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RESEARCH ARTICLE
Impact of cigarette butts on bacterial community structure in soil
Elizaveta Koroleva
1
&Aza Zizipo Mqulwa
1
&Scott Norris-Jones
1
&Sidney Reed
1
&Zahraa Tambe
1
&Aiden Visagie
1
&
Karin Jacobs
1
Received: 8 September 2020 / Accepted: 22 February 2021
#The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021, corrected publication 2021
Abstract
Cigarette butts contribute significantly to global pollution present on the planet. The filters found in cigarette butts contain a
microplastic, cellulose acetate, as well as toxic metals and metalloids which are responsible for pollution in the environment.
Although cigarette butt litter is prevalent in many soils, research on the effects of these cigarette butts is limited. In this study, we
used Automated Ribosomal Intergenic Spacer Analysis (ARISA) to generate DNA fingerprints of bacterial communities in soil
before and after the addition of cigarette butt leachate treatments. An ICP-MS analysis of the biodegradable and non-
biodegradable cigarette butts revealed the presence of various elements: Al, As, B, Ba, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni,
Pb, Sb, Se, Sn, Sr, V, and Zn. The analysis also specified which metals were present at the highest concentrations in the
biodegradable and non-biodegradable cigarette butts, and these were, respectively, Al (1,31 g/kg and 2,35 g/kg), Fe (2,03 g/kg
and 1,11 g/kg), and Zn (3,18 mg/kg and 15,70 mg/kg). Our results show that biodegradable cigarette butts had a significant effect
on bacterial community composition (beta diversity), unlike the non-biodegradable butts. This effect can be attributed to higher
concentrations of certain metals and metalloids in the leachate of biodegradable cigarette butts compared to the non-
biodegradable ones. Our findings suggest that biodegradable and non-biodegradable cigarette butts can significantly affect
bacterial communities in soil as a result of the leaching of significant quantities of certain elements into the surrounding soils.
Keywords Cigarette butt .Bacterial diversity .Soil .Metals .Metalloids .Microbial ecology .ARISA .Mass spectrometry
Introduction
Cigarette butts are one of the leading causes of environmental
pollution worldwide (Bonanomi et al. 2015;Rebischungetal.
2018;Micevskaetal.2006;Mansourietal.2020), with an
estimated 5.6 trillion butts discarded annually (Smith and
Novotny 2011;Boothetal.2015; Torkashvand and Farzadkia
2019; Slaughter et al. 2011). The detrimental effects of smoking
cigarettes on human health are well-documented (Bernhard
et al. 2005;Lee2018). Some of these include respiratory cancer
(Vineis 2008), several cardiovascular conditions (Ambrose and
Baruna 2004), as well as impacts on the reproductive health of
male smokers (Singh and Kathiresan 2015). Second- and third-
hand smoking have been shown to have an even more signifi-
cant impact on health, with prolonged exposure to cigarette
smoke linked to the development of fibrosis, cirrhosis, and
strokes (Martins-Green et al. 2014). In addition, cigarettes have
a significant potential environmental impact when their key
components, which include nicotine, tar, artificial additives as
well as the filter itself, are discarded into the environment with-
out any prior processing.
Several studies have detected the presence of numerous
metals and metalloids in cigarette butts including varying con-
centrations of aluminium (Al), arsenic (As), barium (Ba), cad-
mium (Cd), chromium (Cr), copper (Cu), cobalt (Co), iron
(Fe), mercury (Hg), lead (Pb), manganese (Mn), nickel (Ni),
selenium (Se), strontium (Sr), titanium (Ti), and tin (Zn)
(Chevalier et al. 2018; Dobaradaran et al. 2016;
Dobaradaran et al. 2017; Koutela et al. 2020;Mansourietal.
2020; Moerman and Potts 2011; Pelit et al. 2013). A number
of these are known for their toxicity to living organisms
(Barakat 2011; Tiquia-Arashiro 2018). Moreover, various
metals and other chemicals were similarly detected during
cigarette butt leaching experiments, providing evidence for
The original online version of this article was revised: The correct
captions of Figures 2 and 3 is shown in this paper.
Responsible Editor: Robert Duran
*Karin Jacobs
kj@sun.ac.za
1
Department of Microbiology, Stellenbosch University, Private Bag
X1, Stellenbosch, South Africa
Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-021-13152-w
their dissemination (Dobaradaran et al. 2016,2017;Koutela
et al. 2020; Mansouri et al. 2020). The consensus of these
studies is that there is a high similarity in metal content across
different kinds of cigarettes, though none of these studies ex-
amined biodegradable cigarettes.
The presence of the above-mentioned elements in cigarettes
can be traced to several potential sources which include the soil
in which the tobacco was grown, which fertilisers, herbicides,
and pesticides were used as well as the cigarette manufacturing
process itself (Moerman and Potts 2011; Slaughter et al. 2011;
Dobaradaran et al. 2016;Mansourietal.2020). During smoking,
the filter of the cigarette prevents metal inhalation by the user by
trapping these and other harmful compounds (Dobaradaran et al.
2020). However, as a result of the decomposition of the cigarette
butt and filter, these components are subsequently released into
the environment where the butts are discarded and can become
toxic to human health, animals, and the environment (Joly and
Coulis 2018;Mansourietal.2020). Metals and other toxic res-
idues contained in the butts can affect plant growth (Chibuike
and Obiora 2014; Green et al. 2019), cause the environment
around it to become toxic (Singh and Kalamdhad 2011;Joly
and Coulis 2018), and potentially disrupt the soil ecology.
Several studies have shown that metals can have a significant
measurable impact not only on the composition of microbial
communities but also the metabolism and therefore, the activity,
and/or function of these microbes (Prakash Bansal 2019;
Quéméneur et al. 2020; Xie et al. 2011). Beattie et al. (2018)
observed a decrease in the bacterial population after long-term
exposure to heavy metal (such as Al, Cd, Pb, Zn) decades after a
mining operation. However, prolonged exposure to heavy metals
can also favour the growth of metal-resistant bacteria (Moerman
and Potts 2011) including members of the genus Streptococcus,
Arthrobacter,andBacillus, the presence of which was found to
be highly correlated with Pb concentrations (Prakash Bansal
2019). Exposures to these heavy metals may also result in the
growth of antibiotic resistant bacteria (Lemire et al. 2013).
