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Abstract

Introduction:

Use of sea water as a diluent for disinfectants has been of practical interest for control of aquaculture disease outbreaks in sea where fresh water is limited. This study evaluated the use of natural sea water (NSW), artificial sea water (ASW), or standard hard water (SHW) as a diluent for preparation of accelerated hydrogen peroxide (AHP) solutions against an avian influenza virus, a surrogate for the infectious salmon anemia virus.

Methods:

AHP solutions containing 0.18%, 0.35%, or 0.44% (w/w) of hydrogen peroxide (H₂O₂), corresponding to 1/40, 1/20, and 1/16 dilutions of the disinfectant concentrate, were evaluated at −20°C, 4°C, and 21°C.

Results:

When NSW was used as the diluent, a 0.35% H₂O₂ concentration was required to inactivate ∼6 log₁₀ virus at 21°C in a 5-min contact time. When temperature dropped to 4°C, 0.44% H₂O₂ in NSW was required to obtain a similar inactivation within a 5-min contact time. At −20°C, supplemented with antifreeze agents, the 0.44% H₂O₂ in NSW solutions produced complete inactivation of 5.4 log₁₀ virus within a 10-min contact time. In comparison, lower H₂O₂ concentrations and/or shorter contact times were needed to inactivate equal amounts of the virus at the same temperature when using SHW or ASW as a diluent to prepare disinfection solutions.

Conclusion:

The results suggested that NSW could be used as a diluent in disinfection solutions for virus inactivation as long as disinfectant concentrations and/or contact times are properly increased.

Introduction

Infectious salmon anemia (ISA) is a notifiable fish disease to the World Organization for Animal Health.¹ ISA primarily affects marine-phase farmed Atlantic salmon, which can result in high levels of mortality. ISA first emerged in Norway in the 1980s and subsequently occurred in Canada, the United States, and other countries.² The disease has continued to threaten the Atlantic salmon farming industry. ISA transmission occurs through horizontal routes. The ISA virus, shed by infected fish through blood, mucus, feces, urine, skin, or carcasses, spreads between aquaculture sites through transfer of live infected cultured fish, wild fish, untreated wastes and water, and shared equipment or gear that has not been properly disinfected.³ Mitigation measures to control the spread of the disease include, without limiting to, movement restrictions on fish, waste and water treatment, and decontamination of equipment and harvesting vessels.

ISA virus belongs to the genus Isavirus in the Orthomyxoviridae family, which also includes Influenzavirus genera.⁴ ISA virus genome contains eight single-stranded RNA segments of negative sense. Virus particles are pleomorphic and enveloped. ISA virus can be inactivated by external heat (>56°C), extreme pH (<4 or >12), UV, ozone, and chemical disinfectants containing chlorine, iodine, hydrogen peroxide (H₂O₂), or potassium peroxymonosulfate compounds.^5–7 Fresh water is usually used as a diluent to prepare solutions of chemical disinfectants.⁸ However, as fresh water may not be readily available at saltwater sites, there has been an interest in using sea water as a diluent for preparation of disinfection solutions for disease control during outbreaks. Since data are not available on the use of sea water as a diluent for disinfectants, this study evaluated the virucidal activity of disinfectant solutions prepared with natural sea water (NSW), artificial sea water (ASW), or standard hard water (SHW). A commercially available accelerated hydrogen peroxide (AHP) disinfectant concentrate was used for the evaluation as H₂O₂ breaks down into water and oxygen and has minimum negative impacts to the environment. An avian influenza (AI) virus was used as a surrogate for the ISA virus in this study, as both viruses belong to the Orthomyxoviridae family and are of similar susceptibility to external heat, extreme pH, and a variety of chemical disinfectants.^5–7,9 As ISA outbreaks in Canada have occurred in warm and cold seasons, this study conducted the evaluation at −20°C, 4°C, and 21°C, representing freezing, cold, and warm temperatures, respectively.

