Aquacultural Engineering 34 (2006) 377–388

www.elsevier.com/locate/aqua-online

Reporting standards for biofilter performance studies

John Colt a,*, Jonathan Lamoureux b, Richard Patterson c, Gary Rogers d
a
Northwest Fisheries Science Center, National Marine Fisheries Service,
2725 Montlake Blvd. East, Seattle, WA 98112-2097, USA
b
Engineered Agricultural Technologies, Torvmyrveien 2A, 4362 Vigrestad, Norway
c
Food Science and Technology, Dalhousie University, Halifax, NS, Canada B3J 2X4
d
Aquatic Eco-System, Inc., 2395 Apopka Blvd., Apopka, FL 32703, USA

Abstract

The development of standardized rating and design procedures for biological filters will require that filter performance be
evaluated and reported in a standardized manner. This article recommends draft reporting standards for biofilter performance
studies. It is important that critical parameters are defined and reported in a standard manner, both in terms of definition,
variable names, and units. Depending on the type and scale of an experiment, reporting of certain parameters will be either
mandatory or optional. Basic principles of experimental design, statistical analysis, and randomization must be followed.
Experimental protocols are recommended to ensure the accuracy of measured or computed parameters. The development of
this reporting standard is being organized through the Standards and Reporting Committee of the Aquaculture Engineering
Society (AES). It is anticipated that a revised version of these standards will be incorporated into the Guide to Authors for
Aquacultural Engineering.
Published by Elsevier B.V.

Keywords: Reporting standards; Biofilter performance; Reuse systems

1. Introduction are selected and sized on the basis of personal

A bewildering number of different types of
biological filters has been used in research and
production aquaculture systems. Because of differ-
ences in performance, operating characteristics, and
system requirements, it is difficult to rationally select
the ‘‘best’’ filter for a given application. Many systems
* Corresponding author. Tel.: +1 206 860 3243;
fax: +1 206 860 3467.
E-mail addresses: john.colt@noaa.gov (J. Colt),
jolamoureux@hotmail.com (J. Lamoureux), dick.patterson@dal.ca
(R. Patterson), garyr@aquaticeco.com (G. Rogers).
experience, marketing promises, and other non-
technical criteria.
The development of standardized rating and
design procedures for biological filters will require
that filter performance be evaluated and reported in a
standardized manner. Detailed information on the
physical size of the filter, media characteristics (type,
density, size, and specific surface area), water flow
rates, and specific loading rates will be required. It is
anticipated that filter performance will be evaluated
at several total ammonia nitrogen concentrations
under standardized conditions (temperature, salinity,

0144-8609/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.aquaeng.2005.09.002
378 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388

