Bactericidal , protistocidal and nematodicidal properties of mixtures of alkyldimethylbenzyl ammonium chloride , didecyldimethyl ammonium chloride , glutaraldehyde and formaldehyde

Oles Honchar Dnipro National University, Gagarin av., 72, Dnipro, 49010, Ukraine. Tel.: +38-050-93-90-788. E-mail: brigad@ua.fm Zazharskyi, V. V., Davydenko, P., Kulishenko, O., Chumak, V., Kryvaya, A., Biben, I. A., Tishkina, N. M., Borovik, I., Boyko, O. O., & Brygadyrenko, V. V. (2018). Bactericidal, protistocidal and nematodicidal properties of mixtures of alkyldimethylbenzyl ammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde. Regulatory Mechanisms in Biosystems, 9(4), 540–545. doi:10.15421/021881


Introduction
An obligatory component in the system of veterinary-sanitary measures for the objects of livestock farming is performance of disinfection. Prevention of diseases of infectious etiology conditioned by conditionnally-pathogenic microflora requires disruption of the epizootic chain of distribution of diseases from sources of infection. A leading role in provision of stable veterinary well-being of livestock farming and healthcare of the population is played by the conducting of effective disinfecttion measures which also cause the least possible harm to the environment. Therefore, disinfection preparations are tested using laboratory analyses with methods of biotesting, particularly with ciliates. By toxicity for the ciliates, the substances are divided into four classes: 1 (LC over 0.001%), 2 (LC over 0.1%), 3 (LC over 1%), 4 (non-toxic) (Kotsumbas et al., 2006).
Correlation between the parameters of toxicity during comparative study of acute toxicity for the laboratory animals, ciliates б indicates that the ciliate T. pyriformis can be used an alternative model in predicting acute toxicity of pharmaceutical substances at the stage of their screening and pre-clinical study (Zhmin'ko et al., 2006).
The results of studies using the express-method of toxicity on ciliates indicated that solution of benzalkonium chloride, alkylbenzyldimethylammonium chloride ("Geocyd") in 0.03-0.50% concentrations and 1-10 min exposure exhibited no toxic effect on the culture of T. pyriformis ciliates (Kovalenko et al., 2014).
The extent of acute toxicity at endogastric introduction of LD 50 "Univait" preparation to mice equaled 5200 mg/kg of the animal's body weight. According to the results of the studies, a preparation was developed, which belongs to the fourth class by the classification of chemical substances in relation to the extent of toxicity. "Univait" disinfecting preparation in 0.1-0.5% concentrations during 10 min exposure was insignificantly toxic to the cultures of T. pyriformis ciliates (Zasekіn et al., 2016). For predicting toxicity of aromatic aldehydes for T. pyriformis, mathematical models are proposed, particularly the linear and nonlinear models (Ousaa et al., 2018). At the same time, there are data on the impact of aromatic aldehydes on nematode parasites of agricultural animals. At the impact (24h) of 1% solution of cinnamaldehyde, there was observed death of 100% of eggs of Ascaris suum (LD 50 = 2437 ± 864 mg/l) (Boyko & Brygadyrenko, 2017a). Larvae of Strongyloides ransomi, nematodes of pigs, also died over 24 hours at the impact of 0.1% solution of benzaldehyde. LD 50 for benzaldehyde -142 ± 64 mg/l (Boyko & Brygadyrenko, 2017b). The literature contains a large amount of data on the impact of alkylbenzyldimethylammonium chloride, didecyldimethylammonium chloride, formaldehyde, glutaraldehyde and other certain substances on microorganisms (Braoudaki et al., 2005;Blondeau et al., 2007;Fazlara & Ekhtelat, 2012;Vaerewijck et al., 2012;Ivancovic et al., 2013;Lasemi, 2017). Therefore, the objective of our study was to perform a comparative assessment of bactericidal, protistocidal and nematocidal properties of mixtures of alkylbenzyldimethylammonium chloride, didecyldimethylammonium chloride and glutaraldehyde; alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde; sodium dodecyl sulfate (SDS), oleum terebinthini and glutaraldehyde, and also formaldehyde and glutaraldehyde.

