Introduction

Disinfectants have been used extensively for topical and hard surface applications in healthcare and non-healthcare environments and remain an important tool in reducing microbial infections [8, 12, 18, 23]. In the past 20 years, there has been an increase in the number of antibacterial products and disinfectants sold commercially. This increasing trend has been attributed to the attention of the media towards the increase in healthcare-acquired infections and food poisoning incidents in the 1980–1990s together with the diffusion of domestic hygiene care [15]. To reduce the diffusion of microbial infection, improved cleaning strategies have been adopted in small environments, such as the domestic setting, but also in larger spaces, such as hospital settings where the uptake of infections is much higher and more frequent.

Widely used disinfectants in nosocomial and domestic settings are based on chlorine solutions.

Chlorine, in fact, has been used as a disinfectant for the treatment of drinking water for more than 100 years and its effectiveness as a microbicide has been widely assessed [1, 7]. Up to now, isocyanurates have been chiefly used, besides as sanitizers in the treatment of drinking water and sewage, also in the disinfection of water for swimming pools and industrial cooling towers, in the cleaning of sanitary products, including baby bottles and contact lenses, and in the disinfection of environmental surfaces, of irrigation system for flowers, medical equipment and laundry [21]. The use of chlorine solutions in dentistry deserves a special mention. These solutions have been reported to be employed in the disinfection of dental unit water lines and dental impression materials; moreover, because of their broad antimicrobial activity, they have been widely adopted as root canal irrigants, dissolving the smear layer and preventing its formation [16, 17, 24].

NaDCC has recently been approved by the United States Environmental Protection Agency (USEPA) and the world health organization (WHO) for the routine treatment of drinking water since many studies, performed to investigate the toxicity and irritancy of this product, have given a positive feedback [5]. These studies have shown that dry chlorinated isocyanurates are not corrosive and only slightly toxic, they are not metabolized by the organism and do not bioaccumulate, and are safe to handle. Chronic and sub-chronic toxicity studies also found no toxicity. Developmental toxicity studies have also established that the compound is not fetotoxic, teratogenic (causing birth defects), mutagenic or carcinogenic [5]. NaDCC produces a well-known oxidizing agent, hypochlorous acid, and offers many advantages in terms of stability, safety, costs and convenience. Moreover, a huge amount of papers has reported the higher efficacy of NaDCC with respect to NaOCl against a wide range of microorganisms, such as Gram-positive and Gram-negative bacteria as well as fungi and viruses that, in many cases, are relevant to hospital settings [2–6, 14]. Many experimental results have recommended NaDCC instead of NaOCl for its biocidal effectiveness, such as its: increased resistance to inactivation by organic material, slow decomposition and release of HOCl, capacity to maintain an appropriate level of available chlorine without affecting the pH of the water, low level of toxicity and lower corrosiveness to metal, plastic and rubber [2, 3, 6, 13].

The exact mechanism by which free chlorine destroys microorganisms has yet to be elucidated. The postulated mechanism of chlorine disinfection is the inhibition of some key enzymatic reactions within the cell involved in the vital processes of cell survival, such as the replication cycle and protein synthesis [9, 10]. In previous works, NaDCC was used as aqueous solution and its antibacterial activity was evaluated after the application of the NaDCC solution on different contaminated surfaces [2, 3].

In this work, we explore the possibility of using solid tablets of NaDCC in order to evaluate its activity in the gas phase, following the released chlorine via hydrolysis.

The main aim of our study is to identify a possible new application for solid tablets of NaDCC in monitoring the management of household waste. Chlorine emitted by the slow hydrolysis of NaDCC was tested for its antimicrobial activity against the Gram-negative bacteria Escherichia coli and pathogen Gram-positive Staphylococcus aureus as well as against the fungi Debaryomyces hansenii and Aspergillus brasiliensis. The tested microorganisms are known for their capability to proliferate in household waste. They are commonly found in fruit and vegetables, undercooked beef, unpasteurized milk and juice, in cellulose-rich materials, etc. and, as consequence, are considered ideal candidates for our study [19]. Moreover, they are potentially very dangerous in hospital settings as well as in clinical and research laboratories.

Avoiding the spreading of microorganisms and bioaerosol in the environment could be an attractive strategy in the handling of hospital and biomedical waste containers that are a potential source of pathogens [11], as well as providing a better protection for physicians and technicians operating in the aforementioned settings.

Materials and Methods

Reagents

Sodium dichloroisocyanurate (NaDCC, Fig. 1) 96 % CAS 2893-78-9 was provided by Sigma-Aldrich (St. Louis, MO, USA). The reagents used to prepare the LB growth media for microorganisms were: Bacto Tryptone, Bacto Yeast Extract and Bacto Agar by BD Bioscience (Franklin Lakes, NJ, USA), and Sodium Chloride by Sigma-Aldrich (St. Louis, MO, USA).

Fig. 1
figure 1

NaDCC is the sodium salt of dichloroisocyanuric acid

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Microorganisms and Media

The bacterial strains of E. coli JM109 (Promega Cat. n. 9451), S. aureus and the yeast D. hansenii were obtained from the collection deposited in G. Vigliotta’s microbiology laboratory at Chemistry and Biology department of University of Salerno. They were deep frozen for storage and grown on LB agar plates at 37 °C overnight to 109 colony-forming units (cfu)/mL.

The fungal strain A. brasiliensis was obtained from the collection of Aedes chimica and ambiente srl.

