonamide antibiotics respond differently to abiotic
factors such as UV radiation (Zessel et al., 2014). Pan and Chu (2016)
ranked five antibiotics in the following order based on their degradation
rates in the soil under aerobic conditions: nor­
flox­
acin > erythromycin > chloramphenicol > sulfamethazine > tetracy­
cline. The analyzed antibiotics were characterized by very similar
degradation rates under anaerobic conditions. Tetracycline and sulfa­
methazine were more stable regardless of the experimental conditions
(aerobic, anaerobic, control soil samples, sterilized soil samples) (Pan
and Chu, 2016). Tetracyclines have the longest half-life in agricultural
soil, and they are most readily adsorbed to soil particles relative to other
antibiotic groups. Therefore, the adsorption of tetracyclines to solids
with microporous and nanoporous structures temporarily decreases
their bioavailability for microbial enzymes in soil (Jechalke et al., 2014).
Antibiotics present in soil undergo many biotic and abiotic mechanisms
that affect removal and degradation of these compounds but still some
amounts of antimicrobials could be absorbed by crops (Łukaszewicz
et al., 2016). Because of wide structural and mass differentiation be­
tween antibiotics, some of them end up in roots or they are transported
through xylem and phloem tissue to seeds, leaves, or fruits (Miller et al.,
2016). The ability to absorb antimicrobials and antibiotic residues also
depends on the plant species. Amoxicillin and tetracycline are easily
taken up by plants such as carrot or lettuce (Pan et al., 2014). Research
presented by Kumar et al. (2005) indicates that chlortetracycline can be
easily accumulated by cabbage, corn, or onion. Due to the large atomic
mass of macrolide molecules, these antibiotics usually are not absorbed
M. Buta et al. Ecotoxicology and Environmental Safety 214 (2021) 112070

Table 2
Concentration of selected NSAIDs detected in wastewater and sewage sludge.
Detected drug Source Concentration [µg kg-1/ µg L-1] References

Aspirin Wastewater influent 100.00 (Feng et al., 2013)

Wastewater effluent 0.05–1.51 (Feng et al., 2013)
Diclofenac Wastewater influent 0.10–4.11 (Feng et al., 2013)
Treated sewage sludge ~ 4.00–8.00 (Li et al., 2016)
Wastewater effluent 13.70–22.90 (Saleh et al., 2011)
Biosolid 39.00 (Sagristà et al., 2010)
< 10–1100.00 (Oluwatosin et al., 2016)
0.04–1.95 (Feng et al., 2013)
22.00 (Cortés et al., 2013)
Fenoprofen Treated sewage sludge ~ 100.00–400.00 (Li et al., 2016)
Ibuprofen Wastewater influent 0.17–83.50 (Feng et al., 2013)
Treated sewage sludge 304.00–588.00 (Saleh et al., 2011)
Wastewater effluent < 60–360.00 (Oluwatosin et al., 2016)
Biosolid < 95 (Feng et al., 2013)
217.00 (Cortés et al., 2013)
Ketoprofen Wastewater influent > 0.32 (Feng et al., 2013)
Treated sewage sludge 51.30–89.60 (Saleh et al., 2011)
Wastewater effluent 0.14–1.62 (Feng et al., 2013)
Mefenamic acid Wastewater influent 0.14–3.20 (Feng et al., 2013)
Wastewater effluent 0.09–2.40 (Feng et al., 2013)
Naproxen Wastewater influent 1.79–611.00 (Feng et al., 2013)
Treated sewage sludge 7.7–14.10 (Saleh et al., 2011)
Wastewater effluent 0.17–33.0 (Feng et al., 2013)
Paracetamol Wastewater effluent 6.90 (Feng et al., 2013)

