Recirculating systems are the feedlot systems of aquaculture

The proposed project’s goals are to 1 to examine several research and commercial recirculating systems to determine if bacteria known to be harmful to human are present in numbers that w

DESCRIPTION

Recirculating systems are the feedlot systems of aquaculture Like their terrestrial

counterparts, they have certain economic advantages and environmental disadvantages Animals are concentrated together for ease of handling and harvest but at the same time the concentrated waste stream can be difficult to manage Another analogous component of aquatic and terrestrial intensive production operations is the use of bacteria to treat the wastes from the animals However, whereas the terrestrial systems can utilize compost or soil-based systems physically isolated from the animals, an aquatic system recycles water through the bacterial colonies and returns it to the fish This provides a path for rapid spread of potential pathogens In addition, large numbers of beneficial nitrifying and heterotrophic bacteria, as well as potential pathogens, bacteria are removed from the system when solids are drained or backwashed from the filters of recirculating aquaculture units

The proposed project’s goals are to 1) to examine several research and commercial

recirculating systems to determine if bacteria known to be harmful to human are present in numbers that would be of concern to consumers; 2) to identify the organisms that constitute the majority of the fecal coliforms found in recirculating systems; 3) to determine if discharge requirements which focus on total coliform counts are an appropriate standard for aquaculture effluents; 4) develop an extension program which will provide research based information to operators of recirculating aquaculture systems to minimize the potential for bacterial

contamination to production staff, handlers, processors, consumers and the environment

An interdisciplinary team with expertise in aquaculture production, water treatment, human pathogen identification and aquaculture extension will work with industry members to conduct the research, report the results and work with industry to implement appropriate operational changes Sampling will be conducted at commercial and research facilities in Texas, New Mexico and Arizona Bacterial enumeration, isolations and identification will be conducted in the Class 2 facilities in the Veterinary/Microbiology complex at the University of Arizona Preparation of operating recommendations to minimize the potential of pathogens entering the food supply will be developed by a team of research, extension and industry professionals These recommendations will be prepared in conjunction with a separate project on solids reduction and effluent management from aquaculture facilities

PERFORMANCE SITE(S)

Environmental Research Lab, University of Arizona, Tucson, AZ 85706

Soil, Water and Environmental Science Dept., University of Arizona, Tucson, AZ 85721

Simaron Fish Farm, Route 1, Hempstead, TX 77445

New Mexico State University, Las Cruces, NM 88003

KEY PERSONNEL

Kevin Fitzsimmons Environmental Research Lab, U of AZ Co-Principal Investigator, Aquaculture

Charles Gerba Soil, Water and Environmental Science, Uof AZ Co-P I., Aquatic Microbiology Casey McKeon Environmental Research Lab, U of AZ Head Technician

Patricia Rusin Soil, Water and Environmental Science, Uof AZ Research Scientist, Human pathogens Charles Sims Simaron Fish Farm Farm operator / owner

Walter Zachritz New Mexico State University Scientist, Aquaculture filters

BACKGROUND AND SIGNIFICANCE

Demand for fish and fishery products has increased rapidly due to changing eating habits, change in demographics of the American population, and the overall increase in population At the same time the supply of wild caught fish and fishery products has been stable and in many cases decreased, due to over-fishing, pollution, loss of wetlands and reservation of certain stocks for sport fishing The price of seafood in many cases was also increased rapidly, not only for the reasons listed above, but also because the costs of fishing have increased Dock space and processing facilities in many ports have been redeveloped into upscale high rent locations Insurance costs have risen for what is considered to be the first or second most dangerous occupation, depending on whether you consider just deaths or deaths and injuries Aquaculture and domestication of aquatic animals is the logical evolution from the hunting and gathering of seafood that we have conducted in the past Aquaculture is frequently cited to be the fastest growing sector of agriculture Within the industry there is an evolution to more intensive production of fish and other aquatic animals The epitome of this intensification is the controlled environment recirculating system Commercial scale systems are now used to rear tilapia, catfish, trout, striped bass, yellow perch, flounder, oysters, clams, crayfish, blue crabs and a host of other species for human consumption In addition to the commercial systems, recirculating systems are used in public aquaria, in research labs and increasingly in high school agriculture education programs

