Bacterial interactions of microplastics extracted from toothpaste under controlled conditions and the influence of seawater

Science of the Total Environment 703 (2020) 135024

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Bacterial interactions of microplastics extracted from toothpaste under controlled conditions and the influence of seawater Gul Sirin Ustabasi, Asli Baysal ⇑ T.C. Istanbul Aydin University, Health Services Vocational School of Higher Education, Sefakoy Kucukcekmece, 34295 Istanbul, Turkey

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Interactions between bacteria and

MPs were investigated in both standard and seawater condition.  Environmental relevancy of the growth media significantly impacts on the toxicity.  MPs were affected different bacterium with the media, while the metabolic pathways were the same.  MPs with higher surface charge induced higher inhibition levels.

a r t i c l e

i n f o

Article history: Received 16 August 2019 Received in revised form 11 October 2019 Accepted 15 October 2019 Available online 3 November 2019 Editor: Jay Gan Keywords: Microplastics Toxicity Microorganism Seawater

a b s t r a c t Microplastics have become a global concern due to their increasing use and discharge into the environment. These ubiquitous particles are known to have extremely low degradation rates and accumulate mostly in the marine environment. The evidence for bioaccumulation and indicators of stress linked to microplastics is also stated in the literature. However, the real environmental impact of microplastics has not yet been revealed. Therefore, it is crucial to understand the interaction mechanisms between microplastics and (micro)organisms under controlled (standard) laboratory conditions and environmentally relevant conditions to reflect the true environmental -situation. In this study, we aimed to understand how microplastics extracted from commercially available toothpaste samples interacted with four types of bacteria under both standard and seawater conditions. For this purpose, bacterial inhibitions were examined, and mechanisms of inhibition were evaluated by biochemical parameters (total protein, lipid peroxidase, total antioxidant capacity, and extracellular carbohydrate levels) of bacteria and physicochemical properties (zeta potential, particle size, surface chemistry) of microplastics. Results showed that gram-positive Bacillus subtilis and gram-negative Pseudomonas aeruginosa were affected in controlled and sea water media, respectively. The inhibition of the bacteria relied on the high zeta potentials of the microplastics, and, biochemically, protein and lipid peroxidase activity of bacteria were important in both media. On the other hand, while biochemical responses were similar in both media, the difference between the cell wall and microplastics surface charge was important only in seawater. Ó 2019 Elsevier B.V. All rights reserved.

1. Introduction Throughout the world, the production of plastics has increased from 1.5 million tons to 335 million tons in the last 80 years ⇑ Corresponding author. E-mail address: [email protected] (A. Baysal). https://doi.org/10.1016/j.scitotenv.2019.135024 0048-9697/Ó 2019 Elsevier B.V. All rights reserved.

(Alimba and Faggio, 2019). This increase has caused higher rates of release into the environment and the frequency of contact of plastics with living systems has also increased. Carbery et al. (2018), stated that over 690 marine species have been affected by the plastic debris that ended up in the seas. Plastic debris has recently been recognized as an emerging pollutant, and due to the urgency of the current situation, European countries have

