Eradication of Pseudomonas Aeruginosa and its Ecotoxicity in Freshwater Utilizing Cold Atmospheric Plasma: Is it Environmentally Safe?

Pseudomonas aeruginosa is a multidrug-resistant bacterial strain with the ability to produce exotoxin A which can pose a serious threat to freshwater ecosystems by having pathogenicity against eukaryotes. Detoxi�cation of exotoxin A and disinfection of P. aeruginosa are the main aims of this study. Using a high dosage of antibiotics might have more toxic effects on ecosystems while cold atmospheric plasma (CAP) can promise reliable, rapid, and environmentally friendly detoxi�cation and disinfection. In this study we produced CAP reinforced by H 2 O 2 /H 2 O cold vapor to detoxify exotoxin A and inactivate P. aeruginosa in freshwater. We used Gammarus roeseli as the indicator of ecotoxicity in freshwater. The mortality of G. roeseli individuals elucidated that 420 s of CAP treatment under a surface dielectric barrier discharge (SDBD) set up can effectively passivize exotoxin A in freshwater by disrupting the protein structure of molecules. Ignorable side effects and changes to the physiochemical properties were observed. On the other hand, 8.2 log reduction of P. aeruginosa viable cells was observed after 300 s of treatment by SDBD. A comparison between the disinfection capacities of SDBD produced CAP and antibiotics revealed that CAP is more effective than most of the common antibiotic agents.