by crops (Pan et al., 2014). Absorption of sulfonamides and trimetho­
prim is only limited to the roots (Tanoue et al., 2012). There are many
variables that influence the susceptibility of plants to the adsorption of
antibiotics, and the results of environmental and lab-scale studies pre­
sent wide differences. Extensive environmental studies with maintaining
natural environmental conditions are still needed.
The persistence and availability of antibiotics introduced to soil with
sewage sludge varies, but even small amounts of antimicrobials can
trigger a strong response from living organisms (Guo et al., 2017). Soil
microbes are subjected to strong pressure, because they have to coexist
with antibiotics that are regularly introduced into the environment
(even if their quantities are small) or gradually released from soil par­
ticles (Cycoń et al., 2019), which leads to frequent genetic mutations
and chromosomal rearrangements as well as the exchange of adaptive
genes between microorganisms with different degrees of phylogenetic
affinity (Felden and Cattoira, 2018). Antibiotic resistance genes play a
specific role in this process as the constitutive genomic elements of
bacteria which are naturally resistant to a given antibiotic or as genetic
elements that are acquired by HGT (Frey et al., 2015). Among all HGT
mechanisms, conjugation plays a crucial role in ARGs transfer from one
bacteria to another, mainly via plasmids or transposons (Giebułtowicz
et al., 2018). This process is strongly promoted by the high abundance of
microorganisms inhabiting soil (Frey et al., 2013). The antibiotics and
ARGs present in biosolids not only contaminate soil but can also reach
surface water and leach to groundwater (Frey et al., 2015; Tyrrell et al.,
2019; Wu et al., 2020). These emerging contaminants pose a particularly
high risk when they are transferred to groundwater which is a source of
potable water, or to surface recreational waters (Ma et al., 2017; Sze­
keres et al., 2018). Rainfall significantly contributes to the infiltration of
micropollutants through deep soil horizons (Pruden et al., 2012).
However, infiltration is not the only process in the water cycle, and
according to recent research, micropollutants also reach plants with the
soil solution. In plants, a key role in the uptake of water and nutrients is
played by the roots that act as passive organs of absorption (Thanner
et al., 2016), and xylem tissues responsible for nutrient and water
transport (Tyrrell et al., 2019). Research has shown that plants
contribute to the elimination of ARGs from agricultural soil through
absorption. In uncropped soil, the copy numbers of selected ARGs were
higher than in cropped soil (Wang et al., 2015). The presence of selected
genes encoding resistance to SAs and TCs was also observed in genetic
material isolated from plants grown on fertilized soils (Table 1) (Zhang
et al., 2019). The above poses a serious risk when crops are consumed in
an unprocessed form (Zhang et al., 2019), and the consumption of raw
vegetables is currently encouraged due to the resulting health benefits
(Shen et al., 2019; WHO, 2019). According to Rahube et al. (2014,
2016), soil fertilization with sewage sludge and biosolids increases the
concentrations and diversity of ARGs in crops harvested in the same
season when fertilizers were applied.
5. Non-steroidal anti-inflammatory drugs
Due to the growing levels of microbial resistance to antibiotics,
current research is focusing on antimicrobial drugs as the main source of
the problem. However, other pollutants of environmental concern (PEC)
are also discharged to the natural environment in massive quantities.
These include non-steroidal anti-inflammatory drugs (NSAIDs) con­
taining more than 100 compounds that have been identified in sewage
sludge in recent years (Bartha et al., 2014). Many of these drugs are
over-the-counter analgesics that are easily available and widely used
(Chen et al., 2015). Due to a high demand for NSAIDs around the world,
their production has been increasing recently at 12% per year (Feng
et al., 2013). The most widely consumed NSAIDs are diclofenac (DCF),
ibuprofen, naproxen and (RS)-2-(4-(2-methylpropyl) phenyl) propanoic
acid (Schmidt and Redshaw, 2015; González-Pérez et al., 2017).
Ibuprofen and (RS)-2-(4-(2-methylpropyl) phenyl) propanoic acid have
been identified in wastewater or even drinking water at a concentration
of 3.7–603.0 µg L-1 (González-Pérez et al., 2017). Diclofenac is one of
the most frequently detected pharmaceuticals in treated wastewater
(Osorio et al., 2014; Haiba et al., 2017). Peysson and Vulliet (2013)
identified DCF in samples of sewage sludge in a concentration range of
35–133 µg kg-1. The concentrations of selected NSAIDs in raw sewage,
treated wastewater, and sewage sludge are presented in Table 2. Due to
its high toxicity, DCF was placed on the European Union’s watch list of
substances for Union-wide monitoring in the field of water policy (Eu­
ropean Parliament and European Council, 2014).
Adsorption and biodegradation are the key processes in the elimi­
nation of NSAIDs in WWTPs. The effectiveness of NSAID degradation is
influenced by the pH of the matrix (wastewater/sewage sludge), sludge
age, and HRT (Yan et al., 2019). Biodegradation is a highly effective
method of removing ibuprofen from raw sewage, but not DFC (Fer­
nandez-Fontaina et al., 2012). The adsorption of hydrophilic compounds
to solid particles is limited, and these substances are not highly

