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Dissertation Jack Ardley May 2015

Contents

Contents 2

Table of Tables 3

Table of Figures 3

Introduction 4

Methods & Materials 6

Samples 6

Treatment 6

Virkon® Aquatic & STERI-7 6

Air Drying 8

Control 8

Serial Dilutions 8

Bacterial Culture 9

Bacterial Counting 10

Aseptic Technique 10

Results 10

Discussion 15

Conclusion 17

Bibliography 18

PROTECTION BETWEEN CLEANS

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Table of Tables

Table 1: Table showing the Colony forming units (C.F.U.) present for each piece of treated net at different dilutions (U/C means uncountable, Results in red are presumed contaminated) 11

Table 6: The wet weight, dried weight and total moisture removed from pieces of net via kiln drying and air drying 15

Table of Figures

Figure 1: Layout of equipment during net treatment process 7

Figure 2: Basic Method of Serial Dilution used for the Experiment 9

Figure 3: Stirring hotplates mixing the agar 9

Figure 4: Probability plots for Virkon AquaticAquatic1:100 (P=0.609), Virkon Aquatic Aquatic1:200 (P=0.091), STERI-7 Xtra 1:10 (P=0.200) and STERI-7 Xtra 1:50 (P=0.596) 11

Figure 5: Analysis of Variance with Virkon AquaticAquatic1:100, Virkon AquaticAquatic1:200, STERI-7 Xtra 1:10 and STERI-7 Xtra 1:50 at 0.01 dilution (P= 0.004) 12

Figure 6: Tukey Test comparison for STERI-7 and Virkon AquaticAquaticsolutions at 0.01 dilution 12

Figure 7: Analysis of Variance between Virkon Aquatic 1:100, Virkon Aquatic 1:200, STERI-7 Xtra 1:10 and STERI-7 Xtra 1:50 at 0.001 dilutions (P=0.019) 13

Figure 8: Tukey test of Virkon AquaticAquaticand STERI-7 Xtra solutions at 0.001 dilutions 13

Figure 9: Analysis of Variance between Virkon AquaticAquatic1:100, Virkon AquaticAquatic1:200, STERI-7 Xtra 1:10, STERI-7 Xtra 1:50 and Air Drying at 0.0001 dilution (P=0.002). 14

Figure 10: Bar chart showing the average C.F.U. on 10cm2 pieces of net after treatment with 1:100, Virkon AquaticAquatic1:200, STERI-7 Xtra 1:10, STERI-7 Xtra 1:50 14


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Introduction

It is readily recognised that disinfection is important in both the angling and aquaculture industries (Jorquera et al. 2002; Barbour et al. 2013, Lakeh et al. 2013; Lahnsteiner & Kletzl, 2015). It is vital in stopping the spread of opportunistic and obligate pathogens and also invasive and non-native species (Summerfelt et al. 2009; Oidtmann et al. 2011). A paper by Gunn et al. (2008) describes disinfection as a “key component” in the practises, procedures and policies used in biosecurity to prevent the introduction and spread of pathogens and invasive species. In the biosecurity plan for Calverton fish farm (See Appendix), a large coarse fish farm run by the Environment Agency (EA), disinfection is heavily represented with nothing being allowed on the fish farm before disinfection (A Henshaw, 2014, Pers. Comm.). Pathogen risk analysis is an internationally accepted method for analysing and lowering the risks of commodity trading and movement, a large risk is the movement of aquatic pathogens on aquaculture equipment and therefore disinfection is an important countermeasure to lower this risk (Diggles & Arthur, 2010).

According to Anderson et al. (2014) angling is one of biggest potential pathways for the spread of invasive and non-native species (INNS) and pathogens – it was also found that many INNS and aquatic pathogens can survive for up to a fortnight in damp environments. Other studies have also found angling equipment that can retain moisture which can act as an effective vector for a number of aquatic pathogens (Cliff & Campbell, 2012). Kilian et al. (2012) claims that many pathogenic introductions have been caused by anglers. Research shows that under certain conditions the influence of introduced pathogens can lead to virtual extinction of the host species in the region (Blanc, 2001). Data collection carried out by Carlton & Ruiz (2003) showed that in South Africa angling was the biggest pathway for the introduction of INNS and pathogens in the past, present and the foreseeable future. Although the above evidence shows the risks associated with angling equipment that hasn’t been disinfected, many fisheries still don’t use disinfectants and those that do normally don’t do it efficiently or effectively – with short exposure times and old solutions (Fraser et al. 2006).

