Study Number: AEH-12-RASHP-01; INAD Number: 10-023

Literature Review of the Potential Effects of Hydrogen 

Peroxide on Nitrogen Oxidation Efficiency of the Biofilters 

of Recirculating Aquaculture Systems (RAS) for 

Freshwater Finfish

By Kim T. Fredricks

Open-File Report 2015–1096

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

SALLY JEWELL, Secretary

U.S. Geological Survey

Suzette M. Kimball, Acting Director

U.S. Geological Survey, Reston, Virginia: 2015

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Suggested citation:

Fredricks, K.T., 2015, Literature review of the potential effects of hydrogen peroxide on nitrogen oxidation efficiency 

of the biofilters of recirculating aquaculture systems (RAS) for freshwater finfish: 

U.S. Geological Survey Open-File Report 2015–1096, 21 p., http://dx.doi.org/10.3133/ofr20151096. 

ISSN 2331-1258 (online)
iii

Acknowledgments

This literature review was funded through an interagency agreement with the U.S. Geological Survey Upper 

Midwest Environmental Sciences Center (UMESC) and the U.S. Food and Drug Administration (FDA) 

(number 224-12-7017) in response to the FDA Funding Opportunity Announcement RFA-FD-12-003. The author

also acknowledges Lisa Hein, UMESC librarian, for her work in conducting the searches and obtaining the papers. 

 
 
v 

Contents

Acknowledgments.......................................................................................................................................................iii

Contents ..................................................................................................................................................................... v

Summary .................................................................................................................................................................... 1

I. Overview of Aquaculture.......................................................................................................................................... 1

II. Recirculating Aquaculture Systems (RAS).............................................................................................................. 2

A. Overview of RAS ................................................................................................................................................ 2

B. Advantages of RAS ............................................................................................................................................ 3

C. Challenges ......................................................................................................................................................... 4

D. Current Work with Biofilters ................................................................................................................................ 5

E. Bacteria That Process Nitrogen.......................................................................................................................... 9

III. Diseases.............................................................................................................................................................. 10

IV. Use of Hydrogen Peroxide in RAS ...................................................................................................................... 10

A. Current Label.................................................................................................................................................... 10

B. Application of Hydrogen Peroxide in RAS–Effects on Biofilter Bacteria............................................................ 10

V. Conclusions.......................................................................................................................................................... 15

References Cited...................................................................................................................................................... 15

Tables

Table 1. Summary of papers that tested the effects of hydrogen peroxide on freshwater recirculating 

aquaculture systems................................................................................................................................................ 12
vi 

Conversion Factors

International System of Units to Inch/Pound

Multiply By To obtain

Area

square meter (m2) 10.7639 square foot (ft2)

square meter (m2) 1.19599 square yard (yd2)

Length

Micrometer (μm) 3.937 x 10-5 inch (in.)

Volume

liter (L) 33.814 ounce, fluid (fl. oz)

liter (L) 2.11338 pint (pt)

liter (L) 1.05669 quart (qt)

liter (L) 0.264172 gallon (gal)

cubic meter (m3) 264.172 gallon (gal)

Flow rate

centimeters per second (cm/s) 0.0328 foot per second (ft/s)

Mass

milligram (mg) 3.5274 x 10-5 ounce, avoirdupois (oz)

kilogram (kg) 2.20462 pound, avoirdupois (lb)

metric ton (tonnes) 0.0984207 pound, avoirdupois (lb)

metric ton (tonnes) 1.016 ton, long (2,240 lb)

Density

kilogram per cubic meter (kg/m3) 0.062428 pound per cubic foot (lb/ft3)

kilogram per cubic meter (kg/m3) 0.008345 pound per US gallon (lb/gal)

kilogram per square meter (kg/m2) 0.20482 pound per square foot (lb/ft2)

Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as °F = (1.8 × °C) + 32.

Supplemental Information

Concentrations of chemical constituents in water are given in milligrams per liter (mg/L).
vii 

Abbreviations

CaCO3 calcium carbonate

C/N carbon nitrogen ratio 

CO2 carbon dioxide

DO dissolved oxygen

FDA U.S. Food and Drug Administration

HP hydrogen peroxide

NH3-N un-ionized ammonia-nitrogen

NO3-N nitrate-nitrogen

PAA peracetic acid 

PVC polyvinyl chloride

RAS recirculating aquaculture systems

rRNA ribosomal ribonucleic acid

TAN total ammonia nitrogen

UMESC Upper Midwest Environmental Sciences Center

USGS U.S. Geological Survey
 
1 

Literature Review of the Potential Effects of 

Hydrogen Peroxide on Nitrogen Oxidation 

Efficiency of the Biofilters of Recirculating 

Aquaculture Systems (RAS) for Freshwater 

Finfish

By Kim T. Fredricks

Summary 

A comprehensive literature review was done for the effects of hydrogen peroxide

(HP) on biofilter function in recirculating aquaculture systems (RAS) using these 

databases: ISI/Web of Knowledge, Scopus, and Pubmed. Inclusion and exclusion criteria 

were developed as the literature review was conducted. The initial search produced 

5,748 potential citations. Once the literature search was complete, these 5,748 titles were 

screened for applicable papers using the inclusion and exclusion criteria. If the title 

contained any of the inclusion terms, it was retained. Titles of the remaining papers were 

then screened for exclusion terms. If the title contained one or more of the exclusion 

terms, it was eliminated from further consideration. This refined search produced 

1,405 papers. 