Materials and methods
The research was conducted in the laboratories of the departments of Epizootology and Infectious Diseases of Animals, Physiology and Biochemistry of Agricultural Animals, Parasitology and Veterinary-Sanitary Examination of the Faculty of Veterinary Medicine of Dnipro National Agrarian-Economic University, and also in the Bacteriological Department of Dnipro Regional National Laboratory of Veterinary Medicine in 2017-2018. Bacteria. The cultures of microorganisms of standardized strains (Table 2), cultivated on a dense growth medium over 18-24 hours were washed out with sterile isotonic solution of sodium chloride at temperature of 37 ± 2 ºС. The weighed microbial amounts were processed to 5 · 10 8 CFU/ml of McFarland turbidity standard for optical standartisation of bacteria using a Dilushaker III Digital densitometer, France. The solutions of disinfectants in the working concentration (0.9 ml) were poured into sterile test tubes. To the test tubes with disinfectant solutions (1, 5, 10, 25%), 0.1 ml of weighed microbial amounts were added, mixed, and then the tubes were shaken for a few seconds (Table 1).
Then, 0.5 ml of solution of the neutralizer was added (Tvin-80 -3%, saponin -3%, histidine -0.1%, cysteine -0.1%) and the tubes were shaken. The inoculations were made on to a specific differentialdiagnostic medium by 0.1 ml of the mixture, and the cups with inoculated cultures were put in a thermostat for 24 hours. The methods are descrybed in detail in the articles by Zazharskyi et al. (2018aZazharskyi et al. ( , 2018b. The incubation was performed in accordance with the passport for the growth media. After the time necessary for the cultivation of the studied microorganisms, we assessed the number of the microorganisms that grew in the Petri dish. Distinctive typical colonies were reinoculated to beef-extract agar and incubated for 24 hours at 37 ºС. The cultivated colonies underwent microscopy. If necessary, an additional identification of microorganisms was conducted in accordance with EN ISO 11133: 2014, IDT (Table 2).
Ciliates. Comparative analysis of the impact of disinfecting preparations on criogenic strains of microorganisms was performed in accordance with the generally accepted methods. The cultivation of P. caudatum and T. pyriformis ciliates was done in lactic media. The culture was maintained at room temperature (18-20 ºС). For the biotesting, we used a 24-hour culture which was in the phase of exponential (active) growth. To conduct the toxicological study, we prepared a series of dissolved preparations (Table 1): 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001% and 0.000001%.
In 5 micro-aquarium cavities 20 µl of the medium with ciliates (10-20 individuals) were put. Then 20 µl of aquatic solution of the studied preparations of different concentrations was added to each cavity and the number of cells in each aquarium was assessed. After 1 hour exposure, we again assessed the number of P. caudatum in each cavity of the aquarium and determined the percentage of their survival. During use of T. pyriformis culture, due to the small sizes of the cells and the impossibility of counting them precisely, the assessment of the biotest results was performed in relation to death of ciliates and the pattern of changes in movement. For most substances, we determined almost complete similarity in the decline angle of the straight line of lethality for ciliates and laboratory animals. This allows us to extrapolate the results of studies on protozoans to animals and humans. The values of LD 50 for all studied substances, obtained using the method of expressive biotesting are within confidence intervals for LD 50 values obtained experimentally (Miyoshi et al., 2003;Venkateswara et al., 2007). Nematoda. The larvae of nematodes in feces of ruminants were found using the Baermann test (Zajac et al., 2011). Then, 1 ml of the studied mixtures of the substances in different concentrations (1%, 5%, 10%, 25%) was added to each culture of H. contortus nematode larvae (in five times replication). The experimental exposure equaled 24 hours. We determined the number of vital and dead larvae. The methods are described in detail in articles by Boyko & Brygadirenko (2018a. Statistical analysis. The extrapolation of the data on acute toxicity of the studied substances, obtained for T. pyriformis, to animals was implemented in accordance with the recommended methods of express biotesting. For this purpose, effective dose of a certain substance, obtained in the experiment in determining acute toxicity, was expressed as probit which was placed in the graph of the lethality line of T. pyriformis ciliates and LC 50 value was calculated. The results are satisfactory if LC 50 value obtained using the method of express biotesting is within the confidence interval (error). Value of LC 50 for ciliates was determined using probit-analysis of lethality curves. Probit-analysis is recommend-ded by OECD Guidelines for the Testing of Chemicals for assessment of harmful impact of different toxicants. The statistical analysis of the results with H. contortus was performed through a set of Statistica 8.0 (StatSoft Inc., USA), the figures show the median, 25% and 75% quartiles, minimum and maximum values. LD 50 (%) was calculated as mean ± standard deviation (x ± SD).