Materials and Methods

Virus

An H6N2 strain of AI virus (A/Turkey/Mass/3740/65) was used as a surrogate for the ISA virus in this study. Virus stock was prepared with 10-day-old embryonated chicken eggs (ECEs) from a specific pathogen-free flock of White Leghorn chickens maintained by the Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency, and tested negative for AI virus. The amount of infectious virus was determined by limiting dilutions in ECEs¹⁰ and expressed as the 50% embryo infectious dose (EID₅₀). The use of ECEs conformed to guidelines established by the Animal Care Committee at the Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield).

Disinfection Solutions

AHP solutions were prepared from PreVail™ Concentrate (Virox Technologies, Oakville, Ontario) using SHW, ASW, or NSW as a diluent. The AHP solutions contained 0.18%, 0.35%, or 0.44% (w/w) H₂O₂, corresponding to 1/40, 1/20, and 1/16 dilution of the disinfectant concentrate. SHW was prepared to have a standard hardness of 400 parts per million of calcium carbonate.¹¹ ASW (cat#MISC-CHEM-LAB, VWR, Mont-Royal, Quebec, Canada) was prepared according to ASTM D1141-98.¹² NSW was collected in early spring using a bucket and rope over the side of a wharf at Witless Bay, Newfoundland, and Labrador. For tests at −20°C, to prevent disinfectant solutions from freezing, anhydrous CaCl₂ (Sigma-Aldrich, Oakville, Ontario, Canada) was added to the disinfectant solutions for a final concentration of 20% (w/v). Neutralizer solution used for stopping the activity of the disinfectant was a mixture of nine volumes of Difco™ D/E neutralizing broth (Thermo Fisher Scientific, Ottawa, Ontario, Canada) and one volume of antibiotic–antimycotic (Invitrogen Canada, Burlington, Ontario, Canada).

Quantitative Carrier Test

The second-tier quantitative carrier test¹³ was used to evaluate the virucidal activity of the disinfectant solutions. Disks (1 cm in diameter; 0.75 mm thick) of brushed stainless steel (AISI no. 430; Muzeen & Blythe, Winnipeg, Manitoba, Canada) were washed three times with distilled water and sterilized at 121°C for 25 min before use. Single time point experiments using a contact time of 5 or 10 min were carried out. Triplicate sample and control disks were prepared by dropping 10 μL of the virus inoculum onto each disk. The inoculum contained ∼8 to 9 log₁₀ EID₅₀ of AI virus per milliliter of allantoic fluid. The disk was air dried in a class II biosafety cabinet for 1 h and then, with the inoculum side up, was placed in a 30-mL polypropylene straight side vial, and the vials were placed into wells of custom-made metal blocks preconditioned to maintain the test temperatures. For the sample disks, disinfectant solution (50 μL) preconditioned to the test temperatures was added to cover the dried inoculum. For the control disks, phosphate-buffered saline (PBS, pH 7.0, 50 μL) was added. The vials containing the disks were incubated for 5 or 10 min at −20°C, 4°C, and 21°C. At the end of each contact time, a neutralizer solution (950 μL) was immediately added to each vial (including vials with control disks) to stop the activity of the disinfectant. The suspension from each vial was then 10-fold serially diluted and tested for AI virus infectivity in ECEs.¹⁰ Before testing of the disinfectant solutions, preliminary experiments were conducted to test the effect of the antifreeze agents and the neutralizer on survival of the virus and the ECEs and the effect of the neutralizer on the activity of the disinfectants.

Statistical Analysis

The data presented are the mean values of the difference between the infectious virus recovered from triplicate sets of sample and control disks in duplicate experiments. One-way analysis of variance¹⁴ was used to determine significant differences in the amount of virus recovered from disks incubated at the same temperature for the same contact time. The critical level for significance was set at p < 0.05.

Results and Discussion

ISA virus like other infectious agents may spread through contaminated equipment or gear shared among infected and uninfected fish farms. To expedite decontamination of equipment or gear in sea, the use of sea water as a diluent for disinfectants has been of practical interest when fresh water is limited. However, there is a lack of information on the effectiveness of disinfection solutions prepared with sea water. Hence, this study compared the use of NSW, ASW, and SHW as diluents for preparing disinfection solutions and evaluated the virucidal activity of the solutions at various temperatures using an AHP concentrate as disinfectant and an avian influenza virus as a surrogate for the ISA virus.