Nomenclature VBCE volumetric biomass capacity efficiency

(kg biomass/(m3 kWh)); 6d
Across cross-sectional area of filter (m2); 2e VFC volumetric feed capacity (kg feed/
Amedia total active surface area of media (m2); (m3 d)); 6a
2f VFCE volumetric feed capacity efficiency
ARE ammonia removal efficiency (mg TAN/ (kg feed(m3 kWh)); 6c
kWh); 5aa Vmedia media volume (m3); 2g
AREsys ammonia removal efficiency (system) VNR volumetric nitrite conversion rate
(mg TAN/kWh); 5bb (mg NO2-N/(m3 d)); 5r
BH0 bed height—no flow (cm or m); 2j VNRA apparent volumetric nitrite conversion
BHop bed height—operating (cm or m); 2k rate (mg NO2-N/(m3 d)); 5q
CFB cumulative feed burden (mg feed/L or VOCRhet volumetric oxygen consumption rate
ppm); 3e for hetertrophic bacteria (mg oxygen/
CL cumulative loading (kg/lpm); 3g (m3 d)); 5v
COC cumulative oxygen consumption VOCRnit volumetric oxygen consumption rate
(mg/L); 3f for nitrifying bacteria (mg oxygen/
D submergence depth (%); 2m (m3 d)); 5u
FR feed rate (kg/d); 3b VOCRtot volumetric oxygen consumption rate
FSR filter system ratio (%); 5j (mg oxygen/(m3 d)); 5t
Hdischarge water discharge height (m); 2c VTR volumetric TAN conversion rate
Htank filter height (cm or m); 2a (mg TAN/(m3 d)); 5p
Hwater water height (cm or m); 2b Greek symbols
Lhyd hydraulic loading rate (m3/(m2 d)); 2h DpH DpH (pH units); 5m
Lmedia hydraulic media loading rate DCO2 DCO2 (mg/L); 5l
(m3/(m2 d)); 2i DO2 DO2 (mg/L); 5k
LS loop strength (mg/L or ppm); 6e v rotational speed (rpm); 2n
NO2gen nitrite-nitrogen generation (%); 5n
NO3gen nitrate-nitrogen generation (%); 5o
OCR oxygen consumption ratio (%); 5w
OUE oxygen utilization efficiency (mg O2/
carbon/TAN or BOD/TAN concentrations, dissolved
kWh); 5cc
oxygen, and alkalinity).
P total power (average daily) (kW); 5z
Biofilter performance studies are difficult to
pHe equilibrium pH (pH units); 3o
conduct due to the large number of parameters that
PTR percent TAN removed (%); 5i
must be controlled and the number of measurements
Qfilter biofilter flow (lpm); 2o
that must be completed. Special care must be taken to
Qmu make-up flow (lpm); 2p
ensure that the ‘‘steady-state’’ condition is achieved
Qru rearing unit flow (lpm); 2q
before detailed data collection is started. Special
Qreuse reuse flow (lpm); 2r
protocols are proposed for cleaning of influent and
Qout system discharge flow (lpm); 2t
effluent piping to eliminate nitrification on these
SG specific gravity of media (unitless); 1g
surfaces. This problem is of special concern on bench-
SSA specific surface area (m2/m3); 1f
scale and pilot-scale filters. In larger systems, it may
STR surface TAN conversion rate (mg TAN/
be impossible to remove nitrifying bacteria and an
(m2 d)); 5s
estimate of non-filter nitrification is needed. It is
V0 filter volume—no media (m3); 2d
important that the principles of experimental design
VBC volumetric biomass capacity (kg bio-
and statistical tests are followed. Standard parameters
mass/m3); 6b
of interest, abbreviations, and units are proposed for
biofilter studies and reporting.
 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388 379

Table 1
Classification of the three basic types of biofilter studies
Type of study Typical biofilter size in liter Typical objectives ‘‘Wastewater’’ characteristics
Kinetic 1–50 Determination of the kinetics of ammonia Typically use a synthetic ‘‘wastewater’’
removal under very controlled conditions of defined composition, commonly
of temperature, substrate, etc. without significant BOD component
Pilot-scale 5 to >100 Evaluation of new media or filter May use synthetic wastewater or
configuration under controlled conditions waste from an experimental system
System 100–10000 Evaluation of new media or filter Use waste from large
configuration under production conditions production system

The objectives of this article are to present draft
reporting standards that will form the basis for the
future development of standardized rating and design
procedures for biological filters. These draft standards
are being developed for conventional fixed film
biofilters. Their application to high-intensity ponds
and photosynthesis driven production systems should
be made with care. The development of these
reporting standards is being organized through the
Standards and Reporting Task Force of the Aqua-
culture Engineering Society (AES). Once approved by
the general membership, these standards will be
incorporated in the Guide to Authors for Aquacultural
Engineering on a provisional basis. These standards
will be updated and modified as more experience is
gained in their use. Advances in technology and new
applications may also require modification and
additions to these standards. It is anticipated that
use of an updated version of these standards will
become mandatory for filter manuscripts submitted to
Aquacultural Engineering in the future.
2. Parameters of interest
Experimental biofilter studies can vary signifi-
cantly based on differences in size, scope, and
objectives. Three basic types of experiments in terms
of size, objectives, and wastewater characteristics are
presented in Table 1. This classification should be used
as a general guide only—not all biofilter studies will fit
into this simple classification.

