Prototype Egg Disinfection Unit

Final Report

Newfoundland Cod Broodstock Company Inc.
AIMAP-2010-N08

Table of contents

1. Project Overview
2. Available technology
3. Current egg disinfection chemicals
4. Newfoundland Cod Broodstock's egg disinfection unit

1. Project Overview

In the course of Newfoundland Cod Broodstock's involvement with aquaculture, we have had to overcome many disease challenges that are associated with broodstock fish and can be passed vertically through eggs to progeny.

Following research by the Company and others associated with the development of cod broodstock in the region, as well as other marine fish farmers, the Company was instrumental in installing ozonation equipment for egg disinfection in 2004 in the Ocean Sciences Centre hatchery facility.

The company also established the protocols for effective egg disinfection to remove the threat of viral infection by Nodovirus (VVN), which is a vertically transmitted virus present on the outside of the egg. Nodovirus had been responsible for massive cod fry mortalities in 2002. Since the introduction of these protocols in Newfoundland and elsewhere in the Maritimes and New Hampshire, cod production has remained Nodovirus free since 2004, even though in the first years Nodovirus positive parents were used in the production cycle.

This project's goal was to address the design and construction of a prototype unit “plug and play” egg disinfection unit to be used in the aquaculture industry sectors currently plagued by vertically transmitted viruses, bacteria and fungus.

There is a significant body of research that has addressed the benefits and difficulties of using ozone as a disinfectant in aquaculture. In Europe, the bans on using most chemicals make it all the more important to provide an alternative means of disinfection.

In Canada, ozone has been evaluated for disinfection of eggs of most cold water marine species that have been produced either experimentally or commercially, and since 2004 has been routinely used in Newfoundland. However the current unit is not packaged in a way that makes it easy and foolproof to use. Furthermore this work has not been transferred to warm water marine species, freshwater fish such as trout and salmon and invertebrates such as shrimp, a two million ton industry that has suffered from egg born viral diseases.

A great deal of technical knowledge is required to build, maintain and operate an ozone disinfection unit. There are many providers of ozonation equipment, companies that manufacture and supply and others, such as Atlantech engineering in Canada and Dryden Aquaculture, who have created applications with the equipment. These companies have focused on water disinfection, of primarily fresh water in either aquaculture settings or for wastewater treatment. A few companies, such as the Tropical Marine Centre, which supplies marine aquarium fish worldwide, supplies ozonation equipment for marine and fresh water, aquarium systems. Their marine systems contain Instant Ocean, in which the salts do not have halides such as bromine, unlike natural seawater.  As with the other two examples the company supplies equipment for water disinfection.

Newfoundland Cod Broodstock's ozone disinfection unit's design is focused specifically for egg disinfection. The product is scaled to deal with egg volumes up to two litres and disinfection durations of up to two minutes. The unit is also designed to be simple to use and complete with all the equipment mounted in such a way that it will be "plug & play".

2. Available technology

The following information was compiled through various literatures searches and demonstrates the need for a commercially available ozone disinfection system for the aquaculture industry. By completion of the first "Newfoundland made" unit, Newfoundland Cod Broodstock Company is poised for quick market entry to hatcheries in Canada and abroad.  While most organizations realize the importance of ozonation as a disinfection method, "make shift" units are pulled together using various equipment, and often require a high skill level to operate and maintain the systems.  NCBC's ozone prototype keeps the end user in mind and is specifically built so that it is easy to use for everyone.

Ozone Disinfection of Various Egg Species

Article #1 Summary:

• Haddock eggs exposed to ozone concentration of 3.0 (±0.3) mg/l of total residual oxidants (TRO) of Cl2 for 3.3 to 6.7 minutes was effective for disinfection.
• Ozone dose was calculated as TRO exposure time in minutes, and reported as CT units.
• Egg survival decreased as time of exposure to ozone increased.
• Doses of ozone up to 30 CT units showed no significant differences in survival compared to controls.
• The optimal dosage was 20 CT units (using an ozone concentration of 3.0mg/l, that equates to maximum 6.7 minute exposure time).
• Overall recommendation for haddock was 3.0 mg/l TRO for 3.3 to 6.7 minutes (10 to 20 CT units).