At 21°C, the 0.18% and the 0.35% H₂O₂–NSW solutions inactivated ∼5 and 6 log₁₀ virus, respectively, within 5 min contact time (Table 1). When the temperature dropped to 4°C, a higher concentration (0.44%) of H₂O₂ in NSW was required to inactivate 6 log₁₀ virus within 5 min. Since low temperatures slow down disinfection reactions, increasing disinfectant concentration and/or contact time is reasonable and common options to maintain disinfection effectiveness.^15,16 When the temperature further dropped to −20°C, an addition of antifreeze agents was required to prevent disinfection solutions from freezing. In this study, CaCl₂ was the selective antifreeze agent due to its inexpensiveness and compatibility with the sea environment. Supplemented with 20% (w/v) CaCl₂, the 0.35% and the 0.44% H₂O₂–NSW solutions produced complete inactivation of 5.4 log₁₀ virus within 10 min contact time at −20°C (Table 1). In these H₂O₂–NSW–CaCl₂ solutions, CaCl₂ may also contribute to virus inactivation. As evidence, the 20% CaCl₂ solutions without disinfectant produced 0.4 – 0.6 and 1.5 – 3.7 log₁₀ reduction of the virus at −20°C and 21°C, respectively (Table 2). Similar virucidal effect of the CaCl₂–PBS solutions was also observed in our previous study.¹⁶

Table 1.

Effect of diluents on reduction of avian influenza virus at various temperatures and contact times

H₂O₂% (w:w)	Diluent	Virus reduction (log₁₀ EID₅₀)^a
		5 min		10 min
		21°C	4°C	21°C	4°C	−20°C
0.18%	SHW	5.7	1.9	5.5	3.6	4.3
	ASW	5.5	2.4	5.7	4.1	5.0
	NSW	5.0	2.1	6.0	2.6^*	1.7^*
0.35%	SHW	5.7	5.3	NT	5.8	5.4
	ASW	5.7	2.3^*	NT	5.6	5.2
	NSW	5.7	2.3^*	NT	3.0^*	5.4
0.44%	SHW	6.1	6.1	6.0	6.0	5.4
	ASW	6.1	6.1	6.0	5.8	5.4
	NSW	6.1	6.1	6.0	6.0	5.4

Number in bold indicates complete inactivation of the inoculated virus. Numbers with “^*” are significantly different from those within the column of the same dilution of disinfectant. EID₅₀ = 50% embryo infectious dose, NT = not tested.

w:w = weight of the H₂O₂:weight of diluents.

Number is mean reduction of virus from triplicate samples of duplicate experiments with standard deviation ≤0.5.

ASW, artificial sea water; H₂O₂, hydrogen peroxide; NSW, natural sea water; SHW, standard hard water.

Table 2.

Effect of CaCl₂ addition in various diluents on reduction of avian influenza virus at different temperatures

Diluent	Virus reduction (log₁₀ EID₅₀)^a
	21°C, 10 min		−20°C, 10 min
	20% CaCl₂	0% CaCl₂	20% CaCl₂
SHW	3.7^*	0	0.6
ASW	1.5	0	0.4
NSW	1.5	0	0.6

Numbers with ^* are significantly different from those within the column and the row.

Number is mean reduction of virus from triplicate samples of duplicate experiments with standard deviation ≤0.5.