7
M. Buta et al. Ecotoxicology and Environmental Safety 214 (2021) 112070

effectively removed by sorption. The above compromises the elimina­
tion of hydrophilic NSAIDs in biodegradation processes (Feng et al.,
2013). Highly hydrophilic propionic acid derivatives are removed dur­
ing biological and chemical treatment of wastewater, but the effective­
ness of these treatment methods in eliminating the above
pharmaceuticals can vary considerably (González-Pérez et al., 2017).
According to the literature, conventional wastewater treatment methods
are clearly less effective in removing DFC than other NSAIDs
(González-Pérez et al., 2017), but selected technological systems in
WWTPs (such as systems combining sequencing batch reactors with
activated carbon absorption) can eliminate up to 95% of DFC, but not all
NSAIDs (Yan et al., 2019). As a result, NSAIDs are present in treated
wastewater that is evacuated to surface water bodies as well as in treated
sewage sludge (Cortés et al., 2013; Osorio et al., 2014; Rossini et al.,
2016; Triñanes et al., 2016). Research has demonstrated that NSAIDs are
not completely removed during sewage sludge composting.
Non-steroidal anti-inflammatory drugs are carried by biosolids and can
be released into the environment in concentrations up of to several
hundred ng g-1 DM (Verlicchi et al., 2012; Haiba et al., 2017; Yan et al.,
2019). Similarly to the compounds discussed in the previous sections,
various NSAIDs can be accumulated in the environment (Schmidt and
Redshaw, 2015), because the concentrations of the released compounds
exceed the rate of biodegradation processes. Residual NSAIDs are
characterized by high bioavailability in the environment, and the uptake
of these compounds by living organisms is being extensively studied
(Luque-Muñoz et al., 2017). The results of toxicological studies indicate
that personal care products, including NSAIDs, exert a harmful influence
on earthworms (Kinney et al., 2012), anthropods (Chen et al., 2015),
and fish (Haiba et al., 2017; Luque-Muñoz et al., 2017), but research into
the effects of these compounds on plants has been initiated only recently
(Schmidt and Redshaw, 2015). Plants absorb NSAIDs from the envi­
ronment, and they are capable of accumulating, transporting, and
transforming these compounds inside tissues (Opriș et al., 2020).
Research into the toxic effects of NSAIDs on plants, including crops, has
focused mainly on DCF which is widely consumed around the world.
Hammad et al. (2018) reported that DCF, ibuprofen, and paracetamol
exert cytotoxic and genotoxic effects on the growth and development of
corn (Zea mays). Paracetamol can be transported to corn grain (Hammad
et al., 2018). Other researchers found that the phytotoxic effects of
ibuprofen induce changes in lettuce (Lactuca sativa) roots in the early
stages of growth (Schmidt and Redshaw, 2015). Research conducted on
rice demonstrated that ibuprofen exerts toxic effects and is accumulated
in crops (Aznar et al., 2014). Studies of beans and soybeans revealed that
crops actively participate in the absorption of NSAIDs from
biosolid-amended soil and that these compounds are present in both
underground and above-ground plant parts (Wu et al., 2010).
The absorption of NSAIDs by plants poses a significant risk for
humans and animals consuming these products. However, in the future,
absorption can be used as a phytoremediation technique to reduce the
pharmaceutical contamination of water (Bartha et al., 2014).
6. Heavy metals and heavy metal resistance genes in sewage
sludge - sources, persistence and transfer in the environment
Industrial development leads to an increase in pollutant concentra­
tions, including heavy metals, in wastewater, which directly contributes
to the contamination of sewage sludge (Cheng et al., 2014; Di Cesare
et al., 2016a). The most frequently identified sources of heavy metal
pollution in the environment are presented in Table S3 (Lakherwal,
2014; RoyChowdhury et al., 2018; Vardhan et al., 2019). Heavy metals
are present in sewage sludge in dissolved or precipitated form, and they
can also be adsorbed or associated with solid particles. The limit values
for heavy metals in sewage sludge intended for agricultural use are
regulated by the European Council Directive 86/278/EEC (European
Council, 1986), and they are set by the US EPA in the United States
(RoyChowdhury et al., 2018). The ceiling concentrations of both regu­
lations are presented in Table S4.
In sewage sludge, the most frequently identified heavy metals are Cr,
Mn, Fe, Co, Ni, Cu, Zn, Hg, Cd, Pb, and the highly toxic As and Se. Heavy
metals can be arranged in the following ascending order based on their
concentrations in sewage sludge Zn>Cu>Cr>Ni>Pb>Cd or
Zn>Cr>Pb>Cu>Ni>Cd. Industrial wastewater and soil surface runoffs
are the main sources of heavy metals in sewage sludge (Fijalkowski
et al., 2017). The concentrations of heavy metals in sewage sludge in
selected countries are presented in Table 3. It should be noted that the
heavy metal content of sewage sludge rarely exceeds legal limits, but a
prolonged accumulation of heavy metals in areas where these com­
pounds are deposited with sewage sludge can pose serious environ­
mental risks (Iglesias et al., 2018).
In highly industrialized regions, raw sewage reaching WWTPs is
composed mainly of industrial wastewater, characterized by high con­
centrations of heavy metals (Cheng et al., 2014; Di Cesare et al., 2016a).
The following heavy metals are found mostly in industrial wastewater:
Zn, Ni, Cu, Pb, As, Co, Cd, Hg, and Cr (Olujimi et al., 2012). The content
of heavy metals in raw sewage reaching WWTPs are strictly determined
by their source. Gao et al. (2015) reported differences in the concen­
trations of Zn, Pb, Cu, and Cr in raw sewage with different composition.
Raw sewage containing mostly domestic wastewater was characterized
by the highest concentration of Pb|Cr (440|240 µg L-1) and the lowest
concentration of Zn|Cu (112|37 µg L-1), whereas a reverse correlation
was noted in raw sewage composed mainly of industrial wastewater.
The content of heavy metals in raw sewage is responsible for the heavy
metal contamination of sewage sludge used for agricultural purposes.
Most heavy metals that occur naturally in soil are not available to
plants, and their concentrations are generally low (Z. Song et al., 2017).
Agricultural operations have increased the content of heavy metal in
soil. Tillage, soil liming, and the application of phosphorus fertilizers not
only enrich soil with minerals that are essential for the growth of crops,
but they also increase soil contamination with toxic metals such as Zn,
As, Cd, and Pb (Liu et al., 2017). Soil fertilization with sewage sludge,
excessive use of manure and agricultural chemicals also increase heavy