Results
The mixtures we studied -alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, and also alkyl-benzyldimethylammonium chloride, formaldehyde, and glutaraldehydedemonstrated bactericidal properties even in 1% concentration against cryogenic strains of the following microorganisms: S. аureus, S. typhimurium, E. coli, L. monocytogenes, P. vulgaris, S. marcencens, P. аeruginosa, E. faecalis and Y. enterocolitica. The mixtures of these substances demonstrated a bacteriostatic effect on B. сereus microorganisms: growth was observed in the colonies with addition of 1% of solution of mixture of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, and glutaraldehyde, and also 1%, 5% and 10% solutions of mixture of alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde (Table 3). No negative impact on the mobility of T. pyriformis was demonstrated by the mixtures of sodium dodecyl sulfate (SDS), essential oil, glutaraldehyde, and also formaldehyde, glutaraldehyde with 0.01%, mixtures of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and also alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde with 0.0001% (Table 4). According to the results of our previous studies (Zazharskyi et al., 2018a(Zazharskyi et al., , 2018b, the impact of 0.01 mg/l of mixture of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde, and glutaraldehyde caused the highest death rate of ciliates -26% and 22% respectively (Table 5). LC 50 equaled 1.8 mg/l with use of the mixture of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, 8.4 mg/l -formaldehyde, glutaraldehyde, 27.2 mg/l -alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde, 53.4 mg/lsodium dodecyl sulfate, essential oil, and glutaraldehyde. In the series of experiments on ciliates, the death of different numbers of them was ob-served in interval 0.001-10 mg/l with use of the mixture of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde and formaldehyde, glutaraldehyde, 0.001-100 mg/lalkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde, 0.1-100 mg/l for sodium dodecyl sulfate, essential oil, glutaraldehyde.
The greatest impact on the vitality of nematode larvae in the environment was demonstrated by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride and glutaraldehyde. 100% death rate of H. contortus, nematode larvae of ruminants was observed with use of 1% solution of this mixture. Nematocidal effect was exhibited by the mixture of alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde: nematode larvae of the studied species died at 5% concentration. Mixtures of sodium dodecyl sulfate, oleum terebinthini, glutaraldehyde, and also formaldehyde and glutaraldehyde were the least efficient against invasive larvae of H. contortus. 100% death rate of L 3 larvae was observed only when 25% solution of mixtures of these substances was used (Fig. 2).

Table 5
Influence of the studied substances on P. caudatum (% dead ciliates; n = 5) During usage of mixture of sodium dodecyl sulfate, oleum terebinthini and glutaraldehyde in 5% concentration, the vitality of nematode larvae was observed on average to be 25%, in 10% concentration it was 8% of individuals. When the mixture of formaldehyde, glutaraldehyde was used in 1%, 5% and 10% concentration, on average 1-10% of individuals survived. LD 50 for mixture of sodium dodecyl sulfate, essential oil and glutaraldehyde equals 2.3 ± 0.8%, for formaldehyde, glutaraldehyde it was 0.45 ± 0.16%.

Discussion
The series of studies Takashi & Kei-Ichiro (2007) proved the bactericidal effect of didecyldimethyl ammonium chloride in minimum inhibiting concentration 1.3 mg/l against E. coli. The studies by Shirron et al. (2009) allow us to state that didecyldimethyl ammonium chloride causes a bactericidal effect against S. typhimurium. Walsh et al. (2003) reported that didecyldimethyl ammonium chloride has a bactericidal effect on E. coli, S. aureus, P. aeruginosa and L. monocytogenes. According to Ioannou et al. (2007), alkylbenzyldimethylammonium chloride and dide-cyldimethyl ammonium chloride are at the same time membrane-active agents with subtly different mechanisms of action, which reflect the previous interaction with S. aureus.