Article #2 Ozone System:

• Bench scale ozonation system using 200mg/hr adjustable corona discharge ozone generator.  Ozone was pumped into a 500ml gas-washing bottle modified by the addition of spigots at the top and bottom and dispersed into the water column via an air stone. Seawater was pumped into the washing bottle through the bottom spigot using a peristaltic pump, where is comes into contact with ozone. Water was continuously pumped through the system.  A magnetic stir bar in the beaker ensured an even mixing of water and ozone.   Oxidative power of the ozonated seawater was determined using the DPD colorimetric method using a spectrophotometer reporting units of TRO in mg/l as Cl2.

Summary:

• Disinfection may delay or reduce hatching.
• Nodavirus are shed from broodstock during spawning- probably adhering to the egg surface and infecting offspring at hatching.
• Ozonated seawater effectively inactivates bacteria, however the oxidants formed may also react with compounds in the eggshell (chorion), altering its functional properties and possibly influencing hatchability.
• Eggs were disinfected two days prior to hatching.
• Oxadative power of the ozonated seawater was measured by iodometry according to the method of Franson (1989) and the concentration of total residual oxidant (TRO) was expressed as mg Cl2/l. The oxidative power was converted to mg O3/l).
• Four concentrations of ozone per litter (ranging from 0.2 to 10 mg O3/l) were combined with four different exposure times (0.5, 1,3 and 5 minutes).
• Hatching rates fell as exposure times increased.
• Larvae hatched from ozone treated eggs displayed normal patterns of swimming and development.
• Different levels of ozone had little effect on hatchability, only the highest ozone exposures (4.6 O3/l for 3 minutes and 4.6 O3/l for 5 minutes) showed significant detrimental effects on hatching.

Article #3 Ozone System:

• Seawater was held in a reaction chamber connected to a circulation pump.  Ozone gas, produced by a commercial ozone generator by high voltage corona discharge, was mixed with the seawater using a gas injector connected to the seawater circulation system.

Summary:

• Gluteraldehyde has the ability to kill all pathogens (bacteria, fungus, viruses, and fungal and bacterial spores).  The main concern if the toxicity to humans.
• Ozone generates oxidants (bromine, bromaine, hypobromous acid and hypobromide).
• Concentration of 0.1 mg/1 for 2.5 minutes was required to inactivate striped jack nervous necrosis virus (a nodavirus).
• 0.5mg/l for 3 minutes resulted in reduced hatch rates and decreased survival.
• Atlantic halibut eggs exposed >4mg/l for 1 minute displayed a negative effect on hatching.
• Five different treatments were assessed using freshly spawned Atlantic cod eggs:
  
  

1. Perosan at 3.5 ml/l for 1 minute,
2. Glutaralgehyde at 400 ppm for 10 minutes,
3. Ozone at 800-900 mV for 30 seconds,
4. Ozone at 800-900 mV for 3 minutes, and
5. No disinfectant.
   
   

• Monitoring provides a rapid and single value assessment ORP of the disinfection potential of the water.
• ORP readings are not a linear function of ozone concentrations in water but varies with organic load, pH and temperature.
• 800-900 mV ozonated water for 30 sec was not disinfected (evident by turbidity testing indicating bacterial growth).
• Eggs treated for 1 minute and 3 minutes did disinfect the water (tested by turbidity after 15 days incubation using tryptic soy broth).
• No effect of disinfectant on hatchability.

Article #4 Ozone system:

• Two 30 gal plastic totes connected by PVC pipes. A small pump was attached to the bottom chamber and water was pumped to the upper chamber. An AquaZone Ozone generator (200mg/hr) attached to a tank of pure oxygen was used to convert the oxygen to ozone.  Ozone was injected into the water via a venture injection.  Ozone levels were checked using both a test kit and an ORP meter. The amount of ozone in the water was determined by measuring the amount of ozone in the water using a Lamotte Smart Colorimeter and reading the corresponding mV with a Pinpoint ORP meter.  In these trials, 1 mg/l ozone corresponded to around 800-900 mV ORP.

3. Current egg disinfection chemicals

Under current Health Canada Veterinary Drug Directorate (HC-VDD) regulations for fish egg disinfection, the only approved chemicals for use in Canada are 1) Parasite-S or Formalin-R, and 2) Perox-Aid™ (hydrogen peroxide).  The first product is formalin based, while the second product is an antifungal agent for use on fish eggs.

4. Newfoundland Cod Broodstock's egg disinfection unit

The egg disinfection unit was engineered and manufactured using local people and products where possible.  The unit consists of:

• A platform (a fume hood with all the systems integrated)
• Water system
• Ozone generator
• Ozone monitoring system
• Oxygen manifold and delivery system
• PLC Controls
• All electrical systems