Table 3
Concentration of chosen heavy metals in various sewage sludge samples (µg kg-1 DM) (Cheng et al., 2014).
Cu Zn Pb Cd Ni Cr

China (domestic 5.57 × 104–1.1 × 106 3.43 × 105–3 × 106 1.1 × 104–1.03 × 105 0.71 × 103–7.82 × 103 3.86 × 103–1.24 × 105 2.29 × 104–7.37 × 105
sewage
sludge)
(industrial 6.9 × 104–1.2 × 107 4.02 × 105–3.38 × 106 3.24 × 104–1.09 × 105 0.62 × 103–5.38 × 103 1.19 × 104–2.73 × 105 8.55 × 104–1.44 × 105
sewage
sludge)
Slovenia 1.85 × 105–6.95 × 105 1.03 × 106–2.65 × 106 8.8 × 104–1.75 × 105 1.5 × 103–4.22 × 103 3.72 × 105–9.95 × 105 4.08 × 105–1.235 × 106
France 1.49 × 105–3.4 × 105 5.48 × 105–1 × 106 1.97 × 104–6.2 × 104 0.6 × 103–2.2 × 103 2.64 × 104–4.4 × 104 2.76 × 104–1.2 × 105
Spain 1.31 × 105–4.06 × 105 5.19 × 105–2.47 × 106 4.72 × 104–2.23 × 105 1.68 × 103–9.2 × 103 9.8 × 103–3.66 × 104 2.32 × 104–4.39 × 105
China 1.69 × 105–2.05 × 106 1.69 × 105–6.72 × 106 1.36 × 104–9.37 × 104 0.9 × 103–1.12 × 105 1.58 × 104–2.33 × 105 6.16 × 104–1.844 × 106
Egypt 1.84 × 105–1.38 × 106 3.5 × 105–3.54 × 106 NA 2.3 × 103–3.9 × 103 3.9 × 104–2.71 × 105 1.07 × 105–1.12 × 106

Legend: NA – data not available.