Fig. 1. Example of a process flowsheet for a general reuse system. Not all components maybe present in a specific system nor arranged as shown.
380 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388

A process flowsheet for a general reuse system is The standardized parameters of interest for biofilter
presented in Fig. 1. Important flow include: experiments are presented in Table 2 for the following
general categories:
Biofilter flow (Qfilter): Total flow passing through the biofilter
Make-up flow (Qmu): Amount of new water input to system (1) Media characteristics.
Reuse flow (Qreuse): Amount of recycled water turned to (2) Filter characteristics.
the rearing unit
Rearing unit flow Total amount of water passing through
(3) General influent waste characteristics—culture
(Qmu + Qreuse): the rearing unit system supply.
System discharge flow Amount of discharge water (4) General influent waste characteristics—synthetic
(Qout): waste supply.
(5) Filter performance.
(6) System performance of filter—culture system.
A clear process flowsheet is required for each system
being studied and must show the general movement
of water flow and sampling locations. Photographs For each parameter, the basis or source of the
and highly dimensioned figures are not generally parameters, units, symbols, and priority for the three
needed (EIFAC, 1986) but a cross-sectional view types of studies are listed. For each type of study, the
may be needed to show actual water surface relation- priority of each parameter is listed as mandatory
ships. (MAN), optional (OPT), or not applicable (N/A). Those

Table 2
Parameters of interest for three types of biofilter experiments
Parameter Basis or source Units Symbol Priority of parametera
of parameter
Kinetic Pilot System
study study study
Media characteristics
1a Manufacturer Manufacturer or supplier Information MAN MAN MAN
1b Type Manufacturer or supplier Information MAN MAN MAN
1c Nominal size Manufacturer or supplier mm or cm MAN MAN MAN
1d Material Manufacturer or supplier Information MAN MAN MAN
1e Media dimensions Manufacturer or supplier cm or cm
cm MAN MAN MAN
1f * Specific surface area Manufacturer, supplier, m2/m 3 SSA MAN MAN MAN
or measurement
1g Specific gravity Manufacturer or supplier Value SG OPT OPT MAN
of media
1h Mass density of Manufacturer or supplier kg/m 3 OPT OPT OPT
dry media
Filter characteristics (not all parameters apply to all filters)
2a Filter height Measured; floor to cm or m Htank OPT MAN MAN
top of tank
2b Water height Measured cm or m Hwater OPT MAN MAN
2c Water discharge height Measured with m Hdischarge OPT OPT MAN
respect to grade
2d Filter volume—no media Computed m3 or L V0 MAN MAN MAN
2e Cross-sectional Computed m2 Across MAN MAN MAN
area of filter
2f * Total active surface Computed m2 Amedia MAN MAN MAN
area of media
2g Media volume Computed m3 Vmedia MAN MAN MAN
2h * Hydraulic loading rate 1.44Qfilter/Across m3/(m2 d) Lhyd OPT OPT MAN
 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388 381

Table 2 (Continued )
Parameter Basis or source Units Symbol Priority of parametera
of parameter
Kinetic Pilot System
study study study
2i * Hydraulic media 1.44Qfilter/Amedia m3/(m2 d) Lmedia OPT OPT OPT
loading rate
2j * Bed height—no flow Measured cm or m BH0 MAN MAN MAN
2k * Bed height—operating Measured cm or m BHop MAN MAN MAN
2l Type of water Manufacturer or supplier Product MAN MAN MAN
distribution system information
2m * Submergence Measured % D MAN MAN MAN
2n * Rotational speed Measured rpm v MAN MAN MAN
2o * Biofilter flow Measured or calculated lpm Qfilter MAN MAN MAN
2p * Make-up flow Measured or calculated lpm Qmu MAN MAN MAN
2q * Rearing unit flow Measured or calculated lpm Qru N/A MAN MAN
2r* Reuse flow Measured or calculated lpm Qreuse MAN MAN MAN
2t * Discharged flow Measured or calculated lpm Qout MAN MAN MAN
General influent waste characteristics—culture system supply
3a Culture species Operator Information N/A MAN MAN
3b Feed rate and Estimated kg/d and FR N/A N/A MAN
type of feed information
3c Feeding frequency Operator #/d N/A OPT OPT
3d Protein content of Nitrogen
6.25 % N/A OPT MAN
feed (as fed)
3e * Cumulative 106FR/1440Qmu mg/L CFB N/A MAN MAN
feed burden (or ppm)
3f * Cumulative oxygen See text mg/L COC N/A OPT OPT
consumption
3g * Cumulative loading kg fish/Qru kg/lpm CL N/A OPT MAN
3h * Total ammonia nitrogen Measured mg/L as N TANin MAN MAN MAN
3i * Nitrite-nitrogen Measured mg/L as N NO2-Nin N/A OPT MAN
3j * Nitrate-nitrogen Measured mg/L as N NO3-Nin N/A OPT MAN
3k 5-d biochemical Measured mg/L BOD5 MAN MAN MAN
oxygen demand
3l Chemical oxygen Measured mg/L CODin or OPT OPT OPT
demand or total TOCin
organic carbon
3m Alkalinity Measured mg/L as CaCO3 ALKin MAN MAN MAN
3n pH Measured pH units pHin MAN MAN MAN
3o * Equilibrium pH Measured pH pH units pHe OPT OPT OPT
after aeration
(with air)
3p Dissolved oxygen Measured mg/L DOin MAN MAN MAN
3q Carbon dioxide Measured mg/L CO2in OPT MAN MAN
General influent waste characteristics—synthetic waste supply
4a Total ammonia nitrogen Measured mg/L as N TANin MAN MAN MAN
4b 5-d biochemical Measured mg/L BOD5in MAN MAN OPT
oxygen demand
4c Chemical oxygen Measured mg/L CODin or OPT OPT OPT
demand or total TOCin
organic carbon
4d Alkalinity Measured mg/L as CaCO3 ALKin MAN MAN MAN
4e pH Measured pH units pHin MAN MAN MAN
4f Dissolved oxygen Influent DO in mg/L DOin MAN MAN MAN
waste stream
4g Source of ammonia Compound Information MAN MAN MAN
4h Source of BOD Compounds or brand Information MAN MAN N/A
382 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388

