Introduction

Cigarette butts are one of the leading causes of environmental pollution worldwide (Bonanomi et al. 2015; Rebischung et al. 2018; Micevska et al. 2006; Mansouri et al. 2020), with an estimated 5.6 trillion butts discarded annually (Smith and Novotny 2011; Booth et al. 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; Lee 2018). 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 significant 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 without any prior processing.

Several studies have detected the presence of numerous metals and metalloids in cigarette butts including varying concentrations of aluminium (Al), arsenic (As), barium (Ba), cadmium (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; Mansouri et al. 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 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 examined 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; Mansouri et al. 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; Mansouri et al. 2020). Metals and other toxic residues 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, and Bacillus, 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 contamination developed metal resistance, which has been found to correlate 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 potential 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 contents 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 concentration 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 bioluminescence 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) confirmed 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 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.

Fig. 1
figure 1

a Diagram of the artificial lung smoking-simulation apparatus. b Experimental set up of soil samples in the laboratory

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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 cigarettes containing 7 mg of tar and the non-biodegradable cigarettes 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 cigarette butts of each brand into two separate Erlenmeyer flasks containing 500 ml of sterile distilled water each, for a concentration 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 compounds 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 community structure to settle after the leachate introduction (Gill et al. 2018; Moerman and Potts 2011).

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 manufacturer’s instructions (Zymo Research Corp.). The integrity of the extracted DNA was examined with the use of a 1% agarose gel, stained with ethidium bromide (EtBr). The gel was visualised under UV light for detection of DNA fragments.

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 under 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 potential 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 hydrogen peroxide were added to each vessel. The samples were digested using the microwave method with the following settings: power level 1600 W, 100%; ramp time, 25 min; pressure 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 accuracy 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 identified 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 recorded 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’s F test to determine if there were any significant differences 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 significant 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% significance level.

The structuring of bacterial communities, based on the results of ARISA (sample peaks), was visualised by constructing a non-metric multi-dimensional scaling (NMDS) plot using Bray-Curtis dissimilarity distances. In this 2D ordination 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 construction 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 vegan 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 contribution to the variability in the data. The loading plot component, 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 reflect 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).

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 bacterial 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 addition 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 diversity of the soil samples treated with non-biodegradable cigarette butt leachate clustered tightly with the control samples, suggesting that only the biodegradable cigarette butt treatment significantly affected the bacterial diversity.

Fig. 2
figure 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

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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’s F tests, the variances of all data points were not significantly different at a 95 % confidence 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 significant 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 data was 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 quantifying this effect, and the change in beta diversity was found to be significant.

Fig. 3
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a Shannon indices (H) before and after treatment with cigarette butt leachate. b Simpson indices (D) before and after treatment with cigarette butt leachate

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The average pH readings of the soil samples, prior to and following treatment, are presented in Table 1. Statistical analysis showed that there were no significant differences in the pH values, before and after treatment, for all three treatment groups. According to the results of Fisher’s F tests, 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, therefore, 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 acidity or alkalinity of the soil. A study by Quéméneur et al. (2020) demonstrated that both smoked and unsmoked cigarette filters decreased the pH of marine sediment, suggesting that the filters, rather than the leachate, may affect environmental pH.

Table 1 Average pH readings of soil samples treated with control, biodegradable and non-biodegradable leachate
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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 biodegradable 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 biodegradable cigarettes having a significantly higher level of the elements. The metals which were present in the highest concentration 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; Micevska et al. 2006; Moerman and Potts 2011; Moriwaki et al. 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 predominance 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 cigarette 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.

Table 2 Metal concentrations present in the cigarette butts and leachate treatments as determined by ICP-MS analysis
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It has been suggested previously that the toxicity of cigarette 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; Micevska et al. 2006). Though it is known that bacteria require certain essential elements, such as Ca, Co, Cu, Fe, K, Mn, Mo, Na, Ni Se, V, and Zn for normal physiological functioning, excess concentrations of these metals are toxic (Lemire et al. 2013). Moreover, metals such as Hg and Pb are known to be highly toxic to living organisms (Dobaradaran et al. 2017; Tiquia-Arashiro 2018). The presence 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 respective 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) provides additional evidence 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.

Fig. 4
figure 4

Principal component analysis (PCA) biplot of the soil samples and their correlation with the metals in leachate, pH, and diversity indices. The biplot demonstrates how strongly each factor (represented by a vector) 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

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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 cigarette 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 contents 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 nitrogen content), thereby altering the abiotic factors in the soil habitat. Although this was not investigated in this study, further 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 diversity can most likely be explained by the promotion of certain bacterial groups which are resistant to metals, as well as selective pressure against bacterial groups which are susceptible 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 abundant 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 cigarette 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 nicotine, 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 environmentally friendly, the impact which the increased metal concentrations has in these cigarettes is concerning and represents a potential source of toxic pollution and environmental damage.

It should be noted that this study has a number of limitations. First, only two sampling events took place (before and after treatment) which may not accurately depict the changes in bacterial diversity that are brought about by the application of cigarette leachate. Although this was sufficient for the aim of this study, further research should include multiple sampling time points. Second, the experiments were conducted under controlled laboratory conditions, which may reduce the microbial diversity that is found in the natural environment (Stewart 2012). Third, as mentioned above, changes in other soil characteristics, with the exception 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 sampling time frames as well as cigarette butt concentrations may be in need of further investigation and possibly adjustment for a more informative evaluation of the effects of cigarette butts. Future research into this area should, therefore, include a larger sample size, multiple sampling time points as well as a more thorough analysis of the soil characteristics and taxonomic composition of bacterial communities. 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.

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; however, 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 characterisation 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.