Studies done by Dickinson et al. (2019) and Di Cesare et al.
(2016) showed that bacteria exposed to heavy metal contamina-
tion developed metal resistance, which has been found to corre-
late with the development of antibiotic resistance.
Furthermore, the metal content of cigarette butts is not the
only contributor to environmental pollution. Cigarette butts
contain many other contaminants such as nicotine, phenols,
and polycyclic aromatic hydrocarbons (PAHs) (Dobaradaran
et al. 2020; Vu et al. 2015). The latter forming a class of
compounds known for its carcinogenic properties and its po-
tential danger to the environment (Dobaradaran et al. 2019;
Dobaradaran et al. 2020). Moreover, many cigarette filters are
made of cellulose acetate, a type of plastic that is photo- but
not biodegradable due to the presence of the acetate group
(Dobaradaran et al. 2017; Stigler-Granados et al. 2019). The
filter breaks down into smaller pieces (microplastic pollution)
and can remain in this format in the environment for up to 10
years (Benavente et al. 2019; Novotny and Slaughter 2014;
Mansouri et al. 2020). Subsequently, an alternative method of
cigarette filter production has been developed which is far
more environmentally friendly as the filters produced this
way are made of entirely biodegradable pure cellulose—a
polymer that is decomposable by soil microbiota (Eichorst
and Kuske 2012). Consistent with this development, a study
by Joly and Coulis (2017) found that when placed in soil,
cellulose filters decomposed significantly faster than filters
made of plastic. It was, therefore, the hypothesis of this study
that the amount of metal pollution will increase when the
filters are biodegradable, due to an increased rate of filter
degradation leading to a more rapid dissemination of the con-
tents of the butts into the surrounding environments.
Although this paper focused on soil environments, several
previous studies have investigated the lethal impact of butt
leachates in aquatic environments (Dobaradaran et al. 2016;
Quéméneur et al. 2020). Register (2000) found that a concen-
tration of 1–2 cigarette butts/l had a significant effect on
Daphnia magna, a species of water flea, resulting in either lethal
effects or an altered swimming pattern (compared to the usual
hop-sink swimming pattern). A study by Micevska et al. (2006)
found cigarette butts to be toxic to the bacterium Vibrio fischeri
and 7.4 times more toxic to Ceriodaphnia cf. dubia, another
species of water flea. This toxicity caused a 30-min biolumines-
cence in V. fischeri and a 48-h immobilisation in C.cf.dubia.
Cigarette butt leachates were also shown to affect a freshwater
mussel, Anodontites trapesialis, by altering the behaviour and
even the immune system functioning of the organism
(Montalvão et al. 2019a,b). After 14 days, not only did the
authors note the presence of several heavy metals (Cr, Ni, Pb,
Mn, Zn) in the muscle tissue but also abnormalities in burrowing
behaviours of the exposed mussel specimens (Montalvão et al.
2019b). Additionally, a study by Dobaradaran et al. (2017)con-
firmed the presence of toxic metals Hg and Pb in cigarette butts
found near marine environments, and that long-term leaching of
these metals can affect marine life and organisms.
In contrast to aquatic environments, very few studies have
focused on the impact of cigarette litter on soil microbiota. This
is despite that it is the most common environment for cigarette
butt pollution and leaching in terrestrial soil environments. The
objectives of this study were, therefore, to elucidate the impact
of cigarette butt leachates on soil microbial communities as well
as to determine if this impact differed to a significant degree
between biodegradable and non-biodegradable cigarette butts.
Materials and methods
Experimental design
This experiment was carried out under controlled laboratory
conditions, with an ambient room temperature of
Environ Sci Pollut Res
approximately 21 °C. Nine 2-l plastic (polyethylene
terephalate) bottles were divided into control, biodegradable
and non-biodegradable groups, according to the treatment that
was added to the soil. The tops of the bottles were removed,
cut to a depth of 15 cm, and the resulting containers were
washed using distilled water and soap to remove any prior
contaminants. A total mass of 560 g of homogenised, non-
sterile organic potting soil were weighed and placed into each
container. The soil was commercially purchased and had an
average initial pH of 6.5. The top of each container with soil is
closed with cotton wool and covered with tin foil (Fig. 1b).
This allowed for an aerobic environment with no additional
introduction of microorganisms from the outside environment
(Schultz 1964). Containers were labelled accordingly and kept
for 1 week at ambient room temperature to allow the bacterial
communities to adjust.
Cigarette butt leachate solution preparation
Two different cigarette brands were used in this experiment,
one with a biodegradable filter and the other a non-
biodegradable filter. These brands were selected due to the
similarity in both nicotine and tar content, in order to reduce
variation in the results due to these components. Both cigarette
brands contained 6 mg of nicotine. The tar content differed by
1 mg between the two brands, with the biodegradable ciga-
rettes containing 7 mg of tar and the non-biodegradable ciga-
rettes containing 8 mg of tar. A smoke-simulating apparatus is
set up as described by Montalvão et al. (2018) to exclude the
involvement of smokers in generating smoked cigarette butts
(Fig. 1a). A cigarette is attached to a 20-ml syringe and kept in
place with Prestik™adhesive putty (Fig. 1a). The syringe
plunger was then pulled in and out, mimicking the act of
smoking, until the cigarette was burnt to approximately 1 cm
from the filter. The butts were then collected, placed in plastic
bags, and refrigerated for 4 days at 4 °C for later use in the
study.
The leachate solutions were prepared by placing two ciga-
rette butts of each brand into two separate Erlenmeyer flasks
containing 500 ml of sterile distilled water each, for a concen-
tration of 2 butts/l, based on a previous study by Gill et al.
(2018). These flasks were then closed with cotton wool and
foil. The flasks were placed on an orbital shaker (Scientific
Engineering) at a speed of 106 rpm for 1 week, in a 37 °C dark
incubator. Most of the compounds are leached within 1 day
(Gill et al. 2018); however, the flasks in this study were left on
the shaker for 1 week to ensure maximal leaching of the com-
pounds within the cigarette butts.