8
M. Buta et al. Ecotoxicology and Environmental Safety 214 (2021) 112070

Table 4 Heavy metals, even if present in the soil at low concentrations, can be
Heavy metal concentrations (mg kg-1) in plants. readily absorbed by roots and accumulated in the edible plant parts
Plant Cd Cr Cu Ni Pb References (Yang et al., 2009; Jolly et al., 2013). The international threshold values
cde a ade for selected heavy metals in soils and crops are presented in Table S5.
Tomato NA 6.1 10.5 1.6 4.45 (Amin, 2013)
Onion NA 1.05 cd 6.05 2a 2.7ade Shi et al. (2016) reported that the uptake of heavy metals from soil
Brinjal NA 7.5cde 6.85 3a 4.35ade differed in various parts of crop plants (wheat). The rate of absorption
Lettuce < 0.2c < 0.2 8.67 < 0.2 < 0.2 (Castro et al., was highest in roots followed by leaves, whereas the lowest amounts of
2009) heavy metals were absorbed by stems and grain. The concentrations of
Lettuce 14.98acde NA 8.15 NA 3.64ade (Waterlot et al.,
2013)
Cu and Ni were highest, whereas the concentrations of Pb and Cd were
Potato 0.09 0.11 0.06 0.06 NA (Khan, 2013) lowest. Hu et al. (2013) found that vegetables purchased in a super­
Tomato 0.09 0.12 0.03 0.07 NA market were contaminated with Pb (16%), Cd (26%), and Cr (0.56%).
Rice 0.08 NA 0.19 0.23 NA Another research study confirmed the presence of eight heavy metals in
Lettuce 0.049 0.13 NA NA 0.18 (Chang et al.,
edible plant parts. The following abundance pattern was proposed based
Chinese 0.027 0.23 NA NA 0.11 2014)
cabbage on the concentrations of the identified heavy metals:
Cabbage 0.41cde 1.33 cd 2.84 1.09 0.54de (Eliku and Leta, Cr>Zn>Ni>Cd>Mn>Pb>Cu>Fe. The content of Ni, Pb, Cd, and Cr
Onion 0.22cde 1.25 cd 2.01 0.53 0.34de 2017) exceeded the threshold values set by the World Health Organization by
Green 0.25cde 2.63cde 1.92 0.72 0.49de 90%, 28%, 83%, and 63%, respectively (Balkhair and Ashraf, 2016). The
pepper
Bean 0.17ce 0.51c 1.12 0.28 0.26
concentrations of selected heavy metals in crops and exceedance of the
Swiss chard 0.21cde 1.34 cd 2.31 0.86 0.46de relevant threshold values in international standards are presented in
Table 4.
Legend: a Above the permissible limit of India; b Above the permissible limit of
Heavy metals exert selective pressure on environmental microor­
USEPA; c Above the permissible limit of China EPA; d Above the permissible limit
of EU; e Above the permissible limit of FAO/WHO.
ganisms, which promotes the emergence and spread of microbial resis­
tance to these pollutants (Menz et al., 2018). HGT mechanisms
constitute a major driving force in the spread of both HMRGs and ARGs
metal levels in soil (Kelepertzis, 2014). Research has demonstrated that
in the environment. Mobile genetic elements (MGEs) are an integral part
sewage sludge used as fertilizer contributes to increasing the concen­
of the resistant gene spreading process. Conjugative plasmids and inte­
trations of heavy metals in soil (Sieciechowicz et al., 2014; Marguí et al.,
grons are considered as main carriers of HMRGs (Di Cesare et al.,
2016). Mungai et al. (2016) identified eight heavy metals at concen­
2016b). The presence of HMRGs in many environments has been widely
trations approximating the US EPA threshold values for heavy metals in
described in the literature (Table 5). In a study by Di Cesare et al.
agricultural soil in Kenya. In soil samples from China, Cai et al. (2019)
(2016b), the mean concentrations of HMRGs in raw sewage (municipal
detected high concentrations of Cd, Pb, Zn, Ni, Cu, Hg, Cr, and As which
and hospital wastewater) were determined at 5.45 × 104 gene copies
originated mainly from human activities.
mL-1 (czcA) and 7.38 × 105 gene copies mL-1 (arsB). Yuan et al. (2019)
As already mentioned, sewage sludge is used as fertilizer in the
determined the abundance of HMRGs in the range of 103-106 gene
agricultural sector due to its beneficial properties, but it also contains
copies mL-1 in wastewater and 108-109 gene copies g-1 DM in sewage
pollutants that can be absorbed by crops and pose health risks for con­
sludge. In addition to wastewater and sewage sludge, HMRGs have also
sumers. The presence and accumulation of heavy metals in arable soils
been detected in soil and crops. Pitondo-Silva et al. (2016) performed an
fertilized for long periods of time with sewage sludge have been well
extensive analysis of genes encoding resistance to As, Co, Cu and Hg in
documented in the literature (Chen et al., 2016; Marguí et al., 2016;
soil samples from numerous Brazilian plantations. According to Zhou
Iglesias et al., 2018). Heavy metals present in farmland can be accu­
et al. (2016), crops are one of the main sources of heavy metals for
mulated in the soil solution and transferred to groundwater and crops
humans due to the high contamination of edible plant parts. Since heavy
(Kelepertzis, 2014). The persistence of heavy metals in the soil and crops
metals can be accumulated in sewage sludge and transferred to soil and
is determined by many factors, including soil structure, cation exchange
crops, they pose a significant health risk for humans and animals (Gul
capacity, root secretions, and, in particular, soil pH and the chemical
et al., 2015). However, the influence of sewage sludge fertilizers on the
structure of heavy metals (S. Wang et al., 2017). Crop plants are the most
abundance of HMRGs in soil has not been sufficiently investigated to
important pillar of human nutrition and a rich source of nutrients such
date. Further research is needed, because microorganisms exposed to
as carbohydrates, vitamins, minerals, and fiber (Zhou et al., 2016).
heavy metals rapidly develop resistance to these compounds.