Virtually all of these systems incorporate some level of biological filtration to treat the

metabolic wastes of the fish and shellfish There are a variety of forms that biological filters can take including fixed media beds, fluidized media, rotating contactors, vertical screens, and even in-situ activated sludge The common feature in all of these is that the operator wishes to encourage bacterial activity that will oxidize the nitrogenous waste of the aquatic animals, which is normally excreted as ammonia, and oxidize the suspended solids that are missed in the physical removal portion of the water treatment system The nitrification tasks are

accomplished by Nitrosomonas and Nitrobacter The treatment of the vast array of organic

compounds in the system resulting from fecal waste, uneaten food, mortalities, mucous, and all manner of detrital matter that is not physically filtered is left to a host of heterotrophic and chemotrophic bacteria

One of the goals of a biological filtration unit within a recirculating aquaculture system is to maximize the nitrifying bacterial biomass which will in turn more effectively convert ammonia and nitrites to nitrates The other beneficial bacteria that are encouraged are the heterotrophs and chemotrophs that decompose unwanted organic waste materials that would otherwise pollute the production tanks In addition to the proliferation of beneficial bacteria, the number

of pathogenic microorganisms may also increase Bacteria have been identified in

recirculating aquaculture systems that are considered human pathogens, such as fecal coliforms

including Escherichia coli (Ogbondeminu, 1993; Pullela et al., 1998), Clostridium botulinim (Pullela et al., 1998), Pseudomonas species (Nedoluha and Westhoff, 1997), Aeromonas

hydrophila (Nedoluha and Westhoff, 1997) and Salmonella species (Ogbondeminu, 1993) to

name a few Some typical fish pathogens have also been known to cause illness in humans The microflora of the fish gills, skin, and digestive tract have been shown to reflect the microflora

of the water they inhabit and may also pose a threat to humans (Nedoluha and Westhoff, 1997; Ogbondeminu, 1993) A related concern is the linkage of recirculating systems to hydroponic vegetable production Water carrying human pathogens could be directly transferred to readily consumed vegetative matter

HACCP - Hazard Analysis at Critical Control Points has been required for all seafood

processed in the US since December of 1997 One of the tenants of HACCP is to develop a plan to maintain safety in the food product before, during and after processing The presence of pathogens in recirculating systems would be an important characteristic to be addressed in a HACCP plan Obviously, the potential for bacterial contamination within the production system itself would be a critical control point before processing Libey (1996) specifically included analysis of the microbiological flora in a HACCP example plan for recirculating systems

Experiments by Buras et al (1985) have determined threshold levels of bacteria in water that can cause contamination in fish For example, when carp were reared in water containing

Salmonella at concentrations of approximately 105 /ml, the muscle tissue became contaminated,

possibly becoming a health hazard to fish consumers Salmonella and Escherichia coli are

enteric bacteria that are potential human pathogens when contaminated fish are handled or

consumed (Ogbondeminu, 1993) Salmonella has been reported in cultured fish (Reilly and Käferstein, 1997; D’Aoust, 1994) Salmonella and E coli are of particular concern when

human waste products are used as a nutrient source for fish and there is an extensive literature

examining this aspect (D’Aoust, 1994) E.coli, a fecal coliform, has been found in fish from

recirculating and non recirculating aquaculture systems that have been fed commercial diets

(Pullela et al., 1998) In an experiment by Del Rio-Rodriguez et al., (1997) E coli was found

to establish and grow in rainbow trout This indicates that the fish may be increasing the level

of E coli in the water due to the excretion of the organisms into the production tank.