An example of a pathogen that has been spread via angling and has had devastating effects on wild populations of fish, mainly in Norway, is Gyrodactylus salaris (Paladini et al. 2014). A paper by Knudsen et al. (2007) claims that Gyrodactylus salaris is regarded as one of the main threats to maintaining vigorous wild Atlantic Salmon (Salmo salar) stocks in Norway. Between 1975 and 2010 Gyrodactylus salaris spread to over 48 Norwegian rivers, it is thought that angling played a major role in the spread of the parasite (Sviland et al. 2012). There are several papers that describe angling equipment as a potential pathway for Gyrodactylus salaris to be introduced into countries currently free of the parasite (Peeler & Thrush, 2004; Hesthagen & Sandlund, 2007; Inland Fisheries Ireland, 2012; Anderson et al. 2014). As a result of the Gyrodactylus salaris epidemic it is now a requirement to disinfect fishing tackle in Norway and a certificate of disinfection must be presented on demand (CEFAS, 2014).

There are many different forms of disinfectant suitable for use against aquatic pathogens (Fraser et al. 2006). These include chemical disinfectants such as formalin, iodophers, ozone, sodium hydroxide, peroxy compounds, chloramine t, ultraviolet (UV) light and many more (Torgersen & Hastein, 1995; Fraser et al. 2006; Denning, 2008; Jussila et al. 2014; Schroeder et al. 2014). A popular aquatic disinfectant is a monopersulphonate triple salt based disinfectant (Virkon® Aquatic) (Paetzold & Davidson, 2011). Virkon® Aquatic is the most commonly used and highly regarded disinfectant in both angling and aquaculture (Bryan et al. 2009; Mainous et al. 2010; Scott, 2013; Traverse & Aceto, 2014). Steri–7 Xtra is a fairly new, high level, broad spectrum disinfectant with Reactive Barrier Technology (RBT) (STERI-7 Xtra, 2014). The aim of


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this study was to determine if STERI-7 Xtra could be used as an effective chemical disinfectant in the angling industry, with emphasis on bacterial prevention. According to the Environment Agency (2014) “The two best methods for disinfecting fishing tackle are: thoroughly dry equipment for 48 hours… or using Virkon® Aquatic”. Therefore in this study the efficiency of STERI-7 Xtra, Virkon® Aquatic and air drying will be compared.

Virkon® Aquatic is a commercially available aquatic disinfectant that was made available in 2007 and is often considered as the market leader in aquatic disinfection and sanitization (Dupont, 2007; Stockton & Moffit, 2013). Its active ingredients are 21.4% peroxymonosulfate and 1.5% sodium chloride (Yanong & Reid, 2012). It is used in numerous different ways in biosecurity procedures such as net disinfection, foot dips, surface cleaner, vehicle disinfection, water sanitizing and many more (Scott, 2013; Cheeran et al. 2014) A comparative study of aquatic disinfectants carried out by Mainous et al. (2012) found Virkon® Aquatic to be very effective at eradicating two different species of bacteria – reducing the number of bacteria to zero in just one minute contact time. In another study it was also found that a 0.5% solution of Virkon® Aquatic eradicated all bacteria in 5 minutes contact time (Moffitt et al. 2014). In general for net disinfection 0.5% is considered a “standard” solution and 1% is a high-power solution (Dupont, 2007; Bryan et al. 2009; Dvorak, 2009).

Steri–7 Xtra is a Biocidal disinfectant that claims to kill 99.9999% of pathogens and has a unique Reactive Barrier Technology that prevents further contamination after treatment (STERI-7 Xtra, 2015). The primary active ingredient in STERI-7 Xtra is Bardac 22-70 and Barquat 80. It has been documented that STERI-7 Xtra is an effective disinfectant against many pathogens, for example it has been found that it is effective in eradicating bacterial pathogens associated with cystic fibrosis (Moore & Rao, 2011). Work carried out by Lawley et al. (2010) found STERI-7 Xtra also eradicated Clostridium difficile, a bacteria spore that can lead to severe disease. STERI-7 Xtra is listed by DEFRA and CEFAS. The manufacturer’s instructions state that a 1:50 solution is for general power and a 1:10 solution is for high power.