After the initial screening, the remaining 1,405 papers underwent a second 

screening. Titles and abstracts (when available) were again read to verify that the topic of 

the paper was related to RAS. During the second screening, a second person verified that 

the papers proposed for elimination were not related to RAS. A combined reference list 

of the 512 remaining papers was created and submitted to the U.S. Geological Survey 

(USGS) Upper Midwest Environmental Sciences Center (UMESC) librarian in order to 

obtain the actual papers; electronic copies of those citations were obtained and reviewed. 

The UMESC librarian also received weekly updates from Scopus (a bibliographic 

database containing abstracts and citations for academic journal articles) using the search 

terms. Any resulting papers from those updates were screened using the inclusion criteria 

and relevant papers were requested. From those, 86 were cited in the literature review. 

An additional 11 papers from other search methods (e.g., mining references lists) also 

were obtained. 

I. Overview of Aquaculture

Aquaculture may be defined as cultivation of saleable aquatic products under 

controlled conditions using the highest stocking density possible, the highest quality 
2 

feeds, and active water-quality management (Ebeling and others, 2006). Aquaculture 

accounts for about 50 percent of the world’s food fish (Food and Agriculture 

Organization of the United Nations, 2014), particularly in Asia and developing countries 

(Håstein and others, 2006; Crab and others, 2007). By 2030, an estimated 62 percent of 

fish for human consumption will come from aquaculture (Food and Agriculture 

Organization of the United Nations, 2014). In 2004, 17.3 million tonnes of carp 

(Cyprinus carpio), 1.2 million tonnes of tilapia (Tilapia spp.), 1.1 million tonnes of 

salmon (Oncorhynchus sp.), and 0.5 million tonnes each of rainbow trout (Oncorhynchus 

mykiss) and shrimp were produced using aquaculture methods (Håstein and others, 2006). 

Salmon farming is the main economic activity in Chile, valued at about 

US$ 224.4 million (Bravo and others, 2013). Sustained and enhanced productivity are 

major goals of aquaculture, and current technologies have led to the expansion of 

aquaculture production (Panigrahi and Azad, 2007). 

Aquaculture systems can be classified as one of three types: extensive systems 

(which require little daily care by a fish culturist), intensive systems (in which fish 

survival depends completely on the intervention by a fish culturist), and intensive 

recirculating systems (Ebeling and others, 2006). RAS, in particular, are intensive 

because of the additional need to manage water quality in addition to feeding. This brings 

on unique challenges as well as benefits. This review will focus on recirculation systems, 

particularly those that use freshwater. 

II. Recirculating Aquaculture Systems (RAS)

A. Overview of RAS

RAS are controlled environments used to intensively rear fish and other aquatic 

organisms in ecologically sustainable ways (Summerfelt and others, 2001; Wik and 

others, 2009). RAS technology is designed for intensive fish farming where water 

availability is limited (Avnimelech, 2006; Ebeling and others, 2006; Badiola and others, 

2012) or where biosecurity concerns limit water intake (Avnimelech, 2006). Intensive 

recirculating systems recycle 90 to 99 percent of the water used to grow fish or other 

aquatic organisms (Summerfelt and others, 2001; Ebeling and others, 2006; Park and 

others, 2008; Badiola and others, 2012). With greater operator control over water quality, 

an operator can optimize growth conditions for the species being reared (Badiola and 

others, 2012; Carrera and others, 2013). Maintaining water quality for growout systems 

may provide greater economic output for the producer. Critical water-quality variables in 

RAS include dissolved oxygen, carbon dioxide, ammonia, nitrite, nitrate, pheromones, 

endocrine disrupters that may affect fish maturation, and other toxic chemicals that may 

leach from rubber or plastics used in building the RAS (Colt and others, 2006). RAS 

typically have a unit for growing fish, a mechanical filter to remove larger particles 

before biofiltration, an aerobic biological nitrification area to remove potentially toxic 

nitrogenous compounds, and sometimes an anaerobic denitrification filter (Barbu and 

others, 2008). 

Biofilters are used to help control the accumulation of ammonia-nitrogen in RAS. 

The main driver of water quality in RAS is the feeding rate. As fish metabolize protein in 

feed, they release ammonia as a waste product. Oxygen levels within the culture tank are 

reduced as a result of bacterial metabolism of nitrogenous compounds (NH4

+ + O2 →
3 

NO3

−). Oxygenation of culture tanks had a significant impact on reducing the ammonia 

concentration that reached the biofilters (Caraman and others, 2010). Nitrifying bacteria 

are well known for their ability to secrete and embed in a lipopolysaccharide matrix that 

helps create a stable biofilm (Hagopian and Riley, 1998). Fast growing heterotrophic 

bacteria form the outer layers of biofilms where they can use available oxygen. Oxygen 

use by heterotrophic bacteria, however, can limit the nitrification processes because 

oxygen is required for ammonia oxidation (Chen and others, 2006). In one study, 

nitrification was limited to the top 50 micrometers (μm) of the biofilm (Schramm and 

others, 1996). Slow growing nitrifying bacteria are protected under the outer layer 

(Michaud and others, 2006). 

Attached biofilms on biofilter material are advantageous in that there is increased 

efficiency from a large surface area and stability that reduces the chance of biofilms 

being washed out. Nitrosomanas form dense clusters in the upper part of the biofilm and 

Nitrobacter cluster around the Nitrosomanas clusters, though less densely. Nitrifiers in 

the deeper anoxic layers are relatively uncommon (Schramm and others, 1996). 