Introduction
Pseudomonas aeruginosa is a gram-negative and motile bacterium that can be found in soil, freshwater, and marine water [1][2][3][4] .P. aeruginosa is facultative anaerobe bacteria, as it is well adapted to proliferate in conditions of partial or total oxygen depletion.Recently, the endurance of infectious P. aeruginosa in different aquatic habitats has attracted a great deal of attention 5 .On the other hand, P. aeruginosa strains are known to have remarkable antibiotic resistance which makes them well-known multidrugresistant strains [6][7][8] .Some P. aeruginosa strains can secrete a virulence factor called exotoxin A which is a single polypeptide chain protein consisting of 613 amino acids 9 .P. aeruginosa uses its virulence factor to vandalize some important eukaryotic proteins by inactivating eukaryotic elongation factor 2 (EEF 2) which assists protein translation 10 .EEF 2 protein is essential for catalyzing the translocation of tRNA and mRNA through the ribosome during the translation of other proteins and its de ciency causes symptoms and cell death 11 .P. aeruginosa can strongly affect marine and freshwater ecosystems and organisms by producing its toxin 12,13 .Research has proven that P. aeruginosa can widely infect marine vertebrates by proliferating into their bodies 13 .Genetic investigations also fairly revealed that most P. aeruginosa strains found among the infected marine vertebrates are multidrug-resistant and capable of producing exotoxin A 12 .P. aeruginosa infection in marine organisms can lead to bioaccumulation and endanger a whole ecosystem and environmental safety 13 .Therefore, restraining the water pollution caused by P. aeruginosa is becoming a serious environmental challenge in developing societies 2 .Using antibiotics might be considered as a low cost and plain way for inactivating bacteria in aquatic habitats, but effective antibiotics against bacteria such as P. aeruginosa are proven to have toxic effects on marine or freshwater organisms and consequently pose a serious threat to the aquatic ecosystems [14][15][16][17] .
Furthermore, because P. aeruginosa is resistant to common antibiotics, more effective methods are needed to inactivate this pathogenic bacterium and its toxin in freshwater.In the view of mentioned environmental and health hazards created by P. aeruginosa and its toxin, accurate, green, and new strategies are required to inactivate this bacterium.Among the innovative bactericidal strategies, cold atmospheric plasma (CAP) can be a better substitute for antibiotics 18 .Numerous studies have emphasized the CAP's capability of inactivating or passivizing diverse microbes at low temperatures 19- 23 .It was also emphasized by several studies that CAP strategies do not pose any harm to the environment [24][25][26][27] .It is commonly accepted that cold plasma techniques produce reactive oxygen and nitrogen species (RONS) which are believed to have a valuable contribution to the biological effects of plasma [28][29][30][31] .In fact, plasma-induced species (RONS) can cause damage to the cell walls, DNA, and lipids, as well as denature or degrade proteins 19,32,33 .There are various methods for cold plasma generation such as dielectric barrier discharge (DBD), plasma jets, coronas, and microwave discharges 34 .
Among these mentioned methods, DBD enjoys the advantages of producing stable and uniform discharge on a large scale and operating at relatively low temperatures under atmospheric pressure 34,35 .
Besides, surface dielectric barrier discharge (SDBD) can generate a non-thermal plasma (NTP) over a huge area at a low cost, which has given it more commercialization potential than other systems [36][37][38] .It is also con rmed that SDBD can increase the bactericidal effect of some ROS, especially H 2 O 2 36,39−41 .
Plasma-mediated inactivation of P. aeruginosa has been investigated by several researchers.Matthes et al. treated in vitro P. aeruginosa bio lms with plasma jet by using argon/oxygen mixture as gas feed and chlorhexidine digluconate, which is a commercial antiseptic.A comparison between the results proved that plasma jet can be more rapid in disinfection of P. aeruginosa 42  with no additional harm or side effects on normal cells in rat models 46 .Cold plasma has also been widely used for decontamination and detoxi cation [47][48][49][50][51][52][53][54][55] .CAP strategies have been hopefully successful at disinfection of P. aeruginosa but the detoxi cation of its toxin has been disregarded so far.Therefore, investigating the detoxi cation potential of CAP and its possible effects on freshwater organisms may present a new aspect of repelling the ecotoxicity caused by infectious microorganisms.Gammarus species are amphipod invertebrate animals living in fresh or marine waters 56 .Their population has been widely used as an assessment of water quality 56,57 .The death of Gammarus species is a biomarker of toxicity or ecotoxicity of different substances in marine and freshwaters and also their survival can be a sign of successful detoxi cation 58 .Hence we hired Gammarus roeseli, a freshwater living Gammarus species, to indicate the toxicity of our treated or non-treated solutions.
Recently, investigation on the biological safety of food treated with CAP and the cytotoxic effects of plasma therapies in medicine using animal and insect models has begun 59,60 .This re ects the growing attention of researchers around the world to the side effects and potential toxicity of using CAP commercially, taking into account current health standards.Cold plasma is well known as a suitable plan for the inactivation of pathogenic microorganisms as well as organic contaminants in aquatic environments 61 .To our knowledge, CAP has never been employed for inactivating the ecotoxicity of organic compounds such as exotoxin A. Notwithstanding, a prominent novelty of the present study is offering the rst and only available research on the ecotoxicity and possible adverse impacts of CAPtreated aquatic samples on the return to the water cycle of nature.Prior studies have investigated the toxicity of ozone and hydrogen peroxide treatments on organisms in aquatic ecosystems [62][63][64][65][66] .The study of the effects of CAP treatment on the ecotoxicity of freshwater environments, presented here for the rst time, gives our research a unique credit as the sole reference in this regard.A well-known biomarker (G.roeseli) was called for this purpose.
Our aim in this study is to (1): Eliminate the threat of exotoxin A and P. aeruginosa to the freshwater environment utilizing CAP.(2): investigating possible side effects of cold atmospheric plasma on aquatic environments and its ecotoxicity.(3): evaluating the concentration of RONS to clarify their contribution to disinfection and detoxi cation effects of developed CAP-based strategy.(4): comparing the disinfection e ciency of the CAP with common antibiotics against the bacteria.