Application of treatment
The treatment was applied to the samples by adding 100 ml of
the leachate solution into the soil. The samples were then left
in the laboratory for a period of 3 weeks, to allow the com-
munity structure to settle after the leachate introduction (Gill
et al. 2018;MoermanandPotts2011).
DNA extraction and ARISA-PCR
Soil samples for DNA extraction were taken 2 cm under the
soil surface from each container before and after treatment
with cigarette butt leachate. DNA was extracted from each
of the samples using the Zymo Research Quick-DNA™
Fecal/Soil Microbe Miniprep Kit according to the manufac-
turer’s instructions (Zymo Research Corp.). The integrity of
the extracted DNA was examined with the use of a 1% aga-
rose gel, stained with ethidium bromide (EtBr). The gel was
visualised under UV light for detection of DNA fragments.
b
a
Fig. 1 aDiagram of the artificial lung smoking-simulation apparatus. bExperimental set up of soil samples in the laboratory
Environ Sci Pollut Res
The ARISA-PCR mix consisted of the ReadyMix, MilliQ
water, the FAM (carboxy-fluorescein) labelled forward primer
1406f, 5’TGYACACACCGCCCGT 3’(500 nM), the reverse
primer 23Sr, 5’GGGTTBCCCCATTCRG 3’(500 nM), and
the DNA (0.5 μl) (Slabbert et al. 2010; Fisher and Triplett
1999). The ARISA-PCR conditions were as follows: 5 min
at 94 °C; 34 cycles of amplification for 30 s at 94 °C, 30 s at 56
°C, and 1 min at 72 °C; with a final elongation phase for 7 min
at 72 °C. The integrity of the ARISA-PCR was also examined
by gel electrophoresis using EtBr. The gel was visualised un-
der UV light for detection of DNA fragments. PCR for each
sample was performed in triplicate and pooled to eliminate
background noise of the ARISA profile and to reduce poten-
tial errors due to PCR variability.
Physicochemical parameters
The concentrations of specific elements in the cigarette butt
leachate solution and entire cigarette butts for both the non-
biodegradable and biodegradable cigarettes were determined
through elemental analysis via inductively coupled plasma
mass spectrometry (ICP-MS). A total of two artificially
smoked cigarette butts and two leachate samples (15 ml)
(one biodegradable and one non-biodegradable) were sent to
the Central Analytical Facility at the University of
Stellenbosch for ICP-MS analysis. Whole cigarette butts were
analysed as follows: 0.3 g of sample was weighed directly into
microwave Teflon vessels. Subsequently, 6-ml concentrated
ultra-pure nitric acid and 1-ml concentrated ultra-pure hydro-
gen peroxide were added to each vessel. The samples were
digested using the microwave method with the following set-
tings: power level 1600 W, 100%; ramp time, 25 min; pres-
sure 800 psi; and hold time, 10 min. Following digestion,
vessels were cooled, and each sample was made to a final
volume of 50 ml with 1% HCl prior to analysis. The ICP-
MS analysis was then performed using the Agilent 7900
ICP-MS system. The data was quantified using calibration
solutions prepared from NIST traceable standards, and US
EPA quality control guidelines were followed to ensure accu-
racy of the data. The concentrations of Al, As, B, Ba, Cd, Co,
Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, V, and Zn
present in the samples were determined. These metals and
metalloids were chosen for detection as they have been iden-
tified in previous studies as being present in cigarette butt
leachate (Koutela et al. 2020; Moerman and Potts 2011;
Pelit et al. 2013).
Shortly before DNA extractions were performed, soil pH
readings were taken using a Beckman Phi-32 pH metre.
Fifteen grammes (15 g) of soil were weighed for each sample
and mixed with 30 ml of deionised, sterile water. After letting
the soil and water slurry settle for 30 min, readings were re-
corded to the nearest 0.01 decimal on the calibrated pH metre
(Mclean 1982). Measurements were taken before and after
treatment of cigarette butt leachate with a period of 3 weeks
between readings. The DNA extractions and the pH readings
were performed simultaneously in order to ensure that the
microbial communities in the soil from the ARISA analysis
would correlate with a specific pH reading.
Statistical analysis
The data from the pH test and the Shannon and Simpson
indices before and after treatment were statistically tested for
any significant changes. This was done by firstly completing a
Fisher’sFtest to determine if there were any significant dif-
ferences in the variances of the data. Next, normality tests
were performed on the data in the form of a Shapiro-Wilk
and an Anderson-Darling test. The results from these tests
were then used in the decision to utilise an analysis of variance
(ANOVA) test on the data to detect whether there was a sig-
nificant difference between the data before and after treatment
addition. All statistical tests were performed using XLSTAT
2020 (Microsoft Excel add-in programme) at a 5% signifi-
cance level.
The structuring of bacterial communities, based on the re-
sults of ARISA (sample peaks), was visualised by construct-
ing a non-metric multi-dimensional scaling (NMDS) plot
using Bray-Curtis dissimilarity distances. In this 2D ordina-
tion plot, the closeness of two data points is indicative of
higher beta diversity similarity of the microbial communities.
The vegan and ggplot2 R packages were used for the con-
struction of the plot. A stress function was used to assess the
goodness of fit of the ordination. Stress values of < 0.2 are
indicative of a good representation of the data (Clarke 1993).
Ellipses were added, using the ellipse package in R, to each
group of samples before and after treatment and represented a
95% confidence cluster. Alpha diversity indices including
Shannon and Simpson indices were calculated using the veg-
an package in R. Additionally, a principal component analysis
(PCA) biplot was constructed using the ggbiplot2 package to
evaluate the impact of the metals on the observed diversity
indicators. The PCA component of the plot presents clusters
of variables (soil samples) based on their similarity, whereby
closely clustered data points are more similar to each other
than distantly located points. This is done by transforming
the variables into principal components representing their con-
tribution to the variability in the data. The loading plot com-
ponent, in the form of vectors representing metals, pH, and
diversity indices, reflects the contribution of these factors to
the principal component(s). The angles between vectors re-
flect the correlation between variables, with angles closer to
90 or 270 degrees indicative of lower correlation than those
closer to 0 or 180 degrees (Kohler and Luniak 2005). All
statistical plots were created using R (v3.6.3) in RStudio
(v1.2.5001) (R Development Core Team; http://www.R-
project.org).