Nitrification processes also can be inhibited by heterotrophic processes when organic 

carbon is present. Nitrification decreased about 70 percent due to heterotrophic processes 

when organic carbon was present in RAS in a carbon to nitrogen (C/N) ratio of 1 or more 

(Zhu and Chen, 2001). In production, biofilters may operate under an organic load that is 

high enough to inhibit the nitrification efficiency so it is beneficial for RAS operators to 

remove organic matter to maximize nitrification processes. (A more complete overview 

of RAS can be found in the Engineering Assessment by Holder Timmons Engineering 

and Julie Bebak, written commun., 2014). 

B. Advantages of RAS

There are several advantages to RAS. Recirculation of water helps reduce use. 

Compared with a flow-through system, use of a rotating biological contactor in a RAS 

unit reduced water consumption by 90 percent with no negative effects on specific 

growth or condition factors of rainbow trout (d’Orbcastel and others, 2009a; Marin and 

others, 2011). Within the effluent of a non-RAS facility are high levels of phosphorus, 

carbon, and nitrogen from feed waste, which can have a negative environmental impact. 

Food conversion can be increased in RAS, thereby reducing the potential environmental 

impact. The lower water-exchange rates in RAS also allow for temperature control, 

which can lead to year-round production (Gutierrez-Wing and Malone, 2006; Lyssenko 

and Wheaton, 2006) and help reduce energy costs associated with maintaining a 

particular temperature (Summerfelt and others, 2001; Avnimelech, 2006; Gutierrez-Wing 

and Malone, 2006). RAS also allow for greater biosecurity and independence in location 

of production facilities (Summerfelt and others, 2001; Cancino-Madariaga and others, 

2011). Hybrid striped bass, for example, have been reared in the deserts of California 

(Gutierrez-Wing and Malone, 2006). Lastly, RAS allow for higher fish-production rates 

and a higher density of fish per rearing tank (Lyssenko and Wheaton, 2006; Good and 

others, 2009; Gullian-Klanian and Arámburu-Adame, 2013). For example, in one study, 

freshwater eel (Abguilla japonica) were raised at a density of 2.6 percent (compared to 

0.6 percent traditionally) in a nearly closed system that only replaced water lost to 

evaporation (Suzuki and others, 2003). 
4 

C. Challenges

A significant challenge with RAS is that, unlike flow-through systems, 

nitrogenous and organic-waste products accumulate in the rearing tank. RAS thus require 

biofilters to remove the nitrogenous wastes and settling tanks to help remove organic 

wastes. If fish are stocked before the biofilter is fully functional, fish can develop 

methemoglobinemia (Svobodová and others, 2005). Establishing biofilters is a challenge 

because the bacteria that oxidize ammonia and nitrite to less toxic nitrate are slower 

growing. Allowing a natural population of bacteria to colonize a biofilter can take 4 to 

8 weeks (Kuhn and others, 2010). One reason that biofilters take time to establish is that 

the heterotrophic bacteria can out-compete the nitrifying bacteria for space and oxygen 

because of their faster growth rates (Cancino-Madariaga and others, 2011; Michaud and 

others, 2006; Brambilla and others, 2008; Blancheton and others, 2013). Using a starter 

culture can significantly decrease the time needed to establish a biofilter (Gross and 

others, 2003; Kuhn and others, 2010). 

Other technologies are being investigated as a means to remove ammonia from 

RAS that do not have the same challenges as establishing a biofilter. For example, 

reverse osmosis membranes also may help with ammonia removal in RAS (CancinoMadariaga and others, 2011). Ion-exchange resin also can help remove ammonia from 

RAS. These resins are cheaper to use, require a smaller volume than a typical biofilter, do 

not require a long start-up, are not temperature sensitive, and can be turned on and off at 

will. Additionally, the resins convert ammonia directly to nitrogen gas (Gendel and 

Lahav, 2013). 

A second challenge with RAS is that the higher rearing density may stress fish, 

reducing their immune function, which may then provide greater opportunity for 

pathogens to infect fish. If a pathogenic organism is introduced into the system, either 

from addition of new stock or replacement water, it may persist in the biofilms within the 

system (King and others, 2008; Jacobs and Chenia, 2011). This can result in recurring 

fish mortality and economic losses to the facility. Many of the pathogens are 

opportunistic and cause disease only if the fish are compromised or if their environment 

is suboptimal. The organic peroxide peracetic acid (PAA) had the greatest effect on 

inhibiting growth of pathogenic bacteria in biofilms of RAS (King and others, 2008). 

However, there is always a concern that use of disinfectants may impair the nitrifying 

bacteria in the biofilters, resulting in accumulation of ammonia or nitrite, which is toxic 

to fish (Summerfelt and others, 2001; Pedersen and others, 2009). 

A third challenge is off-flavors in fish that can result in economic loss for the 

producers. These off-flavors are produced by bacteria Nocardia cummidelens, 

N. fluminea, Streptomyces roseoflavus, Streptomyces luridiscabiei, and Streptomyces cf. 

albidoflavus that were isolated from RAS biofilters (Guttman and van Rijn, 2008; 

Schrader and Summerfelt, 2010; Auffret and others, 2013) and heat exchangers (Schrader 

and Summerfelt, 2010). 

Biofilter operation and solids management were identified as main issues in RAS 

in a survey conducted in Europe (Badiola and others, 2012). Integration of the current 

technology to manage biofilters and solids within commercial scale RAS is a challenge. 

One reason for this is that much of the research has been at laboratory scale, which does 

not translate well to commercial-scale operations (Badiola and others, 2012). Dissolved 

organic carbon from feed waste or improper mechanical filtration can reduce nitrification 
5 

because the heterotrophic bacteria feed on the carbon and overgrow the autotrophic 

nitrifying bacteria within the biofilms (Guerdat and others, 2011).