CAP treatments
The strategy employed by us in this study is comprehensively and precisely described in our recent research, so readers are encouraged to refer to that paper for more details 36 .As published in our previous work, this innovative strategy manifested great potential and talent for the complete inactivation of pathogenic microorganisms.And we witnessed the synergistic effect of cold plasma and hydrogen peroxide in disinfecting aqueous environments in a short time and large dimensions.All of this puts a clear vision in front of us to establish a sustainable and large-scale CAP strategy in the event of any aquatic toxicity or contamination associated with pathogenic microorganisms in freshwater environments.In particular, this unique approach provided a great chance to completely inactivate P. aeruginosa, a catalase-positive and highly opportunistic bacterium in aqueous media.
In short, here we use a combination of a cold plasma generator at atmospheric pressure (SDBD) and cold hydrogen peroxide vapors to achieve the goals set for this study.The structure of SDBD is schematically depicted in Fig. 1.As stated formerly, the dimensions applied in assembling SDBD along with its electrical characteristics are reported in full detail in our previous work.These details also include the conditions for testing such as argon gas ow, treatment times, and sample speci cations, except that we have aqueous samples here.
Samples were placed in a petri dish and each treatment was performed in triplicate.All treatments with SDBD device were performed without plasma to ensure the contribution of plasma to the biological effects of our strategy.

Growth studies on medium culture
The standard strain of P. aeruginosa (ATCC 27853) was prepared at the concentration of 1.5×10 8 CFU/ml (0.5 Mcfarland) and densely cultured on Muller-Hinton agar media (Ibresco: i23118).Cultured media were exposed to CAP under the SDBD devise for 30 s, 90 s, 180 s, 300 s, and 420 s.The exposed media were incubated at 37°C for 24 h.The approximate percentage of the disinfected area on each medium was pro led after the incubation.

Growth studies in freshwater
In order to observe the disinfecting e ciency of SDBD in freshwater, the concentration of 0.5 Mcfarland of P. aeruginosa was prepared in sterilized freshwater and treated with SDBD for the same treatment times.After nalizing the treatments, remained bacteria concentration was counted with the pour plate method.

Antibiotic disinfection testing
The percentage of the disinfected area caused by antibiotics was obtained with the antibiogram method.Antibiotic discs including ampicillin, amikacin, cefazolin, ceftazidime, cipro oxacin, cefepime, cefotaxime, colistin, erythromycin, gentamicin, imipenem, nalidixic acid, nitrofurantoin, nor oxacin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin were purchased from PADTAN TEB Co.The concentration of 0.5 Mcfarland was cultured on the Muller-Hinton agar media and an antibiogram disc was placed on each culture medium.All culture media were incubated at 37°C for 24 h.The percentage of the disinfected area was pro led after the incubation time was nished.

Ecotoxicity assay
Pure exotoxin A lyophilized powder was obtained from Merk (CAS number: 91262-95-2) (MDL number: MFCD00132134) and in an effort to evaluate the possible effects of SDBD produced CAP on the ecotoxicity of exotoxin A, Gammarus roeseli was used as an indicator and biomarker of ecotoxicity.G. roeseli samples were all collected from the estuary of Safarud river (Ramsar, Mazandaran province, Iran) and kept in an aquarium in the laboratory space under the same conditions including temperature, relative humidity, total dissolved solids (TDS), pH, and day/night hours for a month.The lethal concentration of 50% (LC50) of exotoxin A in 96 h was found after treating G. roeseli individuals with a broad range of concentrations.After performing CAP treatments for the same treatment times, 20 G. roeseli individuals were added to each treated solution and their mortalities were observed after 96 h..To measure the molar concentration of each ROS produced and injected by CAP, special compounds called spin-traps must be in play.Each spin-trap can bind to a speci c ROS and form a complex which can keep long enough for measurements.In order to do so, solutions with proper concentrations of spintraps must be made and exposed to CAP at similar conditions to each freshwater sample.After that, the treated spin-trap solution(s) should be carried to an electron paramagnetic resonance (EPR) device so the concentration of each ROS could be calculated.More details on this method could be found in the review article by Hawkins and Davies 69 .
EPR measurements and detections were done after preparations and treatments.The EPR device used in this study was a Magnettech MiniScope MS 200 spectrometer.All scans with this EPR were repeated three times while the parameters such as frequency, modulation frequency, modulation amplitude, power, sweeping time, and sweeping width were kept the same as the investigations of Privat-Maldonado, et al. 71 .Analyzed samples were prepared in 50 µL glass capillaries for measurements.The results were procured by double integration (SpectrumViewer ver.