Environ Sci Pollut Res
Results and discussion
The differences in ARISA profiles of the soil samples are
visualised in the NMDS plot (Fig. 2). The stress value of the
plot was calculated to be 0.052, indicating that it is a good
representation of the data (Clarke 1993). A clear shift in bac-
terial community composition of all samples after treatment
can be observed, based on the clear separation of samples
before and after treatment. This was expected due to the addi-
tion of water to the soil (in the form of the leachate) which
increased the moisture content. However, as can be seen by
the groupings of the sample clusters in Fig. 2, the beta diver-
sity of the soil samples treated with non-biodegradable ciga-
rette butt leachate clustered tightly with the control samples,
suggesting that only the biodegradable cigarette butt treatment
significantly affected the bacterial diversity.
Alpha diversity indices were calculated for all samples.
The Shannon (H) and Simpson (D) indices are illustrated in
Fig. 3a and b, respectively. The results of the statistical tests
revealed that no significant differences in the Shannon and
Simpson indices were observed for samples before and after
treatment. According to Fisher’sFtests, the variances of all
data points were not significantly different at a 95 % confi-
dence level, with the corresponding p-values being p=0,966
(Shannon index) and p= 0,463 (Simpson index). The results
of the normality tests done at a 5% significance level showed
that the data followed a normal distribution. The p-values for
the Shapiro-Wilk test of normality were as follows: p=0,534
(Shannon index) and p= 0,890 (Simpson index). The p-values
for the Anderson-Darling test of normality were as follows: p
= 0,322 (Shannon index) and p= 0,743 (Simpson index).
Based on the ANOVA analysis, there were no significant
differences in the indices data after the addition of treatment.
Although the statistical tests suggest that there were no signif-
icant changes in the alpha diversity after the addition of the
leachate, it must be taken into consideration that the relative
scale of the study was small, and thus, only a relatively small
amount of datawas generated. As such, future research should
include a larger sample size for a more informative analysis of
alpha diversity changes. Furthermore, it should be considered
that although the statistical analyses suggest no changes in
alpha diversity, it is a single parameter by which the effect
the treatments had on the bacterial communities can be
assessed. Beta diversity represents another dimension of quan-
tifying this effect, and the change in beta diversity was found
to be significant.
The average pH readings of the soil samples, prior to and
following treatment, are presented in Table 1. Statistical
Fig. 2 Non-metric multidimensional scaling (NMDS) plot showing the
beta diversity of the samples, before and after treatment was applied,
based on the Automated Ribosomal Intergenic Spacer Analysis
(ARISA) profiles (stress value: 0.0524). B 1-3, biodegradable cigarette
treatment; C 1-3, control treatment; N 1-3, non-biodegradable treatment
Environ Sci Pollut Res
analysis showed that there were no significant differences in
the pH values, before and after treatment, for all three treat-
ment groups. According to the results of Fisher’sFtests, the
variances of all data points were not significantly different at a
95% confidence interval (p= 0,101). The normality tests
showed that the pH data followed a normal distribution
(Shapiro-Wilk: p= 0,209; Anderson-Darling: p= 0,203).
Based on the results of ANOVA of all three treatment groups,
the data showed no significant differences from before the
treatment, compared to after the treatment (p= 0,121).
Testing of pH was not conducted on the soil samples during
the adjustment period between DNA extractions, in order to
prevent disturbing the soil microbial communities. It is, there-
fore, possible that the treatments did have an initial effect on
the pH of the soil samples, but that this effect was mitigated
during the adjustment period. However, it is more likely that
none of the treatments had any significant impact on the acid-
ity or alkalinity of the soil. A study by Quéméneur et al.
(2020) demonstrated that both smoked and unsmoked ciga-
rette filters decreased the pH of marine sediment, suggesting
that the filters, rather than the leachate, may affect environ-
mental pH.
ICP-MS analysis reveals the presence of numerous metals
and metalloids in the cigarette butt and/or leachate (Table 2).
The concentration of most elements contained in the biode-
gradable cigarette butts was much greater than that of non-
biodegradable cigarettes. This trend holds true with regard to
the different leachates as well, with the leachate of biodegrad-
able cigarettes having a significantly higher level of the ele-
ments. The metals which were present in the highest concen-
tration in both butts as well as both leachates include Al, Fe,
and Zn (Table 2). Most elements detected in this study were
previously found to be present in cigarette butts in several
earlier studies (Dobaradaran et al. 2016;Micevskaetal.
2006; Moerman and Potts 2011;Moriwakietal.2009).
Chevalier et al. (2018) showed similar metals (such as Al,
Mn, Fe, and Zn) being present within the cigarettes, with the
exception of a predominance in Cr in their study and a pre-
dominance of metals such as B, Ba, and Sr and in this study.
The concentrations of Hg and Pb in both types of cigarette
butts were comparable to those reported by Dobaradaran et al.
(2017); however, the level of As in this study was much lower
than was detected in cigarette butts by Mansouri et al. (2020).
The latter may be attributed to different leaching durations,
type of cigarette brand as well as differences in analytical
techniques. Additionally, the concentrations of Cd, Cu, Mn,
and Zn were within the range of those found in several ciga-
rette brands by Pelit et al. (2013), with the exception of Mn in
non-biodegradable cigarette butts, the concentration of which
was much lower in this study.
