Study Number: AEH-11-RASFRM-01; INAD Number: 011-365

Literature Review of the Potential Effects of Formalin on 

Nitrogen Oxidation Efficiency of the Biofilters of 

Recirculating Aquaculture Systems (RAS) for Freshwater 

Finfish 

By Kim T. Fredricks

Open-File Report 2015–1097

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 formalin on nitrogen oxidation efficiency of the 

biofilters of recirculating aquaculture systems (RAS) for freshwater finfish: U.S. Geological Survey Open-File 

Report 2015–1097, 17 p., http://dx.doi.org/10.3133/ofr20151097. 

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

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 ......................................................................................................................................................... 3

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

E. Bacteria That Process Nitrogen.......................................................................................................................... 8

III. Diseases................................................................................................................................................................ 8

IV. Use of Formalin in RAS......................................................................................................................................... 9

A. Current U.S. Label.............................................................................................................................................. 9

B. Application of Formalin in RAS–Effects on Biofilter Bacteria .............................................................................. 9

V. Conclusions.......................................................................................................................................................... 11

References Cited...................................................................................................................................................... 11

Tables

Table 1. Summary of papers that tested the effects of formaldehyde on recirculating aquaculture systems..... 11
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/sec) 0.0328 foot per second (ft/sec)

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

NH3-N un-ionized ammonia-nitrogen

NO3-N nitrate-nitrogen

PAA peracetic acid 

ppm parts per million

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

USP United States Pharmacopeia

1 

Literature Review of the Potential Effects of Formalin 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 formalin 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 5,682 potential citations. Once the literature search was complete, these 5,682 

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 1,287 papers. 

After the initial screening, the remaining 1,287 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 443 remaining papers was created and submitted to the 

U.S. Geological Survey (USGS) Upper Midwest Environmental Sciences Center (UMESC) librarian to 

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

librarian also would receive 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 also were screened using the inclusion criteria, and any relevant papers were requested. From 

those, 82 were cited in the literature review. An additional 10 references were obtained from weekly 

updates or reference mining other sources and were incorporated into the final literature review. 

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 feeds, and active waterquality 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 
2 

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 → 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 
3 

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 ArámburuAdame, 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). 

C. Challenges

A significant challenge with RAS is that, unlike flow-through systems, nitrogenous and organicwaste 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 (CancinoMadariaga 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 (Cancino-Madariaga 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). 
4 

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 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 food-safety 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 
5 

(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). 

Lastly, 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 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 
6 

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 (m

2

) 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 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; 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 
7 

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 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 nitrate-nitrogen (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 gelplate solidified (Watanabe and 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 
8 

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 nitriteoxidizing 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 nitriteoxidizing 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). 

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). 
9 

Heterotrophic bacteria are common in RAS and may cause diseases and out-compete 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 

(Jiménez-García and others, 2012). 

IV. Use of Formalin in RAS

A. Current U.S. Label 

Formalin is approved by the U.S. Food and Drug Administration (FDA) to treat aquatic 

organisms; it is an aqueous solution containing approximately 37 percent (by weight) of formaldehyde 

gas, United States Pharmacopeia (USP). Currently (2015; 

http://www.accessdata.fda.gov/scripts/animaldrugsatfda/details.cfm?dn=140-989), it is approved for 

the following uses: 

1. control of external protozoa (Chilodonella species, Ichthyodobo species, Ichthyophthirius 

species, Epistylis species, Ambiphyra species, and Trichodina species) and monogenetic 

trematodes (Chilodonella species, Dactylogyrus species, and Gyrodactylus species) in 

finfish; 

2. control of fungi in the family Saprolegniaceae on finfish eggs; and 

3. as a parasiticide for Penaeid Sprimp.

B. Application of Formalin in RAS–Effects on Biofilter Bacteria

The literature search did not find many (five total) papers on the effects of formalin on biofilter 

function in RAS. From the data in the few papers that reported effects of formaldehyde on nitrification 

process, no appreciable effect was observed if the formaldehyde concentration was below 40 mg/L. Key 

experimental variables are summarized in table 1. 

One hour formaldehyde exposures at concentrations ranging from 50–167 parts per million 

(ppm) at 13.4–16.5 °C did not affect biofilter function of a RAS that contained rainbow trout. After 

17 trials, nitrite levels were elevated for 9 days, likely caused by the cumulative mortality of nitrite 

nitrifying bacteria. This is not a concern to RAS operators because it is very unlikely that one biofilter 

would be exposed to such a treatment regime. An interesting finding was that a large number of 

oligochaetes were shed from the biofilter at 140 and 167 ppm formalin. Indefinite exposures, defined as 

no flushing of the system after application of formaldehyde, did not affect biofilter function at formalin 

concentrations that ranged from 15–120 ppm at culture temperatures of 16–18 °C. Formaldehyde was 

undetectable at 11 hours after application of 120 ppm formaldehyde, and oligochaetes were again 

washed from the biofilters when formaldehyde was ≥100 ppm. Formaldehyde was lost at a rate that was 

higher than that lost by water exchange, indicating a high amount of degradation within the system. 