Other challenges with aquaculture, both freshwater and marine, include foodsafety concerns. Rearing fish in crowded tanks can increase the risk of disease 

transmission, and may require the use of antibiotics or other therapeutic agents to prevent 

loss of the crop. For example, Streptocossus iniae may cause chronic septicemia in fish. 

It is commonly isolated from cultured species including rainbow trout, tilapia, channel 

catfish (Ictalurus nebulosus), and Japanese flounder (Paralichthys olivenceus) 

(Håstein and others, 2006). Antibiotic use, particularly in countries where regulation is 

not as stringent as it is in the U.S., is a concern because it can lead to the development of 

resistant bacteria. Those resistance determinants may then be transferred to zoonotic 

pathogens which could impact humans or other commercially important animals. For 

example, molecular evidence exists that Aeromanas can transmit determinants for 

resistance to antibiotics with the human pathogen Escherichia coli (Cabello, 2006). 

Horizontal gene transfer of bacterial resistance has been shown from aquaculture to 

humans (Heuer and others, 2009). Antimicrobial use in developed countries is more 

restricted to try to prevent resistance in human pathogens; however, in other countries, 

their use is not as restricted. In 2007, for example, Canada used 0.175 kilograms (kg) of 

antimicrobials/metric ton of salmon produced whereas Chile used 1.4 kg per metric ton of 

salmon produced (Cabello and others, 2013). Other countries are reducing their use of 

antibiotics to reduce the potential for resistance. Norway reduced antibiotic use by 

37 percent from 1992 to 1996 and by 96 percent in fish that were farmed, even though 

biomass of farmed fish increased by 100 percent (Grave and others, 1999). 

Aquaculture produces substantial nutrient and organic matter that must be treated. 

Nutrients can increase phytoplankton (i.e., eutrophication) and potentially decrease water 

quality. Excess organic matter can limit dissolved oxygen, which could negatively affect 

other aquatic life in the area near the discharge of wastewater (Ghaly and others, 2005). 

Higher levels of nitrogen and phosphorus released with wastewater can accelerate 

eutrophication (Hussenot, 2003). Mats created from waste feed and discharged fecal 

matter may decrease benthic biodiversity and increase biological oxygen demand 

(Droppo and others, 2007). Some of these environmental concerns can be mitigated. For 

example, phytotreatment can be used to help remove nitrogen and phosphorus from water 

before it is discharged (Porrello and others, 2003). The plants that are produced can be 

used as feed, which may provide another source of income. Rye, barley, and oats were 

found to be the best plants at removing nitrogen and phosphorus in one study (Ghaly and 

others, 2005). 

Finally, investment costs are higher with RAS compared to flow-through systems. 

In 1998, it cost about US$0.90 per pound (lb) of annual production in a pond system 

compared to US$1.00–4.00 for RAS (Gutierrez-Wing and Malone, 2006). The payback 

period also is longer than other systems, on average about 8 years (Badiola and others, 

2012). Overall energy costs are typically higher with RAS compared to flow-through as 

well (Singh and Marsh, 1996; d’Orbcastel and others, 2009b). 

D. Current Work with Biofilters

With increased biomass in RAS, water quality often becomes a limiting factor for 

growth, mostly due to increased ammonia and nitrites produced as fish metabolize 
6 

protein in feed (Hagopian and Riley, 1998). Water quality can be controlled by 

replacement, which often requires high exchange rates, or by recycling water through a 

biological filter (Avnimelech, 2006). Biological filters, or biofilters, are an important 

component of RAS and are used to reduce total ammonia (ionized and un-ionized 

ammonia) through nitrification processes that oxidize ammonia to nitrite then nitrate. 

Biofilters can be made of a variety of materials but are often divided into two main 

categories. Fixed film biofilters have media for microorganism attachment and growth 

whereas suspended growth biofilters maintain the microorganisms in suspension. 

Suspended growth biofilters require more management and are not commonly used in 

RAS (Gutierrez-Wing and Malone, 2006). Effective biofilters have high populations of 

nitrifying bacteria that help reduce water required for tank exchanges in RAS (Guerdat 

and others, 2010). However, if organic load in the RAS is high, some heterotrophic 

bacteria can migrate to the biofilter and impair function of the nitrifying bacteria (Barbu, 

2012). Carbon dioxide (CO2) is produced by biofilters through respiration of the 

nitrifying bacteria. Summerfelt and Sharrer (2004) estimated that the biofilter accounted 

for 37 percent of the total CO2 produced in a salmonid RAS and suggested that a CO2

stripping unit be placed immediately after the biofilter, where CO2 is at its highest level, 

to minimize the CO2 fish are exposed to as the water recirculates. Currently (2015), there 

is no standardized method to evaluate biofilter performance, though several authors have 

suggested the need for developing standards for the industry to help plan for media and 

volume of media used in RAS (Colt and others, 2006; Malone and Pfeiffer, 2006). 