Air drying is a large part of the “Check, Clean, Dry” campaign by the Great British non-native species secretariat (NNSS, 2015). The campaign is in place to encourage anglers to dry their angling gear after use, primarily to stop the spread of INNS and pathogens. Inland Fisheries Ireland (2014) claim “The best method of disinfection is drying thoroughly in direct sunlight, optimally for 48 hours”. One study by Ghittino et al. (2003) found that the drying of tanks used to culture fish was not enough to disinfect them and disease causing pathogens were still present after thorough drying. Another study found air drying to be ineffective – it concluded that 96 hours of air drying is insufficient to kill Flavobacterium psychrophilum (Oplinger & Wagner, 2010).On the other hand a study by Moretro et al. (2003) found air-drying to be equally as effective as many chemical disinfectants at eradicating a number of different pathogens, though primarily focusing on salmonella, on a number of different surfaces. Although it is a highly recommended method of disinfection, which is regularly advised to anglers by environmentally concerned organisations – there seems to be very little published evidence to support the claim that air drying is the “best” form of disinfection for angling equipment. There are many published papers on the use of air drying as a disinfectant with varying results, but few are related to aquaculture and even fewer directly related to angling and its equipment.

The final aim of this experiment and research is to create a basic idea of the efficiency of Virkon® Aquatic, STERI-7 Xtra and air drying as methods of disinfection. The study will focus on bacteria as a whole to form a basis in which more specific research can be carried out. The null hypotheses of the experiment is that there will be no significant difference in the efficiency of all three methods of disinfection, the alternative hypothesis is that there will be a significant difference shown.


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Methods & Materials

The data was collected using basic microbiology methods which involved culturing bacteria from treated samples on agar plates and then counting colony forming units (C.F.U.) after a set amount of time incubating.

Samples

There were eighteen pieces of net used in the experiment – all of which were 10cm2. It is important that all net samples were of the same size as uniformity is important in experimental design to ensure accurate and comparable results (Fang & Lin, 2003; Christensen et al. 2014). The net had a mesh size of 5mm, this meant there was enough surface area to gather large quantities of bacteria but it was large enough to allow good water flow.

Each piece of net was submerged in the Clarias gariepinus biological filter system. It is common knowledge that C. gariepinus are a very wasteful species –both with their messy eating and vast quantities of organic waste they produce (Goda et al. 2007; Adewolu et al. 2008; Emikpe et al. 2011). Biological filters work by promoting growth of bacteria on the media inside the filter, this bacteria grows to break down nitrogenous waste – the bacteria includes Nitrosomonas, Nitrobacter and many more ammonia and nitrite oxidising bacteria (Jun & Wenfang, 2009; Kessel et al. 2010; Gregory et al. 2012). With both the organic waste from the C. gariepinus and the beneficial bacteria in the biofilter there was sufficient bacteria present to heavily soil the pieces of net. The nets were hung using a hanger designed to be used on domestic washing lines that evenly spaced the net to ensure equal water flow, it was also ensured that all pieces of net were fully submerged. The net was left hanging in the biological filter for approximately 72 hours (3 Days) to allow for bacterial soilage of the net. According to Moisan et al. (2014) direct exposure to bacteria leads to instantaneous contamination therefore 72 hours should have been more than enough exposure time for the net. After 72 hours in the biofilter the nets were taken to the microbiology lab in sterilised zip lock bags – this is because exposure to the air can lead to contamination (Harrigan et al. 2014)

Treatment

There were five treatments and a control, making a total of 6 different factors. The treatments were 2 different concentrations of both STERI-7 Xtra and Virkon® Aquatic, there was also air drying and a control. Each treatment and the control were triplicated, hence the need for 18 pieces of net. A paper by Vegas et al. (2006) describe replication of an experiment as important to ensure reliable results and minimize errors and data skew, they also say that successful replication enables an individual to grow their body of knowledge. Replication is also important to increase the statistical power of results (Button et al. 2013; Brandt et al. 2014). After 72 hours of hanging in the C. gariepinus biofilter system the net was ready for treatment.