Table 2 (Continued )
Parameter Basis or source Units Symbol Priority of parametera
of parameter
Kinetic Pilot System
study study study
4i Nutrient or trace Compounds added mg/L or mg/L MAN MAN N/A
elements added
Filter performance
5a * Time of acclimation Experimental design d t MAN MAN MAN
before start of data
collection
5b Ammonia flux (1440Qfilter TANin)/Across mg TAN/ MAN OPT OPT
(cross-sectional area) (m2 d)
5c Ammonia flux (media area) (1440QfilterTANin)/Amedia mg TAN/ MAN OPT OPT
(m2 d)
5d BOD5 flux (media area) (1440QfilterBOD5in)/Amedia mg BOD5/ MAN OPT OPT
(m2 d)
5e Temperature Measured 8C T MAN MAN MAN
5f Total ammonia nitrogenout Measured mg/L as N TANout MAN MAN MAN
5g Nitrite-nitrogenout Measured mg/L as N NO2-Nout MAN MAN MAN
5h Nitrate-nitrogenout Measured mg/L as N NO3-Nout OPT OPT MAN
5i * Percent TAN removal (TANin  TANout)/TANin % PTR MAN MAN MAN
5j * Filter system ratio 1440Qfilter(TANin  TANout)/ % FSR OPT OPT MAN
(total TAN removed d)
(see text)
5k DO2 DOout  DOin mg/L DO2 OPT MAN MAN
5l DCO2 CO2out  CO2in mg/L DCO2 OPT MAN MAN
5m DpH pHout  pHin mg/L DpH OPT MAN MAN
5n Nitrite-nitrogen generation (NO2-Nout  NO2-Nin)/TANin % NO2gen OPT OPT OPT
5o Nitrate-nitrogen generation (NO3-Nout  NO3-Nin)/TANin % NO3gen OPT OPT OPT
5p * Volumetric TAN 1440Qfilter mg TAN/(m3 d) VTR MAN MAN MAN
conversion rate (TANin  TANout)/Vmedia
5q * Apparent volumetric 1440Qfilter(NO2-Nout  mg NO2-N/(m3 d) VNRA OPT OPT OPT
nitrite conversion rate NO2-Nin)/Vmedia
* 3
5r Volumetric nitrite VTR + VNRA mg NO2-N/(m d) VNR OPT OPT OPT
conversion rate
5s * Surface TAN 1440Qfilter(TANin  TANout)/Amedia mg TAN/(m2 d) STR OPT OPT OPT
conversion rate
5t * Volumetric oxygen 1440Qfilter(TANin  TANout)/Vmedia mg oxygen/(m3 d) VOCRtot OPT OPT OPT
consumption rate
5u * Volumetric oxygen (3.47VTR + 1.09VNR)(0.92) mg oxygen/(m3 d) VOCRnit OPT OPT OPT
consumption rate (see text)
for nitrifying bacteria
5v* Volumetric oxygen VOCRtot  VOCRnit mg oxygen/(m3 d) VOCRhet OPT OPT OPT
consumption rate
for hetertrophic bacteria
5w* Oxygen consumption ratio VOCRnit/VOCRtot % OCR OPT OPT OPT
5x Pumping power Computed using Qfilter, kW Ppump OPT MAN MAN
(average daily) actual filter head losses,
bed height, and
70% efficiency
5y Other power Measured or estimated kW Pother OPT MAN MAN
(average daily)
5z Total power Computed from kW Ptot OPT MAN MAN
(average daily) above parameters
5aa * Ammonia removal 1440Qfilter(TANin  TANout) mg TAN/kWh ARE OPT MAN MAN
efficiency /24Ptot
5bb* Ammonia removal See text mg TAN/kWh AREsys OPT OPT MAN
efficiency (system)
 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388 383