Fixed film biofilters used in recirculating systems include rotating biological 

contractors (Brazil, 2006; Marin and others, 2011), trickling filters (Kamstra and others, 

1998; Eding and others, 2006; Barbu, 2012), sequencing batch reactors (Fontenot and 

others, 2007; De Schryver and Verstraete, 2009; Luo and others, 2013), and fluidized bed 

biofilters (Skjølstrup and others, 1998; Malone and Beecher, 2000; Timmons and others, 

2006; Weaver, 2006; Davidson and others, 2008). Various media and flow rates have 

been studied, and one study indicated that media characteristics may be more important 

than flow rates at affecting the efficacy of the filter (Yang and others, 2001). The filter 

material should have a surface capable of developing biofilms and capturing organic 

solids (Yang and others, 2001). Media size and surface area often influences maximal 

feed load, which affects the amount of total ammonia nitrogen (TAN) that can be 

removed (Summerfelt, 2006; Pfeiffer and Wills, 2011). Media can vary between 

commercially available media (Pfeiffer and Wills, 2011) to something as simple as 

polyvinyl chloride (PVC) shavings (Prinsloo and others, 1999). Other low cost materials 

that have been used in effective biofilters include lava stones and oyster shells (Ogunlela 

and Ogunlana, 2011), Styrofoam beads (Peng and others, 2003), wood chips and wheat 

straw (Saliling and others, 2007), polystyrene microbeads (Timmons and others, 2006), 

and scrub pads and plastic hair rollers (Al-Hafedh and others, 2003). More surface area 

does not necessarily mean more TAN removal. In one study, commercially available 

Siporax glass beads with a water contact area of 32,000 square meters (m2

) were only 

93 percent efficient at converting ammonia nitrogen (NH3-N) to nitrate nitrogen (NO3-N), 

while the PVC shavings with a water contact area of 1,220 m2

 were 96 percent efficient 

(Prinsloo and others, 1999). 

In rotating biological contractors, media (typically molded disks with a high 

surface area) is attached to a central horizontal shaft. The rotation of the filter partly 
7 

submerges some of the media and rotation allows for shearing of excess biofilm growth 

(Brazil, 2006). Trickling biofilters, on the other hand, use a fixed media through which 

pre-filtered water is trickled down the height of the filter over a thin aerobic biofilm. 

Metabolites from the biofilm are picked up and carried out with the wastewater. Trickling 

allows for oxygenation of the biofilm and degassing of carbon dioxide. Advantages of 

trickling filters include high process stability due to constant high oxygen levels; carbon 

dioxide (CO2) removal by degassing; water cooling in summertime; and simplicity of 

design, construction, operation, and management. Some disadvantages of trickling filters 

are the relatively low volumetric removal rates (with consequently large sized biofilters), 

biofilm shedding, and risk of clogging when not properly designed and operated. For 

certain fish species, additional solids removal is necessary (Eding and others, 2006). 

Sequencing batch reactors incorporate alternating aerobic and anaerobic periods 

to achieve nitrification and denitrification in a single container. The aerobic phase is 

followed by an anaerobic phase, then a second aerobic phase, and a settling phase 

(Fontenot and others, 2007). Lastly, fluidized bed biofilters have a higher surface area per 

unit volume than other fixed film biofilters (Weaver, 2006) and can be filled with 

microbeads (Malone and Beecher, 2000; Timmons and others, 2006) or sand (Weaver, 

2006; Davidson and others, 2008). Fluidized sand biofilters are efficient, compact, cost 

competitive, and relatively easy to manage (Sandu and others, 2002; Summerfelt, 2006; 

Weaver, 2006). Providing adequate oxygen is key to their function as about 60 percent of 

the dissolved oxygen used in a fluidized sand biofilter went toward nitrification 

(Summerfelt and Sharrer, 2004). Biofilm shearing can be done by pumping the top 

particles to the bottom. The released biofilm particles are less dense than the sand and 

simply wash out with the effluent (Davidson and others, 2008). An advantage to sand is 

that it has a high surface area for a relatively low cost. These biofilters can be configured 

to fit the space available and can treat a high volume of water with a relatively small 

footprint, removing 86 to 88 percent of TAN (Davidson and others, 2008). Additionally, 

Sandu and others (2002) found that TAN removal rate increased as column diameter 

increased. They also suggested that the ideal fluidized bed filter would be filled to 40–50 

percent capacity with plastic medium, and upflow velocity would be 1–2 centimeters per 

second (cm/s) at the desired flow rate (Sandu and others, 2002). Microbead filters are a 

low-cost alternative to fluidized sand filters because they can be scaled to large 

production systems. A key advantage of microbead filters is that their cost of operation is 

less than a fluidized sand biofilter because of the ability to use low head high volume 

pumps for their operation (Timmons and others, 2006). 

In a study designed to assess how three different filter types performed under 

production-scale tilapia culture, the floating bead and fluidized sand filters outperformed 

the moving bed bioreactor in terms of ammonia removal, though nitrification rates in 

fluidized bed biofilters were more variable. Nitrite levels were in acceptable ranges for 

the study duration, with the fluidized bed and moving bed bioreactor filters showing 

variability. The floating bead biofilter showed a net nitrite production that was attributed 

to capture and degradation of suspended organic solids at the bottom of the filter. This 

type of filter serves both as a biofilter and a mechanical filter, and captured solids are 

degraded between back washings, which were every 2 hours during the study (Guerdat 

and others, 2010). Another study showed that floating media used less energy to maintain 
8 

flow compared to sand filters, which was seen as an advantage, particularly when both 

filter types had similar solids removal and turbidity reduction (Steicke and others, 2007). 

Short-term shocks to biofilters, such as a change in pH, TAN, or temperature, did 

not negatively affect biofilter operation, and nitrification rates returned to normal within 

2 hours. Trickling filters seemed more sensitive to disturbances, perhaps because of a 

more immediate contact of the water on biofilm. The rate and magnitude of change may 

be important variables in biofilter performance, especially if change is rapid (Lyssenko 

and Wheaton, 2006). 