Virkon® Aquatic & STERI-7 Xtra

The Virkon Aquatic was acquired from the National Aquatics Training Centre (NATC) located at Sparsholt College Hampshire (SCH). To make the solutions the Virkon Aquatic was mixed with distilled water. The distilled water goes through a process in which it is boiled and then condensed into a clean container – this process removes impurities and contaminants (Gutierrez et al. 2009; Deroine et al. 2014). This means that


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there will be nothing in the solutions that may affect such as disinfection – such as chlorine which is present in tap water (Dong et al. 2012). One solution was made to a 1:100 (1%) solution – this is considered a high level solution for net disinfection (Dvorak, 2009). To make this solution 10g of the Virkon Aquatic were added to 1 liter of distilled water. The other solution made was 1:200 (0.5%) which is considered an industry standard solution (Bryan et al. 2009).

The STERI-7 Xtra was supplied directly from the manufacturer to be tested and compared with other disinfection methods. The STERI-7 Xtra solutions were also made with distilled water for the same beneficial reasons as the Virkon Aquatic solutions. One STERI-7 Xtra solution was made to a 1:50 (2%) dilution, according to the manufacturers this is a general use solution. (STERI-7 Xtra, 2014). To create this solution 20ml of STERI-7 Xtra was added to the 1 liter of distilled water. The other solution was a 1:10 (10%) solution which is a high-power solution according to the manufacturers. For this solution 100ml of STERI-7 Xtra was mixed with 1 liter of distilled water.

To treat each bit of net a petri dish was filled with one of the disinfectant solutions, 3 more petri dishes were then lined behind the disinfectant and filled with distilled water. The net was submerged in the disinfectant for 10 minutes. According to Dupont (2007) 10 minutes is enough contact time with Virkon® Aquatic to kill majority of pathogens. According to STERI-7 Xtra (N.D.) STERI-7 Xtra is extremely effective after just 5 minutes. Many environmental organizations recommend 10 minutes disinfection time for angling equipment (Environment Agency, 2014; Inland Fisheries Ireland, 2014). After the 10 minutes disinfection time the net was rinsed in each petri dish of distilled water – this is to stop any disinfectant getting into the test tubes being used for serial dilutions. Once the net is rinsed it goes into a test tube with 10ml of sterilized water from the catfish biofilter system. This was repeated with 3 pieces of net for each disinfectant solution. The tweezers used to pick up and move the net were sterilized in a flame between each use. Flame sterilization is a quick, easy and commonly used method for sterilizing lab utensils (Berovic, 2005). (See Fig. 1 for Setup)


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Distilled Water

Disinfecting Solution

Bunsen Burner

Figure 1: Layout of equipment during net treatment process

Test Tube Rack

Prior to this method being used to treat the net with the disinfectant solutions another method was used: all 3 pieces of net were disinfected at the same time in a large beaker of solution. This method provided inaccurate results and was deemed unsuitable so the results were not used and the method explained above was used instead.

Air Drying

3 pieces of net were air dried after the 72 hour exposure to the catfish biofilter. The net was weighed before and after drying in order to determine how much moisture the net retained after being air dried. It is normally recommended that angling equipment is dried outside in direct sunlight (Environment Agency, 2014; NNSS, 2015). During the time of this experiment the daylight hours were short and the weather was unpredictable. Precipitation would not only have interfered with the drying process but rainwater can also contain very high levels of bacteria (Stephanie & Waturangi, 2011; Joly et al. 2013). Instead the net was hung in a laboratory. At first the nets were being hung in the thesis room of the NATC at SCH but there was much atmospheric contaminants that affected the results, the thesis room was deemed unsuitable so the experiment was carried out again but the air drying was done in a laboratory instead. The nets were hung for 48 hours as is recommended by many environmental organisations (Inland Fisheries Ireland, 2012; Environment Agency, 2014; NNSS, 2015). Once the nets had been air dried for 48 hours they were placed into the test tubes ready for serial dilution.