Antimicrobial Activity of the Mixture’s Vapour Phase with Time

To evaluate the capability of molecular chlorine (Cl2) released by slow hydrolysis to kill the microbial cells, 2 g of NaCCD was inserted in a glass beaker (capacity 100 mL), which was closed in a plastic garbage can (capacity: 10 L). NaDCC was inserted in a glass beaker, which, in turn, was inserted in a garbage can containing LB agar plates on which different dilutions of a known title of the analysed microorganisms (about 106 cfu/mL E. coli, 105 cfu/mL S. aureus, 106 cfu/mL D. hansenii or 105 cfu/mL A. brasiliensis) were spread. Each experimental group was accompanied by a series of control groups composed of plates with a known number of colonies (dilution 10−4 and 10−3) of bacteria or fungal cells incubated in the beaker without antimicrobial mixture.

Each plate was left in the garbage pail at room temperature for 1 week with NaCCD (experimental groups) or without the antimicrobial agent (control groups) and the number of cfu formed was counted at the end. Every week, for four consecutive weeks, the plate, with approximately the same bacterial charge, was replaced to observe the efficiency of the antimicrobial vapour phase with time.

Detection of Active Cl2

Molecular chlorine (Cl2) levels were measured using a iodometric titration (Scheme 1 below). 10 mL of the gas phase was injected in a reactive solution prepared by dissolving 1.0 g of KI (>99.9 %, Sigma-Aldrich, St. Louis, MO, USA) in 10 mL of distilled water and 5 mL of H2SO4/distilled water 1:1 v/v. The formed I2 was determined using a 0.1 N sodium thiosulphate solution (Sigma-Aldrich, St. Louis, MO, USA) using soluble starch as an indicator. Cl2 mass was determined from the volume of sodium thiosulphate and then its concentration in the can is calculated, referring to a volume of 10 L.

Scheme 1
scheme 1

Cl2 detection by iodometric titration

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Analysis was conducted over the 4 weeks of testing to estimate the amount of chlorine emitted by the sodium dichloroisocyanurate hydrolysis. Cl2 levels were than correlated to the antimicrobial activity in the gas phase of NaDCC.

Results and Discussion

In Table 1 the results of 4 weeks of experimentation are reported. Each row reports the number of colony-forming units (cfu) for each microorganism grown in the presence or absence of NaDCC. The results clearly show that, in the presence of NaDCC, a complete inactivation of both bacteria and fungi was obtained up to 4 weeks.

Table 1 Antimicrobial effect of the chlorine emitted by NaDCC during the 4 weeks of experiments. In this table the number of colony-forming units (cfu) is reported for each microorganism tested and compared between the two experimental groups (CNT+ vs. NaDCC)
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Moreover, we monitored the chlorine that developed from solid tablets of NaDCC in a restricted environment (i.e. a garbage can) at the temperature of the experiment (20 °C) by iodometric titration. The results show that, during the 4 weeks of observation, the chlorine concentration remained constant (2.0 g/dm3), thus indicating that this application can be considered an efficacious and lasting solution for the inactivation of microorganisms and consequent diffusion of bad smell in household wastes.

As results from the literature, the domestic setting is increasing in strategic importance since it is considered chiefly responsible for the chain of events that determine the transmission of infection through the community [22]. These indications stimulate to improve the current practice and to promote an increased hygiene in the domestic unit as a strategy to reduce infectious diseases. Creating a greater awareness on the handling of household wastes may provide a better prevention against the spreading of microorganisms in the domestic unit, even more so in recent years in which the culture of waste separation and recycling of wastes has spread in almost all countries. This practice requires that waste persists for a longer amount of time in the house, which favours the spreading of unpleasant smells due to the proliferation of microorganisms that are able to feed on household waste.

We considered useful to inhibit the proliferation of microorganisms in household waste, as it can be potentially extended to the hospital setting where waste containers are frequently in contact with workers and in some cases also with patients having compromised immunological systems. In a previous work the antibacterial activity in the gas phase of a chemical formulation has been demonstrated, based on harmless and natural components, known for their antimicrobial properties [19]. In the present work we propose a new methodology that results to be more effective and inexpensive compared to our previous study.

The experimental data of this study show the duration of the molecular chlorine and its microbicidal efficacy (Table 1), establishing its potential role in waste infection control. NaDCC is largely employed as antimicrobial agent for its efficacy against both bacteria and fungi; however, it has been adopted as disinfectant only as aqueous solution, at direct contact with microorganisms [20].

Previous experimental data record its efficacy principally against vegetative cells of bacteria with a rather reduced effect against spores of fungi and bacilli [3], whereas in our experimental conditions the effect of chlorine emitted by NaDCC was efficient against both vegetative and non-vegetative cells.

In conclusion, the simple humidity-mediated NaDCC hydrolysis gave rise to the production of active chlorine with good microbicidal performance and a long-lasting effect. The antimicrobial activity of the hydrolyzed NaDCC was evaluated against both bacteria and fungi. Bacteria and fungi proliferation was inhibited up to 4 weeks. Chemical analysis was performed in order to quantify the active chlorine present in the garbage can for the entire duration of the experiment and showed that its concentration was constant during the 4 weeks of testing.

From the results reported here we can infer that NaDCC has the potential to be applied for the treatment of both domestic and hospital wastes by controlling microbial proliferation and consequent diffusion of bad smell from the garbage can, thus favouring hygiene in both the household and in larger settings, such as hospital complexes and biomedical laboratories.