Biofilms on biofilter media form layers, and movement of ammonia or nitrite into 

the biofilm is diffusion controlled (Rittmann and McCarty, 1980). The nitrification rate is 

in equilibrium between demand for ammonia or nitrite created by growth of the bacterial 

biomass and the rate of supply of ammonia and nitrite that can be affected by more than 

20 physical, chemical, and biological factors; many of which can be optimized in RAS 

design. Ammonia concentration should be the primary consideration in biofilter design 

because ammonia is toxic to many fish species at low levels. For example, toxic levels of 

ammonia are 0.32 milligrams per liter (mg/L) for rainbow trout and 0.05–0.2 mg/L for 

other salmonids (Summerfelt and others, 2001). The highest water quality that can be 

maintained in RAS is defined by the minimum amount of ammonia that can sustain the 

nitrifier population in the biofilter. Dissolved oxygen should be maintained at >2.3 mg/L 

to sustain nitrification within biofilters (Chen and others, 2006), and a DO/TAN of >2.5 

is recommended (Chen and others, 2006; Summerfelt, 2006). Dissolved oxygen may drop 

rapidly in the biofilm despite sufficient dissolved oxygen in the culture water and may 

become the limiting factor to nitrification if it falls below 2 mg/L. Turbulence can impair 

mass transfer into the biofilm and has the greatest effect on nutrient transfer into the 

biofilm. An alkalinity of >200 mg/L is recommended to maintain optimum nitrification 

rates when water exchange is minimal. Alkalinity ensures adequate buffering capacity to 

prevent pH changes from the H+

 produced during nitrification (Chen and others, 2006). 

Nitrification can consume from 6.0 to 7.4 milligrams (mg) of alkalinity (as CaCO3) and 

about 4 mg of oxygen for every 1.0 mg of ammonia nitrogen that is converted to nitratenitrogen (Summerfelt and others, 2001). 

Additional research on biofilters focused on use of other nitrogen oxidizing 

organisms and immobilizing bacteria on various substrates to help start the nitrification 

processes. Aspergillus niger, a fungus, was introduced into a laboratory-scale (1 liter [L]) 

fixed slab reactor and was found to use ammonia at low temperatures (22 degrees Celsius 

[°C]), but switched to carbon at higher temperatures (35 °C). If used in a production scale 

biofilter, the operator would likely have to know the C/N ratio of waste produced (Hwang 

and others, 2004). A similar study examined the ammonia removal rates in a continuous 

stirred tank reactor in a RAS. Aspergillus niger was the dominant species that removed 

ammonia at a daily rate of 35 mg TAN/L. It required only 20 hours to reach a constant 

removal rate; however, this system also required ozone treatment (Hwang and others, 

2007). 

A study by Watanabe and others (2004) demonstrated that immobilized ammonia 

oxidizing bacteria, Nitrosomonas europaea, helped remove ammonia from rearing water. 

This technology may help start the nitrification process since the bacteria could be 

present in high amounts from the start. However, they also found that the ammonia 

oxidizers were damaged by light during the time the gel-plate solidified (Watanabe and 
9 

others, 2004). Immobilizing bacteria in a fluidized bed biofilter also was investigated as a 

way to increase fish resistance to disease. The bacteria Rhodopseudomonas palustris, 

which can inhibit and destroy reproduction of viruses, was immobilized on alginate gels 

covered with polyvinyl alcohol film. The resulting beads were then placed in a fluidized 

bed before rainbow trout or common carp (Cyprinus carpio) were added. During the 

study, fish growth and development was normal and survival rate was 98 percent. 

Maximum fish load was 45 ± 3 kilograms per cubic meter (kg/m3

), ammonia nitrogen 

removal rate was 80–95 percent, and nitrite N removal rate was <80 percent (Peirong and 

Wei, 2013). 

E. Bacteria That Process Nitrogen

Two distinct groups of bacteria process nitrogen: (1) the ammonia-oxidizing 

bacteria of the genera Nitrosomonas, Nitrosococcus, Nitrospira, Nitrosolobus, and 

Nitrosovibrio; and (2) the nitrite-oxidizing bacteria of the genera Nitrobacter, 

Nitrococcus, Nitrospira, and Nitrospina. Although growth is slow, doubling time is 

shorter for the ammonia-oxidizing bacteria (about 26 hours) than for nitrite-oxidizing 

bacteria (about 60 hours). Optimal temperature for nitrification is about 25 °C, and 

optimal pH is 7.8 (Hagopian and Riley, 1998). 

Additionally, differences are seen in bacterial populations in RAS biofilters and 

culture water and because each fish species introduces a unique microbiological flora, 

diversity varies from RAS to RAS. Diversity also is affected by use of disinfection (i.e., 

ozone treatments) that can affect culture water but does not affect the deeper biofilm 

layers. Diversity may be measured by determining the 16S rRNA sequences of bacteria in 

the system. Ammonia oxidation is associated with Nitrosomonas sp. in freshwater 

systems. Nitrite and ammonia oxidation occur together in nitrification biofilters, with 

Nitrospira sp. being the dominant nitrite oxidizer (Schreier and others, 2010). In 

anaerobic areas, such as the deeper layers of the biofilm, high nitrate/nitrite levels and 

organic carbon wastes promote heterotrophic denitrification. Pseudomonas and 

Comamonas spp. are likely heterotrophs that participate in denitrification in freshwater 

systems (Schreier and others, 2010). 