Control

The control pieces of net were ones that were exposed to the C. gariepinus biofilter system for 72 hours but received no treatment. They were placed directly into the test tubes ready for serial dilutions. The control pieces of net where used to determine the total number of bacteria present without any form of treatment used. It was also hoped that a control would allow for calculation of % bacteria killed for each treatment.

Serial Dilutions

Serial dilutions had to be made up for each net. This is because bacteria are so small that millions can be living on a 1cm2 space (Berdy, 2012; Mook et al. 2012). The serial dilutions were necessary to dilute the bacterial levels down to viable levels that are countable. Literature by Ben-David & Davidson (2014) says “The objective of the serial dilution method is to estimate the concentration (number of colonies, organisms, bacteria, or viruses) of an unknown sample by counting the number of colonies cultured from serial dilutions of the sample, and then back track the measured counts to the unknown concentration”. Each piece of net is placed in a test tube containing 10ml of sterilised biofilter water. By using water from the biofilter the environmental conditions match that from which any surviving bacteria have come from. The water is sterilised with use of an autoclave – autoclaves are commonly used in laboratories as they are an effective way of sterilising using superheated steam under high pressures to kill of any germs and microbes (Plotino et al. 2012). The test tube containing the net is then shaken vigorously and the assumption is made that 100% of the bacteria on the net are now in the water in the test tube. 1ml of the solution was then added to another tube containing 9ml of sterilised biofilter water, this creates a solution containing 10% of the bacteria present and therefore a dilution of 10-1 (See Fig. 2).This can be repeated as many times as necessary to dilute the solution to the desired level – every time its repeated dilutes the solution by ten-fold. The method used was the same method described by Goldman & Green (2008).


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Bacterial Culture

Tryptic soy agar (TSA) was used as the growth media on which the bacteria was cultured on. TSA is a general use agar that is effective at growing a large number of bacteria (Matsuoka et al. 2013; Rurenga et al. 2013). Many studies have used TSA as an effective culture medium in aquatic and wastewater based studies (Altinok et al. 2007; Gerrity et al. 2012; Aguiler et al. 2013; Del’duca et al. 2013). The TSA was made up using the manufacturer’s instructions which was by mixing 40g of the TSA powder with 1 litre of distilled water. A stirring hotplate was used to mix the agar (See Fig. 3). Once the agar was mixed it was sterilised in the autoclave to ensure there were no contaminants. It was then plated up into petri dishes and allowed to cool and solidify. 3 agar plates were incubated with no solution added to test for contamination – all 3 plates produced no bacteria meaning they were sterile.


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10ml Stock

Solution

9ml 9ml

1ml 1ml1ml

Dilution

10-1 10-2 10-3

Figure 2: Basic Method of Serial Dilution used for the Experiment

9ml

Figure 3: Stirring hotplates mixing the agar

Once the agar had solidified it was ready to be used to culture bacteria. 0.1ml from each stock solution and serial dilution was pipetted onto agar plates. 0.1ml is the same as what Golubev (2013) recommends as it will not flood the plate, can be easily spread and gives a simple dilution factor to work with. The 0.1ml had to be factored in – for example 0.1ml pipetted onto a plate from a solution with a dilution factor of 10-1 would give you an indication of 0.001% of total bacteria present. Once on the agar plate the solution was spread as evenly as possible using a sterilised lawn spreading tool. Other studies have used lawn spreading techniques to help with bacterial colony counting (Sarkar et al. 2015).

Once all plates had the solution added and were labelled correctly they needed to be incubated in order to grow the bacteria. The plates were placed in an incubator upside down. The reason the plates are placed upside is to stop condensation dripping on the agar and effect microbial growth (Estridge & Reynolds, 2011; Pattengale, 2013; Simmers et al. 2013). The plates were placed in the incubator at 23°C (the same temperature as the catfish system) for a period of 48 hours. Bacteria will normally grow within the first 24 hours of incubation (Estridge & Reynolds, 2011; Barer, 2015). A study on aquatic bacteria by Pimpliskar & Jadhav (2014) found that bacterial growth peaked at 48 hours.

Bacterial Counting

After 48 hours in the incubator the colony forming units (C.F.U.) on each plate needed to be counted. At this point human error could have occurred with miscounting (Geissman, 2013). To minimise human error as much as possible a click-counter device and a pen were used to count the C.F.U. on each plate. There is still potential for human error with a click-counter but by using the pen to mark counted units and using the backlight to highlight units that risk is minimised.