It has been suggested previously that the toxicity of ciga-
rette butt leachate can be partially attributed to the metals
contained within the cigarettes, which is why a metal analysis
of the cigarette butts was performed (Abdu et al. 2016;
0.9
0.91
0.92
0.93
0.94
0.95
0.96
0.97
0.98
0.99
1
Simpson indices (D) of samples before
and after treatment
D Before D After
3
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
Control Biodegradable Non-biodegradableControl Biodegradable Non-biodegradable
Shannon indices (H) of samples before
and after treatment
H Before H After
ab
Fig. 3 aShannon indices (H) before and after treatment with cigarette butt leachate. bSimpson indices (D) before and after treatment with cigarette butt
leachate
Table 1 Average pH readings of
soil samples treated with control,
biodegradable and non-
biodegradable leachate
Treatment Before treatment After treatment Standard deviation
Control 5.395 5.445 0.035
Biodegradable 5.523 5.472 0.036
Non-biodegradable 5.592 5.493 0.070
Environ Sci Pollut Res
Micevska et al. 2006). Though it is known that bacteria re-
quire certain essential elements, such as Ca, Co, Cu, Fe, K,
Mn, Mo, Na, Ni Se, V, and Zn for normal physiological func-
tioning, excess concentrations of these metals are toxic
(Lemire et al. 2013). Moreover, metals such as Hg and Pb
areknowntobehighlytoxictolivingorganisms
(Dobaradaran et al. 2017; Tiquia-Arashiro 2018). The pres-
ence of heavy metals has been shown in previous studies to be
toxic to several different microbes including V. fischeri
(Micevska et al. 2006).
When comparing the amount of heavy metals contained
within the cigarette leachate and within the whole cigarette
butt, it can be seen that the whole cigarette butt in both cases
contained more of the heavy metals than the leachate, with the
biodegradable cigarette butts containing more of the respec-
tive heavy metals in most cases. Tobacco plants are known to
absorb certain heavy metals, Cd being the most well-known of
these (Abd El-Samad and Hanafi 2017). This difference in
metal absorption between different tobacco plant varieties,
pesticide dosage or contaminated water use, may be a factor
affecting heavy metal concentrations between the non-
biodegradable and the biodegradable cigarettes.
The PCA biplot analysis (Fig. 4)providesadditionalevi-
dence that the changes observed in beta diversity were most
likely due to the presence of heavy metals contained in the
leachate of the biodegradable cigarette butts. This can be seen
as the biodegradable data points cluster according to the metal
vectors on the graph. A change in the bacterial community
was, therefore, expected to occur as a result of the introduction
of metals into the soil environments. The plot also indicates
the lack of correlation between the presence of most metals
and the change in pH suggesting that these factors are not
linked.
The observed shift in bacterial community structuring may
be attributed to several possible reasons. This study, however,
was limited to the investigation of metal contents of the ciga-
rette butts and the associated pH changes. The addition of
biodegradable cigarette butt leachate is shown to correlate
with a change in beta diversity of bacterial communities
(Fig. 2). Considering the large difference between metal con-
tents of non-biodegradable and biodegradable cigarette butt
leachates, it is likely that the addition of metal residues, at
least partially, accounts for the observed diversity changes.
The addition of these residues in the form of leachate also
likely affected several soil characteristics (e.g. carbon and ni-
trogen content), thereby altering the abiotic factors in the soil
habitat. Although this was not investigated in this study, fur-
ther research into the effects of these compounds is warranted.
The exact mechanism by which the community shifts occur is
not addressed in this paper. However, the shift in beta diver-
sity can most likely be explained by the promotion of certain
bacterial groups which are resistant to metals, as well as se-
lective pressure against bacterial groups which are susceptible
Table 2 Metal concentrations present in the cigarette butts and leachate treatments as determined by ICP-MS analysis
B Al V Cr Mn Fe Co Ni Cu Zn As Se Sr Mo Cd Sn Sb Ba Hg Pb
Whole cigarette buttICP-MSanalysis
LOD (μg/kg) 1639 326.9 1.2 36.4 23.7 84.5 1.2 12.4 93.9 21.3 27.5 4.7 3.0 2.1 0.0 4.5 1.0 0.9 1.8 2.5
Non-Biodegradable (μg/kg) *BDL 1315336 518 537 2991 2033132 225 489 968 3185 81 30 7093 22 475 60 *BDL 2185 *BDL 514
Biodegradable (μg/kg)8355 2353070 1388 1580 51899 1116223 381 1075 4193 15700 91 12 43649 173 109 119 9 41331 3 866
Cigarette butt leachate solution ICP-MS analysis
LOD (μg/l)8.20 1.63 0.01 0.18 0.12 0.42 0.01 0.06 0.47 0.11 0.14 0.02 0.01 0.01 0.00 0.02 0.01 0.00 0.01 0.01
% Accuracy QC 105 115 102 102 99 104 99 100 101 103 102 100 97 95 102 102 101 102 101 104
Non-Biodegradable (μg/l)8.91 25.28 0.07 *BDL 1.53 3.94 0.07 0.14 6.66 38.74 0.32 0.05 6.91 0.07 0.08 0.04 *BDL 3.97 0.60 0.16
Biodegradable (μg/l)31.05 126.26 0.26 0.27 35.53 22.90 0.29 1.07 10.22 145.09 0.33 0.02 56.63 0.20 0.12 0.06 0.02 40.45 0.60 0.29
Heavy metal concentration of the respective treatments of cigarette butt leachate
Non- Biodegradable (μg/100 ml) 0.891 2.528 0.007 0.000 0.153 0.394 0.007 0.014 0.666 3.874 0.032 0.005 0.691 0.007 0.008 0.004 0.000 0.397 0.060 0.016
Biodegradable(μg/100 ml) 3.105 12.626 0.026 0.027 3.553 2.290 0.029 0.107 1.022 14.509 0.033 0.002 5.663 0.020 0.012 0.006 0.002 4.045 0.060 0.029
Environ Sci Pollut Res
to these metals. Microbes benefitting from the presence of
heavy metals were shown in a study by Zadel et al. (2020)
where members of the genus Micromonospora, associated
with the Miscanthus x giganteus plant root, were more abun-
dant during heavy metal stress of Cd, Pb, and Zn.
Despite the observed non-significant effect the non-
biodegradable filter leachate had on bacterial diversity and
pH, the cellulose acetate filters within non-biodegradable cig-
arette butts are still a major source of pollution due to the
degradation of these filters leading to the formation of
microplastics (Bonanomi et al. 2015; Rebischung et al.