Scanning electron microscopy (SEM)
The effect of CAP on P. aeruginosa viable cells was studied using scanning electron microscopy (SEM).0.05 ml of treated and non-treated samples were placed on coverslips and incubated at 40°C for 1 h for dehydration and xation.Dried slips were coated with gold in a Polalis sputter coater and the images were taken using an SEC SNE4500 SEM device at an accelerator voltage of 20 kV and at 10,000 × magni cation.

Statistical analysis
Each experimental condition, preparation, and requirement was performed in triplicate and each experiment was separately repeated three times.Data are presented as mean ± standard deviation (SD).Differences among groups are also reported using the statistical package SPSS 24.0 (IBM, Armonk, NY, USA).The preferred procedure for comparing the data was one-way ANOVA followed by Duncan's multiple comparison test as a post hoc test at a 95% con dence level and a signi cance level of p < 0.05.

Bactericidal effect of SDBD plasma on culture media
The bactericidal effect of the plasma produced by the SDBD device was investigated on Muller-Hinton agar media.The percentage of the disinfected area was compared with antibiotic disc diffusion.As shown in Table 1, there was no signi cant difference between the cleared area caused by ampicillin, erythromycin, tetracycline, nalidixic acid, nitrofurantoin, trimethoprim-sulfamethoxazole, cefazolin, vancomycin, amikacin, and cefotaxime.Also, the same result applies to comparing the area cleared by these antibiotics with the control sample.The most effective antibiotics against P. aeruginosa from the weakest to the strongest are imipenem, cefepime, cipro oxacin, and gentamicin, respectively.These disinfected areas caused by these antibiotics were signi cantly more than others.In the case of the CAP treated samples, the disinfected area was time-dependently increased.There was a signi cant difference in disinfected area between each treatment time from 30 s to 420 s.A comparison between the disinfected area caused by antibiotics and CAP treatments in Table 1 states that the cleared area after 30 s CAP treatment and nor oxacin, colistin, ceftazidime, and amikacin are not signi cantly different and CAP treatment for the 90 s and ceftazidime had almost the same disinfection effects.Disinfection effects of cipro oxacin, gentamicin, cefepime, and imipenem were not signi cantly different from 180 s of CAP treatment.The bactericidal effects of gentamicin and CAP treatment for 300 s were not signi cantly different.Finally, treating the culture medium for 420 s disinfected the whole plate, and complete inactivation was achieved.Unlike 30 s, 90 s, 180 s, and 300 s, 420 s of CAP treatment times had signi cantly more disinfection effects than all of the tested antibiotics.These results indicate that in 420 s or more, SDBD generated CAP can be more bactericidal than every known antibiotic.

Bactericidal effect of SDBD plasma on freshwater
All of our freshwater samples for this particular assay were infected with 8.2-log of P. aeruginosa viable cells and treated under the SDBD set up for the same times.The results are listed in Table 2, and as can be seen, there was no signi cant difference between the remaining log after the treatment times of 30 s and 90 s.After 180 s and 300 s the remaining log signi cantly reduced to 1.63±0.57and 0±0.00 respectively.Summarizing the data in Fig. 2  To avoid duplication, in order that obtains details of physicochemical processes (e.g., chemical reactions involved in the generation and loss of long and short-lived reactive species (i.e., RONS) in the gas/liquid phase or the interfacial layer), in indirect interaction between cold atmospheric pressure plasma and deionized water, refer to articles by other authors in this eld [75][76][77] .Also, the (sub-)cellular mechanisms governing the process of microbial inactivation during water disinfection by cold plasma at atmospheric pressure can be found in already authoritative literature 31,33,78,79 .