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made certain efforts to lower the discharge of plastics into the environment by increasing reusing and recycling practices (Plastics Europe, 2017). Due to several chemical and physical processes, plastics tend to fragment into very small particles that are referred to as microplastics (MPs). MPs can also be manufactured to have a particular size (<5 mm) and shape to serve various purposes in industrial products (Jiang, 2018). Frequently used cosmetic products such as shower gels, facial creams, and toothpastes are known to include MPs (Napper et al., 2015; Lei et al., 2017), and they are released into the marine environment through domestic wastewater streams due to the inadequate ability of current wastewater treatment plants to capture MPs (Roex et al., 2013; Kay et al., 2018). The toxicity of MPs may vary depending on certain parameters such as the type of polymer in their structure, their size, their surface chemistry, and their charge (Prata et al., 2019). Even though MPs are considered bio-inert compounds (Andrady, 2011), there is significant evidence indicating that they toxic behavior toward various (micro)organisms (Guzetti et al., 2018). This toxicity does not necessarily originate solely from the structure of the plastic; it can also be caused by the chemical pollutants absorbed on the surface of the MPs (Wang et al., 2018). Moreover, displayed toxicity may change according to the environment in which MPs reside. This is why toxicity studies conducted under controlled laboratory conditions may not always reflect real environmental outcomes. The importance of revealing the interactions between microorganisms and MPs under environmentally relevant conditions and concentrations have been highlighted in previous studies (Lu et al., 2019; Prata et al., 2019; Carbery et al., 2018). Most research studies conducted in this field tend to focus on microalgae and some other organisms related to the water systems, such as mussels, zebrafish, etc. Nevertheless, interaction mechanisms between MPs and bacteria remain a knowledge gap in the literature (Prokic et al., 2019). In the meantime, a limited number of studies of the interaction between MPs and bacteria mainly include chemical adsorption, colonization, ingestion, etc. (Lu et al., 2019). On the other hand, bacteria are good test models for investigating the toxicity of various particles at the cellular level in the environment, because bacteria play many important roles in the environment, and they may be influenced by natural or anthropogenic contaminants in the ecosystem (Navarro et al., 2008; Jiang et al., 2009; Baek and An, 2011; Baysal et al., 2018a,b). A wide range of studies have investigated the toxic and nontoxic effect of various particulate contaminants on bacteria through gram-positive Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) due to the presence of these bacteria in the seawater from natural or anthropogenic sources (Aruoja et al., 2015; Baysal et al., 2018a,b; Baysal et al., 2019). Moreover, depending on the composition of bacteria cell walls, bacteria can exhibit different behavior (bacterial susceptibilities, attachment, etc.) (Brown et al., 2010; Brown et al., 2013; Yang et al. 2013). For example, gram-positive bacteria (B. subtilis and S. aureus) have different sets of enzymes to make teichoic acid, and they use a different number of phosphate unit to make cell wall teichoic acids (Brown et al., 2010; Brown et al., 2013), e.g., S. aureus have more glycerol and ribitol chains on teichoic acids polymers compared to B. subtilis. The cell walls of gram-negative bacteria (E. coli and P. aeruginosa) are much more complex than grampositive-bacteria cell wall, having an outer membrane situated above a thin peptidoglycan layer (Beveridge, 1999; Silhavy et al., 2010). Their outer membrane includes different saccharide groups, e.g., E. coli have ~ 20–30 disaccharide units, and P. aeruginosa has two disaccharide units (Ramphal et al., 1991; Matias et al., 2003; Huang et al., 2008). Therefore, uses of different bacteria strains containing different cell wall properties are vital for observation of the effect of contaminants.

In this study, we aimed to discover the impact of MPs extracted from toothpastes, on four bacterial strains that are normally found in seawater: S. aureus, B. subtilis, E. coli, and P. aeruginosa. For this purpose, we examined the survival of selected bacteria under two conditions: controlled laboratory condition and under the influence of seawater. Their fundamental physicochemical and biochemical responses were measured in order to understand the underlying mechanism of observed inhibition. The results also provide information about the environmental relevance of the results obtained from toxicity tests conducted in controlled conditions. 2. Materials and method 2.1. Materials MPs used in the experiments were extracted as described in our previous study (Ustabasi and Baysal, 2019). Extraction scheme is presented in Supplementary Fig. 1. These MPs were confirmed to have polyethylene (PE) structure by Fourier transform infrared spectroscopy (FTIR) and they were extracted from four different brands of toothpaste commercially available in Istanbul, Turkey (Ustabasi and Baysal, 2019). Therefore, all four of them were analyzed separately. From now on, they will be referred to as PE1, PE2, PE3, and PE4. Seawater used for modified broth preparation was collected from Florya, Istanbul-Turkey. Its chemical properties are given in Table 1. Four different bacterial strains that are known to be present and able to survive in seawater, obtained from American Type Culture Collection (ATCC), were chosen as models to examine the bacterial toxicity of extracted MPs. Two of these bacteria were grampositive (Staphylococcus aureus ATCC 25,923 and Bacillus subtilis ATCC 11774), while the other two were gram-negative (Escherichia coli ATCC 35,218 and Pseudomonas aeruginosa ATCC 27853). General information on the bacteria strains are given in Supplementary Table 1. Tryptic soy broth (TSB) media and other chemicals were obtained from Merck (Darmstadt, Germany). 2.2. Physicochemical properties of MPs Hydrodynamic sizes and zeta potentials of these MPs were measured by Dynamic light scattering (DLS) using Zetasizer Nano ZS (Malvern Instruments, UK) at 25 °C at a 173° scattering angle with 4 mW He-Ne laser. Sample preparation for these analyses involved weighing approximately 500 mg of each MPs and suspending them in 10 mL deionized water prior to measurements. They were also subjected to a 5 min sonication (Sonorex, Bandelin Electronic, Berlin, Germany) to avoid possible agglomerates, and placed in Standard Malvern zeta potential disposable capillary cells and polystyrene cuvettes (Malvern Instruments Ltd, Malvern, United Kingdom) for zeta potential and size measurements, respectively. All measurements were repeated at least three times. The surface chemistry (FTIR spectrum) of the MPs isolated from tootpastes was given our previous work (Ustabasi and Baysal, 2019). 2.3. Toxicity assessment Growth curves of bacteria were obtained in controlled condition by optical density (OD) measurement method at 600 nm (Pelletier et al., 2010; Padmavathy and Vijayaraghavan, 2011; Lin et al., 2014) with UV–VIS spectrophotometer (Thermo Scientific MULTISKAN GO, Finland). The tests were conducted in Tryptic soy broth culture medium: 2 mL of broth was placed in test tubes and freshly

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G.S. Ustabasi, A. Baysal / Science of the Total Environment 703 (2020) 135024 Table 1 Chemical properties of utilized sea water (N:3, ND: not detected). Chemical property

pH

Na (mg/L)

K (mg/L)

NO3 (mg/L)

NO2 (mg/L)

NH3-N (mg/L)

SO4 (mg/L)

PO4 (mg/L)

Cl- (g/L)

Value range

8.3 ± 0.8

6693 ± 535

231 ± 9

0.26 ± 0.02

ND

0.92 ± 0.06

2283 ± 190

ND

15.9 ± 0.6

cultured bacteria colonies were dispersed in the broth until 0.5 McFarland turbidity was reached. The test units were then placed in an incubator (IGS 100, Thermo Fisher) at a controlled temperature of 37 °C. Each treatment was prepared in three replicates. During the incubation period, optical densities of inoculated broths were measured at 1st h, 6th h, 12th h, 24th h, and 36th h. For this purpose, 200 mL of the inoculated broths were transferred into wells of 96-well plates so that all the measurements can be conducted simultaneously. The above mentioned procedure was replied using seawater as a growth media to mimic the normal growth of the selected bacteria in seawater. The growth curves of each bacterium are in Supplementary Fig. 2. Bacteria model system was based on previous studies which allow the representation of environmental conditions (Baysal et al., 2018a,b). Bacteria were exposed to five concentrations of MPs (0, 10, 50, 100 and 500 mg/L) to understand the dosedependency of the expected toxic behavior. These concentration levels were chosen according to the previous studies in the literature regarding the toxicity of MPs at environmentally relevant concentrations (Prata et al., 2019; Prokic et al., 2019). Moreover, these concentrations reflect the number of particles determined in previous studies from the literature. MPs were added onto 2 mL of Tryptic soy broth media (Merck, Darmstadt, Germany) in which isolated bacteria were suspended. Bacteria exposed to MPs in the form of PE were subjected to two different growth media: (i) first one was a broth that is prepared with deionized water, as instructed by the producer and (ii) the second broth was prepared using the seawater collected from Florya, Istanbul to mimic the chemical composition of real sea environment. Both media were autoclaved before they were used in the tests, as instructed by the producer: for 15 min at 121 °C. Basically, two sets of samples were prepared to reveal how bacteria and MPs interact under controlled and environmental conditions. After freshly cultured bacteria were exposed to MPs in 2 mL of selected media as explained above, they were incubated in a dark oven at 37 °C for 24 h. Controls for the toxicity assessment did not contain any MPs, they only contained isolated bacteria suspended in associated broth. Samples and controls were prepared in five replicates and results were reported as the averages of those replicates. Growth inhibition and biochemical assays were conducted immediately after 24 h. Growth inhibition was measured by OD600 method (Pelletier et al., 2010; Padmavathy and Vijayaraghavan, 2011; Lin et al., 2014) with UV–VIS spectrophotometer (Thermo Scientific MULTISKAN GO, Finland). Incubated bacteria solutions were transferred into wells of 96-well plates so that all the measurements can be conducted simultaneously. Total protein concentration was measured by the Bradford method using bovine serum albumin as the standard. After Bradford Reagent addition, both controls and samples were measured at 595 nm using a UV–VIS spectrophotometer (Thermo Scientific MULTISKAN GO, Finland). Lipid peroxidase (LPO) enzyme activities of bacteria exposed to MPs were measured according to the method explained by Arora et al. (2008). Method involves the addition of 150 mM TRIS buffer (pH = 7.1), 100 mM ferrous sulfate, and 150 mM ascorbic acid to the bacterial solution. After incubating them at 37 °C for 15 min, thiobarbituric acid was added to the mixture and a second 15 min incubation was applied in a 100 °C water bath. Final reaction

mixtures were centrifuged and obtained supernatants were placed in wells and measured at 532 nm with the UV–Vis spectrometer (Thermo Scientific MULTISKAN GO, Finland). Total antioxidant capacities (TAC) were measured according to the CUPRAC method described in the work of Apak et al. (2004). This method involves mixing sample solutions with 0.01 M CuCl2, 7.5  10 3 M Neocuproine, and 1 M CH3COONH4 and measuring at 450 nm, after the 30 min waiting period. Trolox was used as the standard. Lastly, extracellular carbohydrate concentrations were measured according to the method described in the work of Dubois et al. (1956), which requires mixing 25 mL of 80% phenol and 125 mL of concentrated sulfuric acid with 50 mL of sample solutions and incubating at 25 °C for 20 min. Measurements were taken at 480 nm for pentose and at 490 nm hexose structured carbohydrates. 2.4. Statistical analysis ANOVA with post hoc Tukey was used to evaluate the differences between controls samples incubated in respective media, as well as the differences among samples. The significance was accepted at the level of p < 0.05. SPPS 17.0 software was applied for the significance and Spearman correlation (two-tailed) tests. 3. Results and discussion The need for exposure studies under environmentally relevant conditions has been presented in the literature (Carbery et al., 2018). Therefore, we aimed to observe the behavior of MPs against bacteria in both seawater and a standard Tryptic soy broth media. As can be seen in Fig. 1, growth inhibition trends caused by PE MPs were altered when the growth medium was changed. In the controlled (standard) condition, the only bacteria that was significantly affected by the addition of MPs was gram-negative P. aeruginosa with 20–35% inhibition. Also, the other gram-negative bacterium, namely E. coli, was slightly inhibited by the tested MPs, except for PE4. The difference in the inhibition levels between P. aeruginosa and E. coli can be caused by the differences in their cell wall compositions. The cell wall composition of P. aeruginosa has less disaccharide than E. coli, thus the higher number of disaccharide units can protect the bacteria from the influence of MP particles (Ramphal, 1991; Huang et al., 2008; Matias et al., 2003; Baysal et al., 2018a,b). However, in the seawater medium the growth of gram-positive B. subtilis was significantly affected by MPs with a 20–30% inhibition, while S. aureus was slightly affected with a 0–30% inhibition with at least two of the extracted MPs. The difference in inhibition levels between gram-positive bacteria can be due to the enzymes responsible for making teichoic acid. S. aureus has more glycerol and ribitol chains on teichoic acids polymers, which resulted in lower inhibition compared to B. subtilis (Brown et al., 2010; Brown et al., 2013; Baysal et al., 2018a,b). In the seawater media, samples with MPs concentration of 100 mg/L and 500 mg/L showed higher inhibitions compared to the samples with 10 mg/L and 50 mg/L MPs concentration; this indicated a dosedependent behavior that was not observed in the standard media. Overall, inhibition levels were relatively high considering the inert structure of PE type MPs. In a previous study conducted by Lagarde et al. (2016), inhibition levels observed for high-density PE against

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Fig. 1. Optical density ratios of samples (A) to controls (A0). Letters a, b, c, and d in parenthesis represent the results for S. aureus (dark blue), B. subtilis (light blue), E. coli (dark green), and P. aeruginosa (light green), respectively. Numbers 1 and 2 show the responses obtained in standard media and seawater media, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

microalgae Chlamydomonas reinhardtii was insignificant, whereas it was nearly 20% for polypropylene. PE type MPs are generally reported not to affect the growth of microorganisms (Prata et al., 2019; Prokic et al., 2019), however, the PE type MPs that we extracted from toothpastes had AF and ACl attached to their surfaces according to the obtained signals around 1000–1400 and 700 cm 1, respectively (Ustabasi and Baysal, 2019). This surface modification is probably because of the fluorine and chlorine found in the formulations of toothpastes, and as a result, this modification enhanced the toxicity of MPs. One of the factors determining the impact of MPs on an organism is its size. Lusher states that the smaller the size of MPs, the greater their impact on cellular level (2015). Therefore, we also examined the particle sizes and zeta potential of the extracted