Anaerobic ammonium-oxidation or anammox bacteria in the taxon Planctomyces 

were discovered in the late 1990s. They convert ammonia and nitrite to nitrogen gas and 

water in the absence of oxygen. These bacteria are slow growing organisms (doubling 

times >11 days, Schreier and others, 2010), whose growth is inhibited by oxygen so they 

are likely found in the low oxygen areas of the biofilm on biofilters. The likely source of 

these anammox bacteria is fish feces (van Kessel and others, 2011). In a RAS culturing 

Scortum barcoo, the Nitrospira formed only a small part of the overall bacterial 

community and could not account for all the oxidation of nitrite to nitrate. It was thought 

that some of the Planctomycetales-related clones were participating in the ammonia 

removal process after the biofilter stabilized (Zhu and others, 2012). In another study, 

anammox-like bacteria were detected by 16S rRNA analysis in a RAS containing Koi 

carp (Cyprinus carpio Koi) but anammox activity was not sufficient to account for 

significant ammonia removal (van Kessel and others, 2010). 
10 

III. Diseases

Bacterial, viral, fungal, and parasitic pathogens, both opportunistic and obligate, 

cause disease in RAS. Obligate pathogens require a host to survive and do not live long 

outside of the fish. The major fish pathogens include Flavobacteria, Saproeignia, 

Aeromonas, Vibrio, and Yersinia (Blancheton and others, 2013), and these can potentially 

infect fish cultured in RAS. In one study, sea bass and mullet reared in recirculation 

systems showed signs of infection by several bacterial species including Aeromonas ssp, 

Myxobacteria, and Saprolegnia. Parasites also infested the same species including 

Ichthyophthirius sp, Chilodonella cephalus, and Diplectanum aequans (Athanassupoulou 

and others, 2004). Furunculosis was found in a recirculation system growing Senegalese 

sole (Solea senegaleus) held at a stocking density of 25 kilograms per square meter 

(kg/m2) (Magariños and others, 2011). In another study, Actinomycetes were isolated 

from a drum filter and heat exchanger in a RAS (Schrader and Summerfelt, 2010). 

Heterotrophic bacteria are common in RAS and may cause diseases and outcompete autotrophic bacteria. They also produce significant quantities of bacterial 

biomass that may clog biofilters and limit nitrification (Michaud and others, 2006). Foam 

fractionation had variable success in removing heterotrophic bacteria (Brambilla and 

others, 2008). 

Although it may seem that RAS would be at higher risks for disease outbreak, 

there were no papers found that indicated well-managed RAS had higher incidences of 

disease. RAS tend to be biosecure; water replacement rates are low, which may limit 

invasions by pathogens; and they may include a disinfection unit that reduces the risk of 

disease (Blancheton and others, 2013). However, if water quality is not maintained, fish 

may develop infections. Nile tilapia showed a higher incidence of parasitic infestation by 

Trichodina and monogeneans when nitrogen compounds in the RAS increased (JiménezGarcía and others, 2012). 

IV. Use of Hydrogen Peroxide in RAS

A. Current Label

At time of publication, 35 % Perox-Aid® (Western Chemical, Ferndale, 

Washington) is the only HP product approved by the U.S. Food and Drug Administration 

(FDA) for use in U.S. aquaculture. It is specifically approved to control mortality of

freshwater finfish eggs due to saprolegniasis (fungi of the family Saprolegniaceae). It 

also is approved to control bacterial gill disease in all freshwater-reared salmonids, and to 

control external columnaris disease on all coolwater finfish and channel catfish. Detailed 

information can be found on their Web site:

http://www.accessdata.fda.gov/scripts/animaldrugsatfda/details.cfm?dn=141-255.

B. Application of Hydrogen Peroxide in RAS–Effects on Biofilter Bacteria

With antimicrobial resistance becoming an increasing concern in human and 

veterinary clinical practice, associated with the use of antimicrobials in agriculture 

(including aquaculture; Defoirdt and others, 2007; Guglielmetti and others, 2009; 

Fernández-Alarcón and others, 2010; Dang and others, 2011; Defoirdt and others, 2011), 

other treatments to control bacterial infections must be developed. HP has been approved 
11 

for use in flow-through systems and can be used in RAS if the user contacts the Western 

Chemical for proper procedures regarding flushing of the drug from the RAS. Some 

drugs have been shown to inhibit the nitrification process in RAS, including ampicillin, 

chloramine T, and erythromycin (Nimenya and others, 1999). Additionally, there are 

concerns about worker safety with the use of formalin, or release of higher saline water if 

sodium chloride is used (Pedersen and Pedersen, 2012). 

HP is an oxidizing agent that is approved as a fish therapeutant. It decomposes to 

water and oxygen and does not accumulate (Pedersen and others, 2006). HP also 

conforms to many of the principles of green chemistry: the reduction or elimination of the 

use of hazardous materials in the design, manufacture, and use of chemicals (Pedersen 

and Pedersen, 2012). A summary of some key variables in RAS exposed to HP is 

summarized in table 1.
12 

Table 1. Summary of papers that tested the effects of hydrogen peroxide on freshwater recirculating aquaculture systems. [kg/m3

, kilogram per cubic meter; °C, degrees Celsius; mg/L, milligrams per liter]

Citation Scale

Fish biomass 

(kg/m3)

Water 

temperature 

(°C)

Hydrogen 

peroxide dose 

(mg/L)

Duration 

(hours) Biofilter type

Pedersen and others,

2006 Pilot 7.5 15.5 13

Single 

addition Submerged upflow

Pedersen and others,

2006 Pilot 7.5 15.5 26

Single 

addition Submerged upflow

Pedersen and others,

2006 Pilot 7.5 15.5 39

Single 

addition Submerged upflow

Møller and others,

2010 Pilot 20–30 20 ± 1 15

Single 

addition Submerged

Møller and others,

2010 Pilot 20–30 20 ± 1 10–13 3 Submerged 

Pedersen and 

Pedersen, 2012 Commercial

20–23.3 

(calculated) 16.5 64

Single 

addition Not reported

Pedersen and 

Pedersen, 2012 Commercial

20–23.3 

(calculated) 16.5 45

Single 

addition Not reported

Pedersen and 

Pedersen, 2012 Commercial

20–23.3 

(calculated) 16.5 20 3 Not reported

Pedersen and others,

2012 Pilot 24 18 15

Single 

addition Submerged upflow

Pedersen and others,

2012 Pilot 92 18 15

Single 

addition Submerged upflow
13 

When HP was applied to active biofilters in RAS, the HP decay followed first 

order kinetics (Pedersen and others, 2006; Pedersen and others, 2009; Møller and others, 

2010). Decomposition of HP varied with the amount of organic matter and the initial 

dose of HP. If loading rates into a RAS exceed the microbial degradation rate, then 

severe negative effects could occur within the biofilter microbial community (Pedersen 

and others, 2006). 