In comparison with SHW, using ASW or NSW as a diluent to prepare disinfection solutions, higher H₂O₂ concentrations and/or longer contact time were generally required to inactivate equal amounts of virus at the same temperature (Table 1). As evidence, virus reduction produced by the 0.35% H₂O₂ in ASW and NSW solutions was significantly (p < 0.05) lower than that by the 0.35% H₂O₂–SHW solution at 4°C with 5 min contact time (Table 1). Possibly, similar to the reactions between halogen ions and ozone in the use of ozone for virus control in aquacultural seawater systems,⁶ the halogen ions in the ASW and NSW might react with the H₂O₂ and reduce the activity of the disinfection solutions. Furthermore, compared with the 0.18% and 0.35% H₂O₂–ASW solutions, the corresponding H₂O₂–NSW solutions produced significantly (p < 0.05) less virus reduction at 4°C with 10 min contact time (Table 1). In this study, the NSW was collected using a bucket–rope over the side of a wharf without filtration or centrifugation to simulate the simplicity of using sea water to prepare disinfectant solutions onsite. Likely, the biota or the organic load in the NSW also consumed the active ingredients and further reduced the activity of the disinfectant solutions. Nevertheless, according to the Canadian guidance document on the safety and efficacy requirements for hard surface disinfectant drugs,¹⁷ a 4 log₁₀ reduction of virus is the approved disinfectant inactivation standard. Based on this standard, the disinfection solutions prepared from NSW were effective for virus inactivation on steel surface used in this study. Possibly, similar virus inactivation would occur on other nonporous surfaces.¹⁸ Maintaining sufficient contact time with disinfectant solutions would be critical for effective decontamination of boats and equipment with various surface position and shape.¹⁹

Furthermore, the disinfection activity of the 0.35% H₂O₂–NSW and H₂O₂–ASW solutions remained stable within 24 h at room temperature (Table 3). The results suggested that the disinfection solutions prepared from NSW could stay effective for at least a day before use. However, the biota or organic substances in NSW may be oxidized by disinfectants to halogenated organic compounds, such as trihalomethanes.⁶ The dominating trihalomethanes in sea water is bromoform, which is a probable human carcinogen,²⁰ known to be persistent in water and bioaccumulated in aquatic animals.²¹ To minimize potential negative environmental impacts, it is recommended that appropriate amounts of disinfection solutions be prepared freshly based on needs, when using sea water as a diluent.

Table 3.

Influence of storage time of disinfectant solutions prediluted with sea water on reduction of avian influenza virus

Diluent for H₂O₂ solution^a	Virus reduction (log₁₀ EID₅₀)^b
	Storage time (hours)
	0	2	4	8	16	24
ASW	5.9	5.9	6.1	6.1	6.1	6.1
NSW	6.0	6.0	6.0	6.0	6.0	6.0

Number in bold indicates complete inactivation of the inoculated virus.

0.35% (w/w) of H₂O₂ in ASW or NSW.

Number is mean reduction of virus from triplicate samples of duplicate experiments at 21°C and 5 min contact time with standard deviation ≤0.5.

In conclusion, results in this study suggested that NSW could be used as an optional diluent for preparing disinfection solutions for decontaminating steel or potentially other nonporous surfaces during ISA virus outbreaks.

Footnotes

Acknowledgments

Appreciation is expressed to Suzanne Nadeau, Atlantic Area Emergency Coordinator, Business Planning and Management, Canadian Food Inspection Agency, and to Scott Bishop, Dave King, Joe Pickett, Nicholas Christie, Inspectors in the Avalon District, St. John's, Newfoundland and Labrador, Canadian Food Inspection Agency for their assistance in providing sea water for this study.

Authors' Contributions

J.G. and M.C. designed and carried out the experiments and analyzed the data. All authors contributed to the article and approved the submitted version.

Author Disclosure Statement

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding Information

This study was supported by the Canadian Safety and Security Program under Grant No. CSSP-2013-TI-1036, entitled “Evaluation of readily available materials for use as antifreezing agents for subzero decontamination.”

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Evaluation of the Use of Sea Water as a Diluent for an Accelerated Hydrogen Peroxide Disinfectant for Inactivation of Avian Influenza Virus: A Surrogate for Infectious Salmon Anemia Virus

Abstract

Introduction:

Methods:

Results:

Conclusion:

Introduction

Materials and Methods

Virus

Disinfection Solutions

Quantitative Carrier Test

Statistical Analysis

Results and Discussion

Footnotes

Acknowledgments

Authors' Contributions

Author Disclosure Statement

Funding Information

References