The studies by Lasemi et al. (2017) demonstrated the impact of 2% solution of glutaraldehyde on the spores of B. subtilis. The results showed that 102 colonies were present on the 10th minute, 18.6 ± 3.4 on the 15th minute, 6.2 ± 1.4 on the 20th minute, 2.1 ± 0.8 on the 25th minute and no colonies after 30 minutes. Over the first 10 minutes, more colonies were observed, after 15-20 minutes this number significantly reduced. After 30 minutes, growth of the colonies completely stopped. 2% density of glutaraldehyde over 30 minutes was sufficient for eliminating the spores of B. subtilis. The data by Simões et al. (2008) indicate that sodium dodecyl sulfate has an antimicrobal effect on the biomembranes of P. fluorescens. In their studies, Chen et al. (2016) mention that pathogenic strains of E. coli, P. aeruginosa and K. pneumoniae have a FrmRAB regulator, and can be used for eliminating endogenous and exogenous Formaldehyde. Vaerewijck et al. (2012) determined that alkylbenzyldimethylammonium chloride and sodium hypochlorite in concentration of active chlorine of 50 mg/l can inactivate Acanthamoeba and two species of Tetrahymena spp. in 15 minutes. The series of studies by Ivancovic et al. (2013) proved the lethal effect of alkylbenzyldimethylammonium chloride on Paramecium caudatum. The studies by Blondeau et al. (2007) allow us to state that alkylbenzyldimethylammonium chloride in a combination with gatifloxacin and moxifloxacinin in concentration of 0.008-0.125 mg/l exhibited bactericidal effect against polyresistent S. aureus. The studies by Braoudaki et al. (2005) demonstrated that alkylbenzyldimethylammonium chloride in combination with erythromycin has a bactericidal effect against S. typhimurium. Fazlara & Ekhtelat (2012) Hattori et al. (2003) observed resistance to this substance by P. vulgaris. Tiwari et al. (2003) also proved the resistance of Serratia marcescens to this substance. By contrast, Paul et al. (2010) allow us to state that alkylbenzyldimethylammonium chloride shows no bactericidial effect against P. aeruginosa.
There are data on using mixtures of formaldehyde and glutaraldehyde as a disinvasive preparation. The study by Palіj et al. (2018) describes the effect of FAG aldehyde preparation on the eggs of nematodes of agricultural animals. It was determined that the preparation in 6.0% concentration at 24 hours exposition demonstrates a disinvasive effect against eggs of Ascaris suum, Ascaridia galli and Toxocara canis. Mixture of these aldehydes is an efficient preparation for disinfecting the premises of livestock contaminated with invasive helminths.
The highest bactericidal, protistocidal, and also nematodicidal effect were observed for use of mixtures of alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride and glutaraldehyde, and also alkyldimethylbenzylammonium chloride, formaldehyde and glutaraldehyde.
Mixtures of alkylbenzyldimethylammonium chloride, formaldehyde, glutaraldehyde, and also alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, and glutaraldehyde demonstrated bactericidal properties on cryogenic strains of S. аureus, S. typhimurium, E. coli, L. monocytogenes, P. vulgaris, S. marcescens, P. аeruginosa, E. faecalis, and Y. enterocolitica, and also nematocidal properties against H. contortus, nematode larvae of ruminants. Maximum toxicity during use of the studied substances against P. caudatum was demonstrated by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, glutaraldehyde, and also formaldehyde and glutaraldehyde. The least toxic were mixtures of sodium dodecyl sulfate, oleum terebinthinі, and glutaraldehyde (14-15 times safer). The mixture alkylbenzyldimethylammonium chloride, formaldehyde and glutaraldehyde showed a moderate level of toxicity. The least toxicity for T. pyriformis was observed for the mixture of sodium dodecyl sulfate, essential oil, glutaraldehyde, and also formaldehyde and glutaraldehyde, the highest for alkylbenzyldimethylammonium chloride, formaldehyde, and glutaraldehyde. The strongest effect on the viability of nematode larvae in the environment was shown by alkylbenzyldimethylammonium chloride, didecyldimethyl ammonium chloride, and glutaraldehyde. 100% death rate of H. contortus, nematode larvae of ruminants, was recorded already at using 1% solution of this mixture. Nematocidal effect was observed for mixture of alkylbenzyldimethylammonium chloride, formaldehyde, and glutaraldehyde: nematode larvae of the studied species died at 5% concentration. Thus, our observations can be useful for practicing doctors of human and veterinary medicine during preparation of antiseptics, disinfecting and disinvasive preparations with predicted biocidal effect of four ammonium compounds.