Detoxi cation effect of plasma: G. roeseli casualties by remaining exotoxin A
The detoxi cation capacity of plasma has been investigated by several studies before 51,53,55,80−88 .RONS produced by CAP devices, speci cally O 3 , play a vital role in CAP-induced detoxi cation 53 .Despite RONS, UV radiation produced during CAP treatment can also speed up the degradation of toxic compounds 51 .
In fact, CAP can manipulate the protein structure and make it ine cient [89][90][91] .Nevertheless, CAP has never been hired for inactivating the ecotoxicity of organic compounds such as exotoxin A. In our study, the aquatic solution of exotoxin A with the concentration of 300 µgr/lit (LC50) was treated with CAP under SDBD set up for 30 s, 90 s, 180 s, 300 s, and, 420 s.After each treatment 20 G. roeseli individuals were added to each treated sample for investigating the effect of CAP treatments on the ecotoxicity of exotoxin A. All treatments were repeated three times.Table 3 contains the results of treated solutions.CAP treatments for 30 s and 90 s had no detoxi cation effect on toxic solutions since the death toll of G. roeseli individuals was not signi cantly changed after 96 h.The results of 180 s were dissimilar to the lower times and the casualties of individuals signi cantly decreased to 38.33±2.89%.CAP treated toxic solution for 300 s and 420 s respectively caused 10±5.00 and 3.33±2.89% of casualty among exposed individuals.In general, the toxicity of treated solutions continued to decrease during the higher treatment times, which offers a time-dependent manner of detoxi cation caused by CAP.A comparison between the RONS concentrations during treatment times (shown in Fig. 2) and the detoxi cation effect of CAP (shown in Table 3) can explain the detoxi cation effect of CAP.It appears that H 2 O 2 and O 3 have a more important role than other RONS, which is totally in accordance with the results of CAP-treated bacteria in freshwater mentioned above.Hence it can be concluded that plasma-induced RONS can be effective against P. aeruginosa and its toxin.

The effect of CAP on the concentration of exotoxin A
The results of the treated exotoxin A with CAP are presented in Table 4.As can be seen, the control samples were vigorously xed at 300 µgr/lit.The 30 s CAP treatment had no signi cant effect on the concentration of the toxin but after 90 s the concentration diminished to 213.33±41.63µgr/lit.The abatement continued through the 180, 300, and 420 seconds since the concentration of the toxin dropped to 123.33±49.33,53.33±30.55,and 3.33±5.77µgr/lit respectively.Statistical analysis indicated that the difference between the concentrations after 90 s, 180 s, and 300 s was signi cant to each other but the difference between 300 s and 420 s was not signi cant.However, treating samples with our CAP strategy for 420 s resulted in the reduction of exotoxin A to nontoxic concentrations.A comparison between these results and the results of section 3.3 notes that there is a synonymy between the concentration of the exotoxin A and casualties of G. roeseli in freshwater since a reduction in one is followed by a reduction in another.All in all, both above-mentioned reductions and the ampli cation of the ROS concentration are happening simultaneously, which explicates that our CAP treatment strategy is effective at detoxi cation of exotoxin A by disrupting its molecular structure.
Table 4 The effect of CAP on the concentration of exotoxin A

Effects of CAP on conventional physicochemical properties of freshwater
Evaluating the physiochemical properties of water is critical for investigating the possible effects of plasma on aquatic environments.Conventional physiochemical properties of freshwater such as pH, conductivity, TDS and, temperature are major factors for a functional freshwater ecosystem since the life of many freshwater living species depends on them 92,93 .The pH of treated freshwater samples was not signi cantly changed after 90 s but after that, it started to reduce.As shown in Table 5 77,94,95 .Both TDS and conductivity of our treated samples were increased during our treatment times.The change of TDS and conductivity have resulted from the presence of RONS in treated samples.The alteration of TDS and conductivity of water was also reported by Rathore et al. 96 .Due to the cold nature of plasma, the temperature of treated samples was not signi cantly changed during CAP treatments until 300 s.
Table 5 The effect of CAP treatments on conventional physicochemical properties of fresh water