Aseptic Technique

Aseptic technique was very important throughout the experiment, aseptic technique is important to minimise the risk of contamination and therefore unreliable results (Harrigan & McCance, 2014). To maintain sterility all procedures that were at risk to contamination (Serial dilutions, plating of agar etc.) were carried out in sterile fume cupboard. All equipment that was used were sterilised via an autoclave or were single use items. For example all pipette tips were single use and were applied to pipettes without any actual handling.

Results

As expected, the chemical disinfectants where the most efficient method of disinfection – with stronger solutions performing better. Air drying performed poorly. It can be assumed that the results are fairly accurate as the replicates are all generally similar with the exception of 2 results which are presumed to be contaminated (See Table 1 & Table 2). All control results have been excluded from the results section as they were all uncountable down to the dilution 0.00001 with vast quantities of C.F.U. present.


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Table 1: Table showing the Colony forming units (C.F.U.) present for each piece of treated net at different dilutions

Treatment Average C.F.U per 1cm2 net

Control (untreated net) >10000000

Virkon® Aquatic 1:100 163

Virkon® Aquatic 1:200 280

STERI-7 1:10 43

STERI-7 1:50 77

Air Dry 115300

The probability plot for all Virkon Aquatic and STERI-7 Xtra solutions at 0.01 dilution shows the data falls within the bounds of natural variance and all have a P-value >0.05 meaning the data is parametric (See Fig. 3). The probability plots for all other dilutions show the same results (See Appendix).

Figure 4: Probability plots for Virkon AquaticAquaticAquatic1:100 (P=0.609), Virkon AquaticAquaticAquatic1:200 (P=0.091), STERI-7 Xtra 1:10 (P=0.200) and STERI-7 Xtra 1:50 (P=0.596)


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At all dilution levels the test for equal variances had a P-value >0.05 meaning the data was equally varied (See Appendix).

A one-way anova shows that there is significant difference between the STERI-7 Xtra Xtra solutions and the Virkon Aquatic with a P-Value of 0.004 (>0.05) (See Fig. 5). The tukey comparison showed that there was a significant difference between the Virkon Aquatic 1:200 solution and both of the STERI-7 Xtra solutions (See Fig. 6).

Figure 5: Analysis of Variance with Virkon Aquatic 1:100, Virkon Aquatic 1:200, STERI-7 Xtra 1:10 and STERI-7 Xtra 1:50 at 0.01 dilution (P= 0.004)

Figure 6: Tukey Test comparison for STERI-7 and Virkon Aquatic solutions at 0.01 dilution


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The anova and tukey test for the STERI-7 and Virkon Aquatic solutions at 0.0001 dilution rate showed the same results as 0.0001 with a significant difference between Virkon Aquatic a 1:200 solution and STERI-7 Xtra at a 1:10 solution (see appendix).

Figure 7: Analysis of Variance between Virkon Aquatic 1:100, Virkon Aquatic 1:200, STERI-7 Xtra 1:10 and STERI-7 Xtra 1:50 at 0.001 dilutions (P=0.019)

Figure 8: Tukey test of Virkon Aquatic and STERI-7 Xtra solutions at 0.001 dilutions


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An analysis of variance with STERI-7 Xtra 1:10 and 1:50 concentrations, Virkon Aquatic 1:100 and 1:200 concentrations and air drying shows that air drying produced significantly different results to everything else (See Fig. 9).

Figure 9: Analysis of Variance between Virkon Aquatic1:100, Virkon Aquatic1:200, STERI-7 Xtra 1:10, STERI-7 Xtra 1:50 and Air Drying at 0.0001 dilution (P=0.002).

A visual representation of the average Colony forming units per 10cm2 piece of net after each treatment (Calculated using the results from 0.01 dilution - with the assumption that 0.01 dilution will be the most accurate as it has been tampered with least) shows that STERI-7 Xtra at a 1:10 concentration was most effective, then STERI-7 Xtra at a 1:50 concentration, then Virkon Aquatic at a 1:100 solution - Virkon® Aquatic at a 1:200 concentration was least effective (See Fig. 10).