Table 5 7. Relationships between antibiotics, metals and resistance

Heavy metal resistance genes detected in various environment. determinants
Heavy Resistance genes Source References
metal 7.1. Co-selection of resistance to metals and antibiotics
Cd czcA wastewater (Di Cesare et al.,
Co czcA 2016a) As mentioned in the previous sections, heavy metals and antibiotics
Zn czcA are ubiquitous in sewage sludge and the natural environment. The co-
As arsB existence of these pollutants poses a significant epidemiological threat
Hg merB, merD, merR wastewater (Yuan et al., 2019) due to the co-selection phenomenon. In bacteria, the co-selection
Ag silE, silR sewage sludge
Cu corR, copR, ricR agricultural (Thomas et al., 2020)
mechanism involves co-resistance and cross-resistance. In co-resistant
As pstB, arsT, pstC soil bacteria, at least two different resistance genes encoding resistance to
Fe acn, furA, bfrA many toxic compounds are localized on the same MGE, whereas in cross-
Ni hoxN nikE nikD resistant bacteria, a single gene is responsible for resistance to antibi­
Zn zraR/hydH, znuC/
otics or heavy metals (Pal et al., 2015) (Fig. S2).
yebM, troB
Mo modC, modB, modA Unlike antibiotics, which can be inactivated through sorption and
As arsB, arsC agricultural (Pitondo-Silva et al., degradation processes in the environment, metals are not degraded and
Co czcA soil 2016) exert long-term selective pressure (Menz et al., 2018). As a result, metals
Cu copA, copB can exert stronger selective pressure on microorganisms than antibiotics
Hg merA
(J. Song et al., 2017). Wu et al. (2018) identified genes encoding