of CAP in freshwater
High amounts of ROS in CAP-treated water can cause harmful effects on living organisms in the freshwater.ROS can be a leading cause of oxidative stress in freshwater organisms which can be extremely harmful to aquatic ecosystems 97,98 .The contribution of ROS in oxidative stress and its negative effects on aquatic organisms is discussed by Valavanidis et al., Di Giulio et al., and Livingstone [98][99][100] .Also, studies have been conducted on the risks of using the ozonation method and the effect of oxidative stress arising from it on ecotoxicity and animal welfare in aquatic environments 62,65 .On the other hand, H 2 O 2 induced in freshwater during our treatments can lead to oxidative stress as well 101 .
Therefore, the possible toxic effect of CAP treated freshwater was investigated using G. roeseli as a toxicity biomarker.20 G. roeseli individuals were added to each CAP treated freshwater and their death toll was observed after 96 h.As shown in Table 6, treating freshwater samples for 30 s caused absolutely no casualty among individuals.Casualty induced by 90 s, 180 s, and 300 s of CAP treatments is not signi cantly different from control samples.But 420 s of CAP treatment in which, the concentration of ROS was higher than others caused 8.33±2.89% of mortality which is signi cantly higher than lower treatment times.In summary, treating freshwater for lower than 420 s with our CAP strategy causes ignorable toxicity in aquatic ecosystems.

Scanning electron microscopy (SEM)
Figure 3 illustrates the treated and untreated P. aeruginosa cells under SEM.It was observed that the untreated cells remained intact and the cell walls were in their normal, rod-shaped form.On the other hand, it is clear that plasma-treated cells have deformed with notable shrinkage (Fig. 3b).Also, captured images with higher zoom (e.g., Fig. 3b (inset)) showed the fact that CAP-treated samples contained torn apart and disrupted cell walls.The debris of disintegrated cells can be seen in Fig. 3b.It is worth noting that the rope-like structures near the bacteria in Fig. 3a, are some parts of the medium culture that stuck on the coverslips during the sample preparation.Our observation in this study agrees well with the previous investigations about the e ciency of the CAP in disinfection 102 .In conclusion, SEM revealed that our CAP treatments caused serious loss of viability in bacterial cells by disrupting their cell walls.

Conclusion
In this study, we investigated the disinfection and detoxi cation activity of H 2 O 2 /H 2 O associated CAP produced by SDBD against P. aeruginosa and its toxin in freshwater.The main purpose of this study was to present a low-cost, large scale and environmental-friendly CAP strategy to be used if any pollution related to P. aeruginosa and its toxin occurred in freshwater ecosystems.We studied the e ciency of SDBD produced CAP in eliminating P. aeruginosa in freshwater and also we compared the bactericidal capacity of CAP with common antibiotics.The detoxi cation effect of our new CAP strategy was veri ed in freshwater by G. roeseli as a marker of ecotoxicity.The in uence of CAP on the concentration of exotoxin A was also investigated.The effect of CAP on conventional physicochemical properties of freshwater and also the possible toxic effects of CAP treated freshwater were separately investigated as side effects.Our results pointed that plasma is likely more effective against bacteria than common antibiotics.Also, 8.2 log reduction of P. aeruginosa viable cells was observed after 300 s of treatment and SEM images con rmed that disruption of the cell walls is the reason for this loss of viability.Mortality of G. roeseli individuals indicated that treating freshwater for 420 s can remarkably detoxify exotoxin A and our following analysis on the concentration of the exotoxin A elucidated that disrupting the protein structure on molecules could be the reason.But CAP treatment can also induce negligible toxicity due to concentration of ROS however it can't noticeably alter the physicochemical characteristics of water.In conclusion, H 2 O 2 /H 2 O associated CAP produced by SDBD can effectively eradicate P. aeruginosa and exotoxin A in freshwater with causing ignorable toxicity and no harm to the sustainability of freshwater ecosystems.Thus, it can be used if any pollution concerning P. aeruginosa and its toxin occurred in small freshwater ecosystems such as ponds, swamps, and lakes.shows the morphological characteristics observed under SEM of P. aeruginosa cells (a) before and (b) after plasma treatment.Inset: Plasma-treated P. aeruginosa cells at higher magni cation.