2018; Kurmus and Mohajerani 2020). Moreover, the metal
pollution as a result of these cigarette butts is also significant
due to the large volume of butts being discarded (Dobaradaran
et al. 2016). In addition, other toxic compounds such as nico-
tine, tar, and PAHs released from the butts may also affect
microbial life (Dobaradaran et al. 2019; Dobaradaran et al.
2020; Joly and Coulis 2018). Cigarette butts may also release
larger amounts of PAHs into the environment if left exposed
for longer periods of time (Dobaradaran et al. 2019) and could
be detrimental to living organisms in those environments
(Dobaradaran et al. 2020). A study by Bonanomi et al.
(2020) revealed that long-term decomposition of cigarette
butts (2–5 years) can cause an increase in the toxicity levels.
Although biodegradable cigarettes are marketed as more en-
vironmentally friendly, the impact which the increased metal
concentrations has in these cigarettes is concerning and
represents a potential source of toxic pollution and environ-
mental damage.
It should be noted that this study has a number of limita-
tions. First, only two sampling events took place (before and
after treatment) which may not accurately depict the chang-
es in bacterial diversity that are brought about by the appli-
cation of cigarette leachate. Although this was sufficient for
the aim of this study, further research should include multi-
ple sampling time points. Second, the experiments were
conducted under controlled laboratory conditions, which
may reduce the microbial diversity that is found in the nat-
ural environment (Stewart 2012). Third, as mentioned
above, changes in other soil characteristics, with the excep-
tion of pH, were not evaluated, which may have provided
further insight into the observed changes in diversity.
Lastly, due to the exploratory nature of this study, the sam-
pling time frames as well as cigarette butt concentrations
maybeinneedoffurtherinvestigationandpossiblyadjust-
ment for a more informative evaluation of the effects of
cigarette butts. Future research into this area should, there-
fore, include a larger sample size, multiple sampling time
points as well as a more thorough analysis of the soil char-
acteristics and taxonomic composition of bacterial commu-
nities. It is, however, evident from our results that the used
cigarette butts discarded in soil have an observed effect on
the bacterial communities, with the biodegradable cigarette
butts seemingly having a more pronounced effect.
Fig. 4 Principal component analysis (PCA) biplot ofthe soil samples and
their correlation with the metals in leachate, pH, and diversity indices.
The biplot demonstrates how strongly each factor (represented by a vec-
tor) influences the principal components. Data points N 1-3 before/after
indicate samples before and after treatment with non-biodegradable
cigarette leachate; data points B 1-3 before/after indicate samples before
and after treatment with biodegradable cigarette leachate; data points C 1-
3 before/after indicate samples before and after control treatment with
pure distilled sterile water
Environ Sci Pollut Res
Conclusion
The current study partially confirmed our hypothesis that the
contents leached from cigarette butts have an effect on the
bacterial diversity in the soil, as the biodegradable cigarette
butt leachate was shown to alter the bacterial beta diversity. In
addition, the study provided evidence that smoked cigarette
butts are a source of several toxic metals and metalloids,
which were shown to be present in the cigarette butts itself,
as well as its leachate. Our results suggest that the shift in
community structure is likely attributed to the relatively higher
concentration of metals in the biodegradable cigarettes; how-
ever, additional evidence is needed to confirm this. Future
research is, therefore, needed to investigate the impacts of
other cigarette brands and those containing different metal
concentrations to establish the effects of these elements on
bacterial community structure. Moreover, a taxonomic char-
acterisation of the changes in bacterial diversity as well as the
presence of other potentially toxic compounds in cigarette
butts that may influence microbial communities is warranted.
This will provide a deeper understanding of the environmental
hazards posed by improperly discarded cigarette butts.
Acknowledgements We are grateful to Stellenbosch University
Microbiology Department for the use of their equipment and materials,
the Central Analytical Facility at Stellenbosch University for performing
the ARISA and ICP-MS analyses and Dr B. Loedolff for providing as-
sistance and guidance throughout the project. Thank you to Mr C. Brink
and Ms T. Conradie for assisting in ARISA visualisation and data anal-
ysis and Mrs J. Daniels for preparing the materials and reagents.
Authors’contributions EK, AZM, SNJ, SR, and ZT were undergraduate
students at the time the study was conducted. They were responsible for
the conceptualisation, planning, and execution of the project; they also
produced the draft manuscript. AV is a post-graduate student who
mentored and guided them during the course of the study; KJ is the
lecturer of the course and was responsible for assisting with the writing
and finalisation of the manuscript. The author(s) read and approved the
final manuscript.
Funding The project formed part of the Microbial Ecology module
(MKB364) in the Molecular Biology and Biotechnology undergraduate
programme at Stellenbosch University. Funding for the project was pro-
vided by the Department of Microbiology.
Data Availability Submission of raw data is not applicable to this manu-
script but will be made available to reviewers on request.
Declarations
Ethics approval and consent to participate No ethics approval or con-
sent was required for this study.
Consent for publication All authors agreed to submission of the
manuscript.
Competing interests The authors declare no competing interests.
References
Abd El-Samad M, Hanafi HA (2017) Analysis of toxic heavy metals in
cigarettes by Instrumental Neutron Activation Analysis. J Taibah
Univ Sci. 11:822–829
Abdu N, Abdullahi A, Abdulkadir A (2016) Heavy metals and soil mi-
crobes. Environ Chem Lett 15:65–84
Ambrose J, Baruna R (2004) The pathophysiology of cigarette smoking
and cardiovascular disease. J Am Coll Cardiol. 43:1731–1737
Barakat MA (2011) New trends in removing heavy metals from industrial
wastewater. Arab J Chem. 4:361–377
Beattie RE, Henke W, Campa MF, Hazen TC, McAliley LR (2018)
Variation in microbial community structure correlates with heavy-
metal contamination in soils decades after mining ceased. Soil Biol
Biochem. 126:57–63
Benavente M, Arévalo Caballero MJ, Silvero G, López-Coca I, Escobar
G (2019) Cellulose Acetate Recovery from Cigarette Butts. MDPI
Proceedings 2:1–6
Bernhard D, Rossmann A, Wick G (2005) Metals in Cigarette Smoke.