9
M. Buta et al.
resistance to SAs (sul), aminoglycosides (aph, aac), tetracyclines (tet),
and heavy metals (arsB, copB, ruvB), whereas Besaury et al. (2013) and
Li et al. (2012) reported positive correlations between the content of
heavy metals and the presence of HMRGs in soils and sewage sludge.
Zhao et al. (2018) identified metals that potentially co-select for ARGs,
and found 24 significant positive correlations between them, and no
significant negative correlations. The presence of As, Cd, Co, Cr, Cu, Hg,
Ni, and Zn was correlated with selected antibiotic resistance genes. Zinc
was associated with genes conferring resistance to aminoglycosides
(aadA, strB, aac), beta-lactams (blaCMY), and multidrug resistance
genes (ttgB, acrF, qacEdelta). Copper was found to co-exist with genes
encoding resistance to aminoglycosides (aadA and aac) and MLSB an­
tibiotics (mefA). Cobalt was associated with tetracycline resistance genes
(tetP), and Ni – with vancomycin resistance genes (vanC). Strong positive
correlations between genes encoding resistance to heavy metals and
antibiotics were also determined in samples of treated wastewater.
Genes conferring resistance to colistin (mcr-1) and methacycline (mecA)
were associated with the Cu resistance gene (copA). Correlations were
also found between macrolide resistance genes (mphB) and genes
encoding resistance to Co, Zn, and Cd (czcA) (Yuan et al., 2018).
7.2. Mechanism of co-resistance
The co-selection of resistance to metals and antibiotics, involving the
co-resistance mechanism, has been widely described in the literature
(Table S6). This phenomenon is strongly linked with the presence of
resistance genes on MGEs such as integrons and transposons, which play
a key role in the emergence of bacterial resistance through HGT (Pal
et al., 2017). The changeable region of DNA harbors resistance mecha­
nisms in integrons which induce the co-selection process in bacteria
colonizing environments contaminated with metals, antibiotics, and
other toxic compounds. Rosewarne et al. (2010) reported numerous
class I integrons in bacterial populations colonizing an aquatic envi­
ronment strongly contaminated with heavy metals. The authors
demonstrated that class I integrons contributed to the induction of
bacterial resistance under exposure to high levels of heavy metal stress
(Rosewarne et al., 2010).
Research into bacteria colonizing environments strongly contami­
nated with heavy metals provides interesting examples of co-resistance
to antibiotics in heavy metal-resistant bacteria. The genetic material of
the analyzed bacterial strains contained numerous genes encoding
resistance to heavy metals (As, Cu, Cd, Zn, Cr) and antibiotics (beta-
lactams, FQs) (Farias et al., 2015). These genes were carried by plasmids
(Farias et al., 2015). The co-existence of plasmid-borne genes conferring
resistance to heavy metals (Cd, Zn, Cu, Hg) and antibiotics was also
documented by Jayaprakashvel et al. (2015) and Pal et al. (2015). In
these studies, genes encoding resistance to Zn and Cu and genes
conferring resistance to aminoglycosides and macrolides were identified
in plasmids.
Amachawadi et al. (2013) and Fang et al. (2016) confirmed the
important role of HGT in the co-resistance process. In the work of
Amachawadi et al. (2013), a highly mobile Cu resistance gene was
identified on a plasmid carrying macrolide and glycopeptide resistance
genes. In the study by Fang et al. (2016), genes encoding resistance to
FQs, chlorhexidine, and beta-lactams were detected on plasmids
harboring Cu and Ag resistance operons.
7.3. Mechanisms of cross-resistance and co-regulation
Cross-resistance is yet another mechanism of co-selection for anti­
biotic and metal resistance in bacteria (Table S6). In this process, anti­
biotics and metals initiate identical biochemical pathways, which leads
to the emergence of antibiotic resistance stimulated by metal resistance
(Imran et al., 2019).
Studies investigating cross-resistance to various toxic compounds
have shown that in bacteria, this mechanism is effectively mediated by
10
Ecotoxicology and Environmental Safety 214 (2021) 112070
efflux pumps. An analysis of the genome of Acinetobacter baumannii
strains revealed that they harbor efflux pumps that confer resistance to
heavy metals, metalloids, and antibiotics (Fournier et al., 2006). Nishino
et al. (2007) found that the BaeSR system, which regulates AcrD and
MdtABC efflux pumps in Salmonella Typhimurium strains, confers resis­
tance to Cu, Zn, and beta-lactam antibiotics.
The cross-resistance mechanism was also identified in Enterobacter
cloacae strains isolated from Pb-contaminated soil in a lead battery
manufacturing facility. These strains were resistant to Pb and several
antibiotics, including oleandomycin, ampicillin, lincomycin, cephalori­
dine, co-trimoxazole, and chloramphenicol. The key to their resistance
was the mechanism of extracellular sequestration of toxic substances in
an exopolysaccharide matrix (Naik et al., 2012). Gaidhani et al. (2014)
observed that the cross-resistance mechanism was responsible for
multidrug resistance and resistance to Ag and Hg compounds in clinical
isolates of Acinetobacter haemolyticus.
Co-regulation also plays an important role in co-selection for resis­
tance (Fig. S2; Table S6). In this process, high metal concentrations in
the environment increase bacterial resistance to antibiotics by regu­
lating the expression of the respective genes, which decreases the sus­
ceptibility of bacteria to these compounds (Rensing et al., 2018). This
mechanism was described in detail by Harrison et al. (2009). In their
study, Cr and Cu ions induced oxidative stress in Escherichia coli strains,
which increased the expression of the soxS gene that regulates the AcrAb
efflux pump. Due to the higher expression of this efflux pump, Cr, and Cu
increased bacterial tolerance to chloramphenicol, tetracycline, novobi­
ocin and oxacillin.
8. Conclusions and future perspectives
Despite the fact that biosolids meet current quality standards, their
application in farming could pose a threat to the health and the life of
living organisms that are exposed to contaminants for long periods of
time. Heavy metals and antibiotics present in biosolids and their mutual
interactions contribute to the emergence of bacterial resistance to these
pollutants. The absence of effective methods capable of removing ARGs
from sewage sludge promotes the transfer of genes encoding resistance
to nearly all antibiotic groups, including last-resort drugs, and multidrug
resistance genes. It seems that only a drastic reduction in the use of
antibiotics or the use of alternative treatment agents (active substances
of plant origin, e.g. anthocyanins) could allow the reduction of micro­
pollutants in sewage sludge, and therefore their wider use in agriculture.
Prolonged accumulation of even small amounts of heavy metals exposes
environmental microorganisms to the toxic effects of these compounds,
which leads to the inactivation or death of soil microbes, and lowers the
quality of agricultural soils. The presence of pollutants of environmental
concern in biosolids has not yet been addressed by a large-scale envi­
ronmental research and/or legal regulations to minimize the risk of their
transfer to the environment and their long-term effects on living or­
ganisms. All pollutants described in this article are transferred to crops
and pose a risk for consumers. Therefore, the presence of pollutants in
treated sewage sludge should be taken into account in the scientific
debate concerning the use of sewage sludge in agriculture.
Funding
This study was supported by grant No. 2017/27/B/NZ9/00267 from
the Polish National Science Center and by Project financially supported
by Minister of Science and Higher Education in the range of the program
entitled “Regional Initiative of Excellence” for the years 2019–2022,
Project No. 010/RID/2018/19, amount of funding 12.000.000 PLN.
Martyna Buta has received a scholarship from the Interdisciplinary
Doctoral Program in Bioeconomy (POWR.03.02.00-00-I034/16-00)
funded by the European Social Fund.
M. Buta et al. Ecotoxicology and Environmental Safety 214 (2021) 112070