Formaldehyde loss followed first order kinetics (Heinen and others, 1995). 
10 

Keck and Blanc (2002) tested the effects of formaldehyde on biofilter bacteria in a marine 

recirculation system. They used two 300 L tanks connected to a fluidized-bed biofilter. A commercially 

available 33 percent formalin solution was used in treatments that ranged from 20 to 90 mg/L of 

formaldehyde for 2, 4, and 6 hours. Formaldehyde at these levels did not appear to inhibit ammonia 

oxidizers. However, at concentrations greater than 40 mg/L of formaldehyde for 1 hour, there was a 

significant inhibition of nitrite oxidizers. Keck and Blanc suggested that 1 hour treatments ≥40 mg/L 

formaldehyde not followed by flushing or a 4-hour or more exposure to 60 mg/L formaldehyde be 

considered threshold values for marine systems. Their overall conclusion was that the effect of formalin 

on nitrification was minimal. 

Formaldehyde removal from submerged fixed biofilters was tested in a full-scale recirculation 

system and found to be related to available biofilter surface area. Tanks were stocked with rainbow trout 

(Oncorhynchus mykiss) and fed 1.6 kg of formulated feed daily. Daily water renewal was about 

10 percent of total volume. Prior to formaldehyde application, water was turned off creating a closed 

circuit between the biofilter and reservoir. The biofilter contained Biobloc 150 HD® media with a total 

volume of 0.667 cubic meter (m3

) and a total surface area of 25 m2. A nominal concentration of 40 

mg/L formaldehyde was achieved in the tanks, and this was allowed to pass through the biofilter. 

Formaldehyde was eliminated in 32 to 35 hours at 15 °C, and 60 hours at 10 °C, which approximated 

zero order kinetics. In a similar pilot-scale study, zero order elimination also was observed. In the fullscale experiment, a 13 percent increase in removal was found for every 1 °C increase, resulting in a Q10

(the change in rate of the measured variable that results when temperature is increased by 10 °C) of 3.4 

in the temperature range of 5.7 to 16.2 °C. No appreciable changes to biofilter function were observed 

(Pedersen and others, 2007). 

Breakdown of formaldehyde in a RAS with a trickling filter increased as temperature of the 

system increased (Pedersen and Pedersen, 2006). The estimated half-life of formaldehyde at 5.5 °C was 

16 hours, but only 5 hours at 14.5 °C. The results of the study by Pedersen and Pedersen (2006) indicate

that trickling filters can help remove formaldehyde and that bacterial decomposition played a large role 

in formaldehyde decomposition. 

Repetitive longer term static applications of formalin in a pilot-scale test of a freshwater RAS 

holding rainbow trout also were investigated. Specifically, the authors tested the effects of formalin on 

submerged upflow biofilter function and fish growth. The biofilters contained Biobloc 150 HD® media 

with a total volume of 0.166 m3 and a total surface area of 25 m2. The treatment tanks received 10–

20 mg/L formaldehyde either daily or weekly, and formaldehyde removal was monitored for 10 weeks. 

When low formalin levels were used daily, formaldehyde removal increased and nitrogen removal from 

the RAS was not affected. At intermittent doses, variability was seen in ammonia and nitrate oxidation. 

Overall abundance of ammonia-oxidizing and nitrogen-oxidizing bacteria was higher in the untreated 

tanks. Prolonged additions of low dose formalin resulted in increased removal of formaldehyde without 

affecting biofilter performance. Fish growth was not affected by the formalin treatments. One drawback 

to repeated treatments with formalin is protozoan resistance (Pedersen and others, 2010). 
11 

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

, kilogram per cubic meter; °C, degrees Celsius; mg/L, milligrams per liter; NR, not reported by authors]

Citation

Water 

type Scale

Fish 

biomass 

(kg/m3)

Water 

temperature 

(°C)

Formaldehyde 

dose (mg/L)

Duration 

(hours) Biofilter type

Heinen and 

others, 1995 Fresh NR 19.4–14 13.4–16.5 50–167 1

Fluidized-bed

sand

Heinen and 

others, 1995 Fresh NR 19.4–14 16–18 15–120 Indefinite

Fluidized-bed

sand

Keck and Blanc, 

2002 Marine Pilot 10 18–24 20–90 1

Fluidized-bed 

with 

Biospheres®

Keck and Blanc, 

2002 Marine Pilot 10 18–24 60 2–6

Fluidized-bed 

with 

Biospheres®

Pedersen and 

others, 2007 Fresh Full 8.8 14 40

1 dose, 

followed 

for 96 

hours

Submerged 

upflow

Pedersen and 

others, 2007 Fresh Full 8.8 14 40

1 dose, 

followed 

for 96 

hours

Submerged 

upflow

Pedersen and 

others, 2010 Fresh Pilot 8–14 17 10

2 times per 

week

Submerged 

upflow

Pedersen and 

others, 2010 Fresh Pilot 8–14 17 10 Daily

Submerged 

upflow

Pedersen and 

others, 2010 Fresh Pilot 8–14 17 20 Daily

Submerged 

upflow

1

Fish biomass had to be estimated because only the entire biomass range was reported. The 

estimate was made by dividing range of biomass provided by number of trials, then by the reported tank 

volume of 10 cubic meters. 

V. Conclusions

• The maximal level of formaldehyde that would not impair biofilter function was 40 mg/L or 

less. 

• Although minor alterations in the microbial abundance of ammonia-oxidizing and nitriteoxidizing bacteria occurred, repeated dosing of biofilters at low formaldehyde levels 

(10–20 mg/L or less) appeared to be safe and had no major adverse effects on biofilter function. 

• If a particular biofilter is repeatedly exposed to low levels of formaldehyde over the course of 

several weeks, the nitrite nitrifying bacteria could be inhibited. 

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