IUBMB Life 57:805–809
Bonanomi G, Incerti G, Cesarano G, Gaglione SA, Lanzotti V (2015)
Cigarette butt decomposition and associated chemical changes
assessed by 13C CPMAS NMR. PloS ONEs 10:e0117393. https://
doi.org/10.1371/journal.pone.0117393
Bonanomi G, Maisto G, De Marco A, Cesarano G, Zotti M, Mazzei P,
Libralato G, Staropoli A, Siciliano A, De Filippis F, La Storia A,
Piccolo A, Vinale F, Crasto A, Guida M, Ercolini D, La Storia A
(2020) The fate of cigarette butts in different environments: decay
rate, chemical changes and ecotoxicity revealed by a 5-years decom-
position experiment. Environ Pollut. 261:1–11
Booth DJ, Gribben P, Parkinson K (2015) Impact of cigarette butt leach-
ate on tidepool snails. Mar Pollut Bull. 95:362–364
Chevalier Q, El Hadri H, Petitjean P, Bouhnik-Le Coz M, Reynaud S,
Grassl B, Gigault J (2018) Nano-litter from cigarette butts: environ-
mental implications and urgent consideration. Chemosphere 194:
125–130
Chibuike GU, Obiora SC (2014) Heavy polluted soils: effect on plants
and bioremediation methods. Appl Environ Soil Sci:1–12. https://
doi.org/10.1155/2014/752708
Clarke KR (1993) Non-parametric multivariate analyses of changes in
community structure. Aust J Ecol. 18:117–143
Di Cesare A, Eckert EM, D'Urso S, Bertoni R, Gillan DC, Wattiez R,
Corno G (2016) Co-occurrence of integrase 1, antibiotic and heavy
metal resistance genes in municipal wastewater treatment plants.
Water Res. 94:208–214
Dickinson AW, Power A, Hansen MG, Brandt KK, Piliposian G,
Appleby P, O'Neill PA, Jones RT, Sierocinski P, Koskella B, Vos
M (2019) Heavy metal pollution and co-selection for antibiotic re-
sistance: A microbial palaeontology approach. Environ Int. 132:
105117
Dobaradaran S, Nabipour I, Saeedi R, Ostovar A, Khorsand M,
Khajeahmadi N, HayatiR, Keshtkar M (2016) Association of metals
(Cd, Fe, As, Ni, Cu, Zn and Mn) with cigarette butts in northern part
of the Persian Gulf. Tob Control 26:461–463
Dobaradaran S, Schmidt TC, Lorenzo-Parodi N, Jochmann MA,
Nabipour I, Raeisi A, Stojanovic N, Mahmoodi M (2019)
Cigarette butts: an overlooked source of PAHs in the environment?
Environ Pollut. 249:932–939
Dobaradaran S, Schmidt TC, Lorenzo-Parodi N, Kaizur-Cegla W,
Jochmann MA, Nabipour I, Lutze HV, Telgheder U (2020)
Polycyclic aromatic hydrocarbons (PAHs) leachates from cigarette
butts into water. Environ Pollut. 259:113916
Dobaradaran S, Schmidt TC, Nabipour I, Ostovar A, Raeisi A, Saeedi R,
Khorsand M, Khajeahmadi N, Keshtkar M (2017) Cigarette butts
abundance and association of mercury and lead along the Persian
Environ Sci Pollut Res
Gulf beach: an initial investigation. Environ Sci Pollut Res. 25:
5465–5473
Eichorst SA, Kuske CR (2012) Identification of cellulose-responsive bac-
terial and fungal communities in geographically and edaphically
different soils by using stable isotope probing. Appl Environ
Microbiol. 78:2316–2327
Fisher MM, Triplett EW (1999) Automated Approach for Ribosomal
Intergenic Spacer Analysis of Microbial Diversity and Its
Application to Freshwater Bacterial Communities. Appl Environ
Microbiol. 65:4630–4636
Gill H, Rogers K, Rehman B, Moynihan J, Bergey E (2018) Cigarette
butts may have low toxicity to soil-dwelling invertebrates: Evidence
from a land snail. Sci Total Environ. 628:556–561
Green SD, Boots B, Da Silva CJ, Starkey T (2019) Cigarette butts have
adverse effects on initial growth of perennial ryegrass (gramineae:
Lolium perenne L.) and white clover (leguminosae: Trifolium
repens L.). Ecotoxicol Environ Saf 182:109418. https://doi.org/10.
1016/j.ecoenv.2019.109418
Joly FX, Coulis M (2018) Comparison of cellulose vs. plastic cigarette
filter decomposition under distinct disposal environments. Waste
Manag 72:349–353
Kohler U, Luniak M (2005) Data inspection using biplots. Stata J. 5:208–
223
Koutela N, Fernández E, Saru ML, Psillakis E (2020) A comprehensive
study on the leaching of metals from heated tobacco sticks and
cigarettes in water and natural waters. Sci Total Environ. 714:
136700. https://doi.org/10.1016/j.scitotenv.2020.136700
Kurmus H, Mohajerani A (2020) The toxicity and valorization options of
cigarette butts. Waste Management 104:104–118
Lee PN (2018) Tar level of cigarettes smoked and risk of smoking-related
diseases. Inhal Toxicol. 30:5–18
Lemire JA, Harrison JJ, Turner RJ (2013) Antimicrobial activity of
metals: mechanisms, molecular targets and applications. Nat Rev
Microbiol. 11:371–384. https://doi.org/10.1038/nrmicro3028
Mansouri N, Etebari M, Ebrahimi A, Ebrahimpour K, Rahimi B,
Hassanzadeh A (2020) Arsenic content of cigarette butt leach-
ate of five cigarette brands into water. Int J Environ Health
Eng. 9:13
Martins-Green M, Adhami N, Frankos M, Valdez M, Goodwin B,
Lyubovitsky J, Dhall S, Garcia M, Egiebor I, Martinez B,
Green HW, Havel C, Yu L, Liles S, Matt G, Destaillats H,
Sleiman M, Gundel LA, Benowitz N, Jacob P, Hovell M,
Winickoff JP, Curras-Collazo M (2014) Cigarette Smoke
Toxins Deposited on Surfaces: Implications for Human
Health. PLoS ONE 9:e86391. https://doi.org/10.1371/journal.
pone.0086391
Mclean EO (1982) Soil pH and line requirements. In: Methods of soil
analysis: Part 2 Chemical and microbiological properties 9, pp 199–
224
Micevska T, Warne MSJ, Pablo F, Patra R (2006) Variation in, and
causes of, toxicity of cigarette butts to a cladoceran and microtox.