Figure 10: Bar chart showing the average C.F.U. on 10cm2 pieces of net after treatment with 1:100, Virkon Aquatic 1:200, STERI-7 Xtra 1:10, STERI-7 Xtra 1:50


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Although all control plates were uncountable and so were most off the air drying samples, a visual inspection showed that there was still far less contamination on the air dried samples than on the control samples. It looks as if 48 hours air drying removes as much moisture as 24 hours at 100°C in a Kiln (See Table 6).

Wet Weight (g) Dried Weight (g) Moisture Removed (g)

Air Dry 1.66 0.8963 0.7637

Air Dry 1.78 0.9068 0.8732

Air Dry 1.82 0.9522 0.8678

Kiln Dry 1.79 0.9055 0.8845

Kiln Dry 1.72 0.8406 0.8794

Kiln Dry 1.70 0.8129 0.8871

Table 6: The wet weight, dried weight and total moisture removed from pieces of net via kiln drying and air drying

A test for equal variances showed that the wet weights of all the nets were not significantly different (See Appendix). An ANOVA found there was no significant difference between the moisture removed by both air drying and kiln drying (See appendix).

Discussion

The fact that all of the control samples were uncountable, even down to a dilution of 0.00001, shows that all of the disinfection methods trialled had an effect as they produced countable levels of colony forming units. The control may have been of more use if it could have been counted – this would have made it possible to calculate the percent of bacteria each treatment eradicated. If this experiment was to be replicated then it would be advisable to further dilute the control until levels of bacteria that could be counted were achieved.

A simple eyeball analysis of the results seems to show that STERI-7 Xtra was more effective at eradicating bacteria than Virkon® Aquatic was, and the stronger solutions of each product were more effective – as was expected. This trend dissipates and is not shown as well with the weaker dilutions. A study by Knaide et al. 2012 found that the accuracy of serial dilutions decreases with each dilution – so the 0.01 results can be considered the most accurate. Looking at the data also shows that air drying did work as a method of disinfection, but it was nowhere near as effective as the chemical disinfectants.

It can be seen from the ANOVA and tukey test for all the chemical treatments that there was a significant difference present in the results from the 0.01 dilution. The significant difference is shown to be between the Virkon® Aquatic 1:200 treated samples compared to both STERI-7 Xtra treated Samples – this means that the Virkon® Aquatic concentration recommended for general use is significantly less effective than the STERI-7 Xtra at a general use and high power concentration. At 0.001 and 0.0001 dilutions the significant difference was only between the Virkon® Aquatic 1:200 treated samples and the STERI-7 Xtra 1:10 treated


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samples – suggesting that the STERI-7 Xtra high power solution is significantly more effective than the Virkon® Aquatic general use solution. It was expected that the higher power solutions would be more effective at eradicating bacteria but the results showed that there wasn’t a significant difference between the recommended general use strength of Virkon® Aquatic and the recommended high power dosages of Virkon® Aquatic and STERI-7 Xtra. This suggests that STERI-7 Xtra at its high power dosage may be the most powerful of the chemical disinfectants - a more in depth study would need to be carried out to confirm this. A study with more replicates would have led to more data points so more accurate and reliable statistical tests could have been carried out and there would have been less risk of type 1 and type 2 errors. The statistical power is directly affected by how many data points are present, the fewer data points there are the higher risk of type 1 and type 2 errors occurring (Yi, 2013; Wang et al. 2015).

A very similar study carried out by Jussila et al. (2014) found that at a concentration of 1:200 Virkon® Aquatic was very effective at killing pathogenic microbes on a porous material used to represent net mesh – it was more effective than the other 3 disinfectants it was being compared with: proxitane, wofasteril and hydrogen peroxide. It was also found to be very effective in this study as bacteria was extremely abundant with no treatment and Virkon® Aquatic took it down to minimal levels. Although there is no directly comparable data from other literature, as eradication of total C.F.U. with Virkon Aquatic has not been published elsewhere, several papers are in agreement that Virkon® Aquatic is very effective as a disinfectant (Stockton, 2011; Mainous et al. 2012; Walker et al. 2013).