Other fish pathogens may also be linked to human illness Aeromonas hydrophila and

Plesiomonas shigelloides are both constituents of the microflora of natural waters, but are

considered opportunistic pathogens of stressed fish (Reilly and Käferstein, 1997; Nedoluha and

Westhoff, 1995) There is evidence indicating that A hydrophila and P shigelloides can cause human gastroenteritis (Nedoluha and Westhoff, 1995; Reilly and Käferstein, 1997) and A

hydrophila can possibly produce fatal septicemia in immuno-compromised individuals

(Palumbo et al., 1985; Ward 1989)

In recent years, there has been an increasing concern over Listeria monocytogenes in the aquaculture industry (Garland, 1995) L monocytogenes causes human listeriosis (Fuchs and

Reilly, 1992) and has been isolated from a wide variety of food products including dairy products and seafood (Fuchs and Reilly, 1992; Garland 1995) It is a highly adaptable

microorganism, surviving in many different environments (Fuchs and Reilly, 1992)

Consequently, it is a possible contaminant in aquaculture systems (Ward, 1989)

Flick (1996) reports the presence of Vibrio fluvialis, a human pathogen, from a hybrid striped

bass facility This is especially important considering the large percentage of marketable stripers produced in recirculating systems now The presence of potentially pathogenic

Mycobacterium spp in intensive recirculating aquaculture systems has been noted by Smith

(1996) and in extensive aquaculture by Chen et al (1997a,b)

Strep infections are another instance where fish pathogens may be hazardous to handlers and

consumers Streptococcus iniae is a known fish pathogen that has recently been linked to illness in humans (Getchell, 1998; Weinstein et al., 1996) In 1995 S iniae was determined as

the causative agent of several cases of human illness in Canada (Weinstein et al., 1996;

Getchell, 1998) Most of the infections were a result of injuries obtained while preparing farm raised tilapia and striped bass There have been two recent outbreaks among tilapia in

recirculating systems in the United States without human illness and because no

chemotherapeutic measures have been approved for treatment, this organism can severely

impact fish farmers (Getchell, 1998) Shoemaker and Klesius (1997) discuss the identification

and several methods for partial control of S iniae in recirculating systems.

Considering the number of potential and demonstrated pathogens reported from aquaculture and more specifically recirculating aquaculture, further study seems to be warranted The amount of fish, crabs, oysters and other aquatic animals being grown or stored alive in

recirculating systems is rapidly increasing and the threat to consumers is real The goals of the project proposed here will be to 1) to examine several research and commercial recirculating systems to determine if bacteria known to be harmful to humans are present in numbers that would be of concern to consumers; 2) to identify the organisms that constitute the majority of the fecal coliforms found in recirculating systems; 3) to determine if discharge requirements which focus on total coliform counts are an appropriate standard for aquaculture effluents; 4) develop an extension program which will provide research based information to operators of recirculating aquaculture systems to minimize the potential for bacterial contamination to production staff, handlers, processors and consumers The results and conclusions from the project will be submitted to public scrutiny at professional meetings and by submission to peer-review in technical journals The findings to be extended to the industry will be provided in the context of HACCP planning for the producers and processors

The project will examine several species in recirculating systems, but it would be impossible, within the scope of this effort, to sample all of the species now in production Tilapia are the most commonly produced fish in recirculating aquaculture systems and we will sample several

of these The tilapia production system will serve as the model for development of the outline for how industry can address this issue

We recognize that this a matter of great importance to the industry and will need to be handled

in a professional manner The industry wants to provide the safest and highest quality product possible to its consumers All responsible producers would rather destroy animals and take a financial loss rather than risk making anyone sick At the same time no one wants to unduly frighten customers with exaggerated or premature claims of contamination of the food supply This project will strive to conduct the research and present the results in a constructive and professional manner that will improve the safety for the consumer and improve the ability of the producer to provide the product that the consumer wants

PRELIMINARY STUDIES

The University of Arizona maintains several freshwater and marine recirculating systems for aquaculture research These systems utilize biological filtration units as a part of the water treatment process Past studies (Skeen et al., 1997; Piedrahita et al., 1996) have experimented with several styles of biofilters including up-flow and down-flow sand and gravel bed filters, rotating biological contactors, trickling beds using plastic media, fluidized sand and plastic bead media and constructed wetlands A protocol developed by Piedrahita et al (1996) was developed to allow cross comparison of various filter designs Skeen et al (1997) reported the variations in efficiencies between two floating bead biofilters in a recirculating system