Arch Environ Contam Toxicol. 50:205–212. https://doi.org/10.
1007/s00244-004-0132-y
Moerman JW, Potts GE (2011) Analysis of metals leached from smoked
cigarette litter. Tob Control 20:30–35
Montalvão MF, Chagas TQ, da Silva Alvarez TG, Mesak C, da Costa
Araújo AP, Gomes AR, de Andrade Vieira JE, Rocha TL, Malafaia
G (2019a) Cigarette butt leachate as a risk factor to the health of
freshwater bivalve. Chemosphere 234:379–387
Montalvão MF, Chagas TQ, da Silva Alvarez TG, Mesak C, da Costa
Araújo AP, Gomes AR, de Andrade Vieira JE, Rocha TL, Malafaia
G (2019b) How leachates from wasted cigarette butts influence
aquatic life? A case study on freshwater mussel Anodontites
trapesiali. Sci Total Environ 689:381–389
Montalvão MF, Sampaio LL, Gomes HH, Malafaia G (2018) An insight
into the cytotoxicity, genotoxicity, and mutagenicity of smoked cig-
arette butt leachate by using Allium cepa as test system. Environ Sci
Pollut Res. 26:2013–2021. https://doi.org/10.1007/s11356-018-
3731-2
Moriwaki H, Kitajima S, Katahira K (2009) Waste on the roadside, ‘poi-
sute’waste: its distribution and elution potential of pollutants into
environment. Waste Management 29:1192-1197
Novotny TE, Slaughter E (2014) Tobacco Product Waste: An
Environmental Approach to Reduce Tobacco Consumption. Curr
Environ Health Rep 1:208–216. https://doi.org/10.1007/s40572-
014-0016-x
Pelit FO, Demirdöğen RE, Henden E (2013) Investigation of heavy metal
content of Turkish tobacco leaves, cigarette butt, ash, and smoke.
Environ Monit Assess. 185:9471–9479
Prakash Bansal O (2019) The Influence of potentially toxic elements on
soil biological and chemical properties. In: Metals in Soil-
Contamination and Remediation. IntechOpen. https://doi.org/10.
5772/intechopen.81348
Quéméneur M, Chifflet S, Akrout F, Bellaaj-Zouari A, Belhassen M
(2020) Impact of cigarette butts on microbial diversity and dissolved
trace metals in coastal marine sediment. Estuar Coast Shelf Sci. 240:
106785
Rebischung F, Chabot L, Biaudet H, Pandard P (2018) Cigarette butts: a
small but hazardous waste, according to European regulation.Waste
Manag 82:9–14
Register K (2000) Cigarette butts as litter—toxic as well as ugly.
Underwater Naturalist 25:23–29
Schultz JS (1964) Cotton closure as an aeration in shaken flask fermen-
tations. Appl Microbiol. 12:305–310
Singh CR, Kathiresan K (2015) Effect of cigarette smoking on human
health and promising remedy by mangroves. Asian Pac J Trop
Biomed. 5:162–167
Singh J, Kalamdhad A (2011) Effects of heavy metals on soil,
plants, human health and aquatic life. Int J Res Chem
Environ 1:15–21
Slabbert E, Kongor RY, Esler KJ, Jacobs K (2010) Microbial diversity
and community structure in Fynbos soil. Mol Ecol 19:1031–1041
Slaughter E, Gersberg RM, Watanabe K, Rudolph J, Stransky C,
Novotny TE (2011) Toxicity of cigarette butts, and their chemical
components, to marine and freshwater fish. Tob Control 20:i25–i29.
https://doi.org/10.1136/tc.2010.040170
Smith EA, Novotny TE (2011) Whose butt is it? Tobacco industry re-
search about smokers and cigarette butt waste. Tob Control 20:2–9.
https://doi.org/10.1136/tc.2010.040105
Stewart EJ (2012) Growing unculturable bacteria. J Bacteriol 194:4151–
4160
Stigler-Granados P, Fulton L, Nunez Patlan E, Terzyk M, Novotny TE
(2019) Global health perspectives on cigarette butts and the environ-
ment. Int J Environ Res Public Health 16:1858. https://doi.org/10.
3390/ijerph16101858
Tiquia-Arashiro SM (2018) Lead absorption mechanisms in bacteria as
strategies for lead bioremediation. Appl Microbiol Biotechnol. 102:
5437–5444. https://doi.org/10.1007/s00253-018-8969-6
Torkashvand J, Farzadkia MA (2019) Systematic review on cigarette butt
management as a hazardous waste and prevalent litter: control and
recycling. Environ Sci Pollut Res. 26:11618–11630 https://doi-
org.ez.sun.ac.za/10.1007/s11356-019-04250-x
Vineis P (2008) Smoking and impact on health.Eur Respir Rev. 17:182–
186
Environ Sci Pollut Res
Vu AT, Taylor KM, Holman MR, Ding YS, Hearn B, Watson CH (2015)
Polycyclic Aromatic Hydrocarbons in the Mainstream Smoke of
Popular U.S. Cigarettes Chem Res Toxicol. 28:1616–1626. https://
doi.org/10.1021/acs.chemrestox.5b00190
Xie X, Liao M, Ma A, Zhang H (2011) Effects of contamination of single
and combined cadmium and mercury on the soil microbial commu-
nity structural diversity and functional diversity. Chin J Geochem
30:366–374
Zadel U, Nesme J, Michalke B, Vestergaard G, Płaza GA, Schröder P,
Radl V, Schloter M (2020) Changes induced by heavy metals in the
plant-associated microbiome of Miscanthus xgiganteus.SciTotal
Environ. 711:134433. https://doi.org/10.1016/j.scitotenv.2019.
134433
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