CRediT authorship contribution statement health risks in the Pearl River Delta, South China. Environ. Monit. Assess. 186 (3),
1547–1560. https://doi.org/10.1007/s10661-013-3472-0.
Chen, Q., An, X., Li, H., Su, J., Ma, Y., Zhu, Y.-G.G., 2016. Long-term field application of
Review concept and design, M.B.; Drafting of manuscript (review sewage sludge increases the abundance of antibiotic resistance genes in soil.
and editing), M.B., J.H. and W.Z.; Critical revision, M.B., E.K., M.H.; Environ. Int. 92–93, 1–10. https://doi.org/10.1016/j.envint.2016.03.026.
Supervision, E.K. All authors have read and agreed to the published Cheng, M., Wu, L., Huang, Y., Luo, Y., Christie, P., 2014. Total concentrations of heavy
metals and occurrence of antibiotics in sewage sludges from cities throughout China.
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Christodoulou, A., Stamatelatou, K., 2016. Overview of legislation on sewage sludge
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Declaration of Competing Interest https://doi.org/10.2166/wst.2015.521.
Cortés, J.M., Larsson, E., Jönsson, J.Å., 2013. Study of the uptake of non-steroid anti-
inflammatory drugs in wheat and soybean after application of sewage sludge as a
The authors declare that they have no known competing financial fertilizer. Sci. Total Environ. 449, 385–389. https://doi.org/10.1016/j.
interests or personal relationships that could have appeared to influence scitotenv.2013.01.061.
Cycoń, M., Mrozik, A., Piotrowska-Seget, Z., 2019. Antibiotics in the soil environment –
the work reported in this paper. degradation and their impact on microbial activity and diversity. Front. Microbiol.
https://doi.org/10.3389/fmicb.2019.00338 (10).
Di Cesare, A., Eckert, E.M., Corno, G., 2016a. Co-selection of antibiotic and heavy metal
Appendix A. Supporting information
resistance in freshwater bacteria. J. Limnol. 75, 59–66. https://doi.org/10.4081/
jlimnol.2016.1198.
Supplementary data associated with this article can be found in the Di Cesare, A., Eckert, E.M., D’Urso, S., Bertoni, R., Gillan, D.C., Wattiez, R., Corno, G.,
online version at doi:10.1016/j.ecoenv.2021.112070. 2016b. Co-occurrence of integrase 1, antibiotic and heavy metal resistance genes in
municipal wastewater treatment plants. Water Res. 94, 208–214. https://doi.org/
10.1016/j.watres.2016.02.049.
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