The common element in all of the systems tested has been the use of biofilms of bacteria and algae as part of the treatment process Biofilters, especially the floating media bed filters, are now being incorporated into recirculating aquaculture systems all over the US The industry recognizes the beneficial bacteria in these filters but is also aware of the potential hazards presented by pathogenic bacteria

In a review of diseases found in tilapia recirculating systems at the University of Arizona (Lightner et al., 1988) reported the presence of bacterial infections in tilapia, but none were determined to be potential human pathogens A study (McKeon, 1998) was performed at the University of Arizona Environmental Research Laboratory to determine the level of fecal coliforms in recirculating fish production systems One of the systems tested was a 200 cubic meter system consisting of 100 fiberglass tanks treated by a settling tank and a vertical screen biofilter The system is stocked with several varieties of tilapia that are fed a variety of

commercial and experimental diets Samples from this system were found to contain total coliform levels as high as 104 CFU/100 ml and fecal coliforms as high as 103 CFU/100 ml The second system is used to rear catfish and grass carp in a 25 cubic meter raceway The water treatment systems includes a settling tank, a rotating biological contactor and a second

biological filtration unit using multiple vertical screens This system was also found to harbor total coliform levels as high as 104 CFU/100 ml and fecal coliforms as high as 103 CFU/100 ml These levels indicate the possible presence of human enteric pathogens that could cause illness

in fish handlers and consumers if proper precautions are not observed The source of the coliforms is unknown Additional studies are underway to identify the fecal coliforms

discovered in the systems

RESEARCH DESIGN AND METHODS

Phase 1 - Researchers from the University of Arizona will examine the biofilter and fish from the recirculating systems at the Environmental Research Lab of the UA in the first phase Three separate systems are currently in operation that will be available for sampling One system is inside a greenhouse and consists of 16 fiberglass production tanks stocked with either

tilapia (Oreochromis niloticus) or channel catfish (Ictalurus punctatus) The treatment system

consists of a settling tank followed by one of three biofiltration units The units are

commercial floating media models (A-1 Aquaculture and Armant) A second system to be tested consists of 100 fiberglass production tanks, also in a greenhouse, stocked with tilapia and treated by a settling tank and a multiple vertical screen biofilter (Red Ewald , Inc.) A third system to be tested consists of one production raceway treated with a settling tank, a rotating biological contactor, and multiple screen biofilter The third system is entirely enclosed inside

a windowless prefabricated building and is stocked with triploid grass carp (Ctenopharyngodon

idella).

Samples will be collected from the water in the production tanks, from the biofilter media and from the skin and fillets of fish harvested from the production tanks Since many of the

organisms of concern require specialized media and incubation, the samples will be split in order to accommodate the testing procedures For example,

INSERT Particular methods for isolating, identifying and counting the microbes of interest Phase 2 - In the second phase of the project we will visit a research facility in New Mexico for sampling The facility at New Mexico State University has two recirculating systems which will be tested One system utilizes production tanks in a greenhouse treated with a settling tank and a floating media filter stocked with tilapia The other system, also stocked with tilapia, has a settling tank followed by a constructed wetland Samples collected at N M S U will be prepared in Las Cruces and then transferred by automobile for incubation, identification and enumeration at the University of Arizona

Phase 3 - In the third phase we will sample from commercial facilities in Texas and Arizona The facility in Texas is one of the major tilapia producers in the US with sales throughout the Central and Eastern US Samples collected at the site will be prepared and returned to Tucson for incubation, identification and enumeration Special permits for air transport of potential pathogens will be obtained A recirculating trout system will be sampled from Northern Arizona This system rears rainbow trout in a two phase system using well water fed to fiberglass tanks The filtration system uses a mix of settling tanks, fluidized and fixed media biofilters The water and fish samples will be returned to the University of Arizona labs by automobile