There is no literature available on the use of STERI-7 Xtra as a disinfectant for aquatic bacteria, meaning this study has formed a basis on its viability as an aquatic based product. The results suggest that it is very viable for STERI-7 Xtra to be brought to the aquatic market – it proved to be a very effective disinfectant and shows potential to be a better disinfectant than Virkon® Aquatic although further research would need to be carried out in order to determine that. Exposure to high power 1:10 concentration STERI-7 Xtra actually led to total eradication of bacteria on some of the plates – 100% disinfection rate is a sign of very efficient disinfection (Moreto et al. 2013). If this test was repeated then testing more concentrations of STERI-7 Xtra should be considered to try and establish a minimum concentration in which it loses its efficiency.

There were two noticeable errors in the results. A 0.0001 dilution that had been treated with STERI-7 at a concentration of 1:10 and grew an uncountable amount of bacteria. As this was the net treated with the high power STERI-7 solution and the sample was at its weakest dilution it can assumed this was an error caused by a contaminant. The other 2 samples of the same parameter contained 0 C.F.U which acts as further evidence that the uncountable plate was contaminated. The other error was an uncountable plate from a net treated with Virkon® Aquatic at a 1:200 strength – the dilution factor was 0.001. The other plates of the same parameter yielded 9 and 15 C.F.U, so it seems likely that if there were significantly more C.F.U to an uncountable level then it is an error via contamination. Errors may have occurred due to poor aseptic technique, more reliable results may have been obtained if it was carried out by someone formally trained and competent in aseptic techniques.

From an economic view point and to fully establish which product is the most cost effective method of disinfection a further study would have to be undertaken to determine the potential weakest solution of each product that still worked effectively. Air drying is obviously the most cost effective method of disinfection as it doesn’t cost anything, but it is much less effective than chemical disinfection. A paper by Hillock & Costello (2013) also found air drying to be the most cost effective method of disinfection but


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recommended the use of chemical disinfectants as the efficiency they offer makes them worth the extra expense. The costs involved in biosecurity in aquaculture are not a priority due to the huge potential risks affiliated with poor biosecurity practise (Cliff & Campbell, 2012).

The results show that air drying is definitely not the “best” method of disinfection of angling gear as some organizations state (Environment Agency, 2014; Inland Fisheries Ireland, 2014). All of the air dried samples at the 0.01 dilution produced uncountable results, although they were uncountable you could see they were not as heavily infected as the control plates and therefore did have an effect. An ANOVA of all the results at 0.0001 dilution showed that air drying produced significantly different results to all the other treatments, with it being significantly less effective. No significant difference was found in the weight removed via air drying and kiln drying suggesting that the air drying removed most of the moisture, although it can be assumed the kiln dried net would have been fully sterilized via the heat exposure. Air drying may have potentially been more effective if it was left for longer as aquatic pathogens will eventually die off in dry conditions (Pradhan et al. 2011).

Conclusion

In conclusion STERI-7 Xtra has huge potential to be introduced into the angling and aquaculture markets for the use of disinfection. This study has proved that it acts as an effective disinfectant against aquatic bacteria. In its high power form it has the potential to be a more effective disinfectant than the current market leader: Virkon® Aquatic. This study has formed a basis on the efficiency of Ster-7 as an aquatic disinfectant but further study could expand on its efficiency against specific pathogenic organisms and get a better comparison against other disinfectants such as Virkon® Aquatic. Virkon® Aquatic also performed very well as a disinfectant – this was expected due to its reputation in the market. The only significant difference was between general use Virkon® Aquatic (1:200) and high power STERI-7 Xtra (1:10). Virkon® Aquatic has a slight cost advantage over STERI-7 Xtra but another study would have to be undertaken to study effective concentrations and longevity of each product to accurately determine their cost effectiveness. Air drying is not as effective as is suggested by some organizations, it was the least effective of the three methods of disinfection trialed in this study. Air drying was significantly less effective than all forms chemical disinfection trialed. More research should be carried out in air drying as it does work as a form of disinfection and may be the only method anglers have access to. In conclusion the alternative hypothesis is accepted as there was a significant difference in the efficiency of the three disinfection methods – with more research STERI-7 Xtra could become a very effective an well utilized disinfectant in all aspects of aquatic industry.


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