When biofilters from two pilot-scale RAS were exposed to 0, 5, 10, 20, 25, or 

30 mg/L HP, the decay followed first order kinetics, and the nitrogen oxidation processes 

recovered quickly after a single dose of up to 30 mg/L. An average dose of 10–13 mg/L 

over 3 hours of repeated HP spiking had a moderate inhibitory effect on nitrification in a 

pilot-scale biofilter with a high organic load but had a severe inhibitory effect on a pilotscale biofilter with a low organic load. The biofilters of the RAS with the low organic 

load had not recovered by 24 hours post-exposure indicating potential long-term 

perturbations of the biofilter (Møller and others, 2010). 

HP also was applied to a production-scale freshwater RAS that produced rainbow 

trout. Normal aquaculture practices were followed during the study. Throughout the 

experimental time period, the farm had about 30–35 metric tonnes of rainbow trout. Two 

studies were performed that evaluated biofilter performance: (1) high dose (64mg/L), 

single application to a closed biofilter section, and (2) multiple doses to the raceways 

with evaluation of biofilter response. Water temperature was about 16.5 °C during the 

experiments. Results from part 1 showed an increase in oxygen, indicating that 

heterotrophic and nitrifying bacteria were immediately inhibited. Ammonia oxidation 

rates were significantly less than controls for 7 days after exposure. Nitrite oxidation also 

was negatively affected, but to a lesser extent. Addition of HP resulted in liberation of a 

large amount of organic matter, which was controlled by enclosing the biofilter section 

and redirecting COD-enriched water to the sludge compartment. In the second study, 15 

mg/L HP was applied using the standard practice of adding the chemical to one end of the 

raceway. The concentration was maintained at about 15 mg/L for 3 hours by addition of 

HP. Fish were observed aggregating downstream of the HP pulse. The half-life of HP 

was found to be 1.5 hours. The biofilter elements were initially by-passed (which was not 

standard practice) for 3 hours then exposed to residual HP for 45 minutes, resulting in a 

slight decrease in TAN removal rates compared to control sections. No negative effects 

on fish were seen during the 3-hour treatment (Pedersen and Pedersen, 2012). 

In another pilot-scale RAS study using rainbow trout, the effects of low dose 

(10–20 mg/L) HP on biofilter function was investigated under these conditions: low 

intensity (24 kg/m3

) and high intensity (92 kg/m3) culture conditions. Water temperature 

in the six 1,700 L units was maintained at about 18 °C. Water renewal rate was fixed at 

80 liters per day (L/d) for both groups. Before HP was added, the inflow and outflow 

from the culture tanks were stopped, resulting in two closed systems: the rearing unit, and 

the biofilter unit to achieve a target of 15 mg/L HP. HP was then added to either the 

culture tank or to the biofilter. Water samples were analyzed over 4 hours for HP. Halflife for HP added to the biofilter of high intensity units was 13 minutes, and half-life for 

the low intensity group was 63 minutes. HP decay also was different when it was added 

to the culture tanks. About 95 percent of the added HP had decayed in 1.5 hours from the 

high intensity culture tanks; HP concentration was essentially 0 by 2 hours. Only 25 

percent was removed in 1.5 hours in the low intensity culture tanks, and concentration 
14 

leveled out at about 10 mg/L for the remainder of the experiment. Feed rate was the same 

for all culture tanks until the day after the exposure when feed rate to the low intensity 

culture RAS tanks was increased by 4 times (to the same level as the high intensity 

tanks). Nitrite concentration increased after the increase in feed rate and remained 

elevated for about 8 days in the low intensity RAS, after which nitrite levels stabilized at 

low levels. Because nitrite went over 1 mg nitrite-nitrogen per liter, feed rate was reduced 

on day 2 post-exposure. Conversely, nitrite levels were not changed in the high intensity 

RAS units. Ammonia concentration was not affected in either type of RAS (Pedersen and 

others, 2012).
15 

V. Conclusions 

• HP may be a useful tool to help mitigate disease occurrence in RAS, particularly at lower doses 

of HP. 

• In a system with a lower organic load, the nitrite oxidation step may be negatively impacted for a 

prolonged time (Møller and others, 2010; Pedersen and others, 2012). 

• In systems where biofilters are exposed to prolonged higher levels of HP, the nitrite oxidation 

step may be negatively impacted for a prolonged time (Møller and others, 2010; Pedersen and 

others, 2012). 

• Because of the rapid decay of HP in culture water, it may be possible to treat fish if the biofilter 

can be bypassed temporarily, or if the biofilter can be bypassed such that it receives only a low 

residual concentration of HP. A potential negative of bypassing the biofilter is the potential for it 

to become a parasite reservoir (Pedersen and others, 2012). 

• Based on the studies reviewed for this literature review, it is likely that pilot-scale data would be 

very different from commercial-scale data. Commercial systems have higher carbon loads and 

mature bacterial populations in the biofilter, which may result in even faster rates of decay. 

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