Phase 4 - Analysis - Statistical analysis of the data will be conducted with the use of chi square tests The Statistical Analysis System (SAS) software will be utilized to facilitate analysis Phase 5 - The findings and conclusions will be presented to the industry partners immediately should any potential pathogens be discovered at their location Should this be the case,

suggestions will be provided to the producer on how the contamination may be removed or reduced to safe levels Beyond that aspect, the results and conclusions of the research will be presented at professional meetings and submitted to technical journals for critical peer review After receiving the appropriate peer review, the findings and an outline of suggestions

regarding methods for improving the microbial safety of the recirculating systems will be developed and extended to the industry

Several of the research and commercial systems tested are rearing tilapia Tilapia will also be the most common fish tested when taking fillet and skin samples However, several other species, including catfish, grass carp and trout from recirculating systems will be tested Tilapia is the most frequently reared fish in commercial recirculating systems and will serve as the model for the operating recommendations we expect to develop around the findings

Considering the variety of potential pathogens that we anticipate may be discovered in the analysis of recirculating systems we expect that several methods of control will be required Treatment and control recommendations will be developed for each potential pathogen

identified Since bacterial release from dead individuals is frequently the most important source of infection within the fish (Kitao, 1993) rapid removal of moribund individuals would

be the most important recommendation Improved water quality and health management are also critical (Plumb, 1994) in cases where fish may be carriers In cases where the source water may be the site of introduction, pre-treatments may be appropriate Feeds are another potential source of contamination and have been shown to be capable of infecting trout with

E.coli (Del Rio-Rodriguez et al., 1997) So recommendations might include checking for feed

contamination and improving feed handling

The approved use of antibiotics in aquaculture is limited and their use within recirculating systems will be of even greater concern as the potential for misuse is heightened Careful review of which antibiotics are approved for use in particular systems and particular species will be required before recommendations are provided This is a very fluid situation with only

a few compounds approved, others under INAD’s and still others considered for extra-label use under emergency conditions

The use of compounds to treat the production water in a recirculating system is only slightly less confusing A greater number of chemicals are available for various water treatments, but how these would affect the various bacteria in biofilters is not very well understood pH, alkalinity, hardness, temperature, dissolved oxygen level, total dissolved solids, presence of copper will all affect bacterial populations The recommendation guides will address all of

these water quality interactions and how these may be adjusted to impact each potential pathogen

The competition for resources by beneficial and potentially pathogenic bacteria will be

considered There are several products now on the market that claim to favor beneficial bacteria and reduce levels of “problem “ bacteria There seems to be little technical literature

to support these claims, but that may change in the future If a sufficient scientific basis can be demonstrated these products and methods would be included in the operating suggestions CONCLUSION

There has been a rapid increase in the use of recirculating aquaculture production systems in the last few years Most of these systems utilize biological filters to treat the water in the production system These biofilters utilize large colonies of bacteria to treat the water There

is a recognized danger that by encouraging rapid growth of beneficial bacteria and degrading water quality under high production operations, bacteria that are known human pathogens may also multiply and contaminate the water and aquatic animals being reared Preliminary studies

at the University of Arizona demonstrated that total coliforms levels of 104 CFU’s/100ml and fecal coliform levels of 103 CFU’s/100ml can be found in two separate recirculating systems rearing grass carp and tilapia

This project will sample and analyze water and fish skin and fillets for bacterial contamination Samples will be collected from commercial and research facilities rearing trout, tilapia, catfish and grass carp using several varieties of biofilters Levels of total coliforms, fecal coliforms, and several bacteria known to be human pathogens will be determined for locations within the recirculating systems and the fish product The findings will be presented for peer review and evaluation at professional meetings and in the peer reviewed literature Then using the results and other available information, operating recommendations will be prepared providing

suggestions on how to best maintain and control the levels of unwanted bacteria for

recirculating systems and their products

The research team committed to this project is multi-disciplinary, from several states and includes research, extension and commercial members The members of group have worked together in the past, successfully completing research in the performance of aquaculture biofilters, water quality and microbiology of drinking water supplies

LITERATURE CITED

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Buras, N., L Duek, and S Niv 1985 Reaction of fish to microorganisms in wastewater Applied and Environmental Microbiology 50:992-994

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No NRAES-98

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