Table 2 (Continued )
Parameter Basis or source Units Symbol Priority of parametera
of parameter
Kinetic Pilot System
study study study
5cc * Oxygen utilization (1440Qfilter DDO)/24Ptot mg O2/kWh OUE OPT MAN MAN
efficiency
System performance of filter—culture system
6a * Volumetric feed capacity (kg feed/d)/Vmedia kg feed/(m3 d) VFC N/A N/A MAN
6b* Volumetric biomass kg biomass/Vmedia kg biomass/m 3 VBC N/A N/A MAN
capacity
6c Volumetric feed VFC/24Ptot kg feed VFCE N/A N/A MAN
capacity efficiency (m3 kWh)
6d Volumetric biomass VBC/24Ptot kg biomass VBCE N/A N/A MAN
capacity efficiency (m3 kWh)
6e Loop strength 106(kg/d feed)/1440Qru mg/L LS N/A N/A OPT
(or ppm)

Parameters superscripted with an asterisk (*) are discussed in more detail in the text.
a
MAN, mandatory; OPT, optional; N/A, not applicable.

parameters listed with a mandatory classification must
be reported for a specific type of study. Metric units are
used for all parameters (ASTM, 1982), but not all units
in Table 2 are SI units. Researchers are encouraged to
use the recommended symbol and must use the standard
units. Uniformity in reporting units will greatly assist in
the comparison of performance of different systems.
The use of English units is strongly discouraged, but
may be presented for special parameters in parenthesis
along side the standard metric units (for example, a flow
of 3.78 lpm (1 gpm)).
Although there are many parameters listed in
Table 2, some parameters are specific for a given
system or waste source and not all parameters are
mandatory. To conserve publication space, it is
recommended that this data be presented in a standard
appendix for biofilter studies, and not described in
detail in the main body of the text. Depending on the
specific parameter and experimental design, a para-
meter may be described as a single value, a mean
 standard deviation, or a range.
Most of the parameters in Table 2 are self-
explanatory. Some additional information is presented
below for selected parameters:
(1f) Specific surface area, SSA: Information is
generally supplied by the manufacturer. The accuracy
of this information is not defined at this time.
(2f) Total active surface area of filter, Amedia: The
total surface area of the filter can be computed from the
specific surface area of the media (SSA) and the media
volume (Vmedia). The active surface area of the filter is
the area of filter media occupied by bacteria. The value
of this parameter will depend on the total surface area of
the media (SSA
Vmedia), waste characteristics,
system type, and operating procedures. Except for
some specialized reactors, the computation of this
parameter is not a simple task and Amedia  S-
SSA
Vmedia. For two sizes of Kaldnes media, the
active surface area of media ranges from 66 to 73% of
the total media area (Rusten and Neu, 1999). The
Kaldnes media are used in moving-bed applications, so
the percentage of active surface area may be lower than
typical stationary plastic media. There does not appear
to be any standardized way of computing the total active
surface area of the filter, and when reported, authors
must document the method used in estimating Amedia.
(2h/2i) Hydraulic loading rate, Lhyd, and hydraulic
media loading rate, Lmedia: The first parameter is equal
to the filter flow in m3/d divided by the cross-sectional
area of the filter vessel (m2). The second parameter is
equal to the filter