MPs, and the results are shown in Table 2. DLS results showed that PE1 and PE2 were nearly half the sizes of PE3 and PE4. However, a size-dependent growth inhibition pattern was not observed among tested bacteria. The reason may be that the size difference between MPs was not significant enough (except between PE2 and others) to exhibit size-dependent inhibition. Moreover, to understand the effect of the surface properties of MPs on their interactions with bacteria, the zeta potentials of MPs were examined. Zeta potentials, which affect the interaction between bacteria and particles, can be used to predict the stability or agglomeration of particles. Higher zeta potentials lead to more stable particles, while lower zeta potentials can lead to agglomeration. Moreover, zeta potentials can be used as a secondary metric to determine surface chemistry changes. Increased negativity or

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G.S. Ustabasi, A. Baysal / Science of the Total Environment 703 (2020) 135024 Table 2 Size and zeta potential results of MPs, obtained by DLS measurements.

Size (nm) Zeta potential (mV)

PE1

PE2

PE3

PE4

390.2 ± 90.2 33.4 ± 10.1

323.3 ± 51.8 18.0 ± 17.1

621.9 ± 155.2 30.9 ± 7.3

656.8 ± 194.4 23.8 ± 7.5

Fig. 2. Total protein content rations of samples (A) to controls (A0). Letters a, b, c, and d in parenthesis represent the results for S. aureus (dark blue), B. subtilis (light blue), E. coli (dark green), and P. aeruginosa (light green), respectively. Numbers 1 and 2 show the responses obtained in standard media and seawater media, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

positivity shows the attachment of negatively or positively charged functional groups on the surface. Furthermore, in order to understand the interactions between bacteria and MPs, the relationship between the bacteria cell wall and the particle surface charge (zeta potential) is crucial. It is known that charge difference between the

particle surface and bacteria cell wall interact each other (Baysal et al., 2018a,b). In our study, in the controlled condition, gramnegative bacteria were affected by the MPs, while gram-negative bacteria and MPs had the same charge. However, in the seawater conditions, the inhibited bacteria were changed and the viability

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of gram-positive bacteria was influenced from the negatively charged MPs. These results showed that the significance of the charge difference between the bacteria and the MPs depended on the exposure media. In the meantime, the results showed that, the two MPs that had the highest zeta potentials (PE1 with 33.4 mV and PE3 with 30.9 mV) showed higher inhibition levels against bacteria compared to the other ones. These results indicated that higher negatively charged functional groups on the MPs surfaces and higher particle stability caused the inhibition. For a deeper understanding of the inhibition mechanism, biochemical responses (protein, carbohydrate, LPO levels) were also examined. As can be seen in Fig. 2, the overall decrease in protein levels did not exceed 20% compared to their controls, which did not contain any MPs. Similar to their growth inhibition values, P. aeruginosa in standard media showed a 0–30% decrease in protein content, whereas B. subtilis in seawater media showed a 15–35%

decrease. All decreases observed in protein content were higher for 100 mg/L, and 500 mg/L than for 10 mg/L, and 50 mg/L MPs added samples, further verifying dose-dependency. On the other hand, even though S. aureus in seawater and E. coli in standard media showed slight inhibitions, they did not show significant decreases in terms of their protein content. Significant increases in extracellular carbohydrate levels were observed for the most cases as displayed in Figs. 3 and 4. Dosedependent behavior was not observed in this case. E. coli was the least affected bacteria in both media and P. aeruginosa was the one most affected. Even though its growth inhibition was lower than B. subtilis in seawater media, its extracellular carbohydrate levels were higher by at least 20%. Similar to our results, Lagarde et al. (2016) found that exposing microalgae to high-density PE caused overexpression in the sugar biosynthesis pathways. An additional noteworthy observation was that carbohydrate levels

Fig. 3. Pentose type carbohydrate concentration ratios of samples (A) to controls (A0). Letters a, b, c, and d in parenthesis represent the results for S. aureus (dark blue), B. subtilis (light blue), E. coli (dark green), and P. aeruginosa (light green), respectively. Numbers 1 and 2 show the responses obtained in standard media and seawater media, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Hexose type carbohydrate concentration ratios of samples (A) to controls (A0). Letters a, b, c, and d in parenthesis represent the results for S. aureus (dark blue), B. subtilis (light blue), E. coli (dark green), and P. aeruginosa (light green), respectively. Numbers 1 and 2 show the responses obtained in standard media and seawater media, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

were much higher for each bacterium in standard media than in seawater media. LPO activity is a significant parameter for measuring the oxidative stress that a microorganism undergoes when exposed to a certain contaminant (Prokic et al., 2019). Changes in the LPO activity levels of bacteria after exposure to MPs are shown in Fig. 5. From the results, P. aeruginosa exhibited a dose-dependent decrease in LPO activity with the occasional exception of 500 mg/L. In seawater conditions, B. subtilis also displayed a dose-dependent decrease in LPO activity, excluding 50 mg/L that had lower LPO values compared to other concentrations. Interestingly, S. aureus in seawater and E. coli in standard medium also showed slight decreases in their LPO activity. According to Ribeiro et al. (2017), a lowered antioxidant defense mechanism may cause destabilization in the lysosomal membranes. Impact on LPO activity is perhaps one of

the main pathways through which PE type MPs manifest their toxicity. Moreover, high levels of LPO activity are also considered as an indicator for reactive oxygen species (ROS) generation. Similar to our study, Sun et al. (2018) did not observe ROS generation in Halomonas alkaliphila caused by the presence of MPs, even though it was seen with nano-sized plastics. Finally, the total antioxidant capacities of bacteria exposed to PE MPs were analyzed. However, no significant changes were observed in both media. Thus we did not show or explain the results. To summarize, gram-negative P. aeruginosa was sensitive to MPs extracted from toothpaste in the standard media. The other tested gram-negative bacteria, E. coli, also displayed a slight inhibition in the standard media (0–15%), while in the seawater media, it was able to remain intact. On the other hand, while gram-positive

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Fig. 5. LPO activity ratios of samples (A) to controls (A0). Letters a, b, c, and d in parenthesis represent the results for S. aureus (dark blue), B. subtilis (light blue), E. coli (dark green), and P. aeruginosa (light green), respectively. Numbers 1 and 2 show the responses obtained in standard media and seawater media, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

B. subtilis was not affected in the standard media, it was inhibited under environmentally relevant conditions (0–40%). Similarly, the other gram-positive bacteria, S. aureus, was also inhibited slightly in the seawater media (0–15%, and nearly 30% with two of the MPs only at 100 mg/L). Our findings suggested that the medium in which toxicity tests are conducted ultimately affects the results. The tests conducted under controlled laboratory conditions may not reflect the real environmental consequences of MPs contamination. Furthermore, for both bacteria types, the main pathways causing the inhibition seem to be protein metabolism and LPO activity. Since overexpression of carbohydrates is observed with all samples (even in the samples with no inhibition), it may not be specifically/ directly involved in the toxicity mechanism. Additionally, the fact that growth inhibition, protein content, and LPO activity exhibited

dose-dependent trends while carbohydrate levels did not, further indicated that carbohydrate metabolism is not a part of the toxicity mechanism. A noteworthy observation was that the slightly inhibited bacteria (S. aureus and E. coli) also showed slight decreases in their LPO activities, while their protein metabolisms were unaffected. This indicated that both protein metabolism and LPO activity should be disturbed to observe significant growth inhibition in bacteria. The diagram that shows the pathways for the toxicity mechanism is in Fig. 6. Finally, the sizes of the tested MPs did not affect the observed inhibition trends even though zeta potentials (surface chemistry of the MPs) seemed to have a role in the interactions of MPs with bacteria. Stable (high zeta potentials) MPs were more effective on the inhibition compared to the less stable (low zeta potential) MPs.

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Fig. 6. Scheme of the inhibition mechanism according to the inhibited bacteria in both standard and seawater media.

4. Conclusion

References

The present study examined the toxic behavior of PE structured MPs against four bacteria that are found in seawater. It also signified the impact of experimental conditions on the results of toxicity tests by revealing the discrepancies observed under controlled and environmentally relevant conditions. The contrast in the trends observed in different media suggested that the toxic behavior of MPs against bacteria may manifest itself in various ways, depending on the media they are in. It was observed that grampositive bacteria were more prone to be damaged by MPs in the seawater environment, while gram-negative bacteria were more likely to be affected by MPs in standard media. This indicated the significance of cell wall properties for the interactions of MPs with bacteria. In both media, observed inhibition against P. aeruginosa and B. subtilis was dose-dependent and also affected by the surface charge of the MPs. Two main metabolic pathways were found to be involved in bacteria-MPs interaction: protein metabolism and LPO activity. Further studies investigating the interactions between (micro)organisms and MPs should pay more attention to the environmental relevancy of selected growth media.

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Data statement Research data is not publicly available. Upon reasonable request, data can be shared by corresponding author, Asli Baysal. Funding declaration This research did not receive any specific funding. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.scitotenv.2019.135024.

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