Figure 3
Figure 3 686.Measurement of toxin protein concentrationIt has been proved that each exotoxin A molecule weighs about 67 KDa 67 .According to the studies of Erickson, a 67 KDa protein should have a diameter between 2.4 and 3.05 nm68.As mentioned in the introduction, CAP can produce RONS which are likely to break down the peptide bonds and rip the protein into smaller pieces.In this study, we ltered the treated samples by an Anisotropic Aluminum oxide membrane lter-2 nm pore size (SPI supplies), so the unbroken proteins stuck and the smaller protein debris together with the water go through.After that, the weight of the stuck molecules could be measured with an Optika SMG series laboratory scale (with 0.01 mgr accuracy).Th concentration of exotoxin A is calculable if the weight is clari ed.2.7.Conventional physiochemical properties of waterPhysiochemical characteristics of the water used for keeping G. roeseli such as temperature, conductivity, TDS, and pH were measured before and after each treatment time.pH and temperature were measured at the same time with AZ 86502 pH and thermometer.TDS and conductivity were measured with NEWCON digital TDS meter and DY-PH-02 digital meter, respectively.
2.8.RONS measurements 2.8.1.ROS Except for H 2 O 2 and ozone, ROS are mostly short-lived ions and radicals that cannot be measured directly in CAP-treated samples.Herein we obeyed the procedure used in a previous study by Sohbatzadeh, et al.
2.6.3) of the respective simulated spectra (NIH P.E.S.T. WinSIM software ver.0.96) of the formed radical adducts.The concentration of induced H 2 O 2 in the liquid samples was measured directly by UV/Vis spectroscopy using a reagent solution of potassium-titanium (IV) oxalate dehydrate in H 2 O/H 2 SO 4 73 .2.8.2.RNS Most RNS are nitrate and nitrite anions that can keep for at least one day so their concentration in treated samples can be measured with ion chromatography columns.Column Metrosep A Supp 10 -250/4.0 was assembled with anion eluent composition of 5 mM Na 2 CO 3 + 5 mM NaHCO 3 and ow of 1 ml/min and pressure level of 14.94 MPa.Metrohm model 881 compact IC pro 1 was put to the recording process for 36 min and manual integration to detect the concentration of nitrate and nitrite in treated and control samples.

Table 1
Percentage of medium culture disinfected area caused by antibiotics and CAP treatments a,b .
illustrates that ROS especially H 2 O 2 , O 3 /O, O 2 •− , and • OH had more contribution in log reduction during our CAP treatments.A comparison between the reduction of log and the concentration of RONS during treatment times indicates that among the ROS, H 2 O 2 had more contribution than others.O 3 /O, O 2 •− , and • OH had a more important role after H 2 O 74 Nitrate anion might also play a bactericidal role until 90 s but after that its concentration had negligible changes.Very low concentrations of RNS during the treatment process using our new plasma-based strategy promise an environmentally friendly solution without causing nitrate contamination in water reserves after the release of aqueous samples into nature74.

Table 2
Log reduction of bacteria in fresh water during treatment times a,b .
a Signi cant difference (p<0.05)within each column was determined by different superscript letters.bValues are means±SD (n=3).

Table 3
Detoxi cation effect of CAP on exotoxin A a,b .
a Signi cant difference (p<0.05)within each column was determined by different superscript letters.bValues are means±SD (n=3).
a,b .
the pH of 300 s and 420 s were signi cantly lower than other treated samples and the control sample.Our ndings in this part follow the previous studies byOehmigen etal., Shainsky et al., and Zhou et al. who reported that a reduction in pH during CAP treatments might be due to the presence of H 2 O 2 and RNS a,b .

Table 6
Toxicity of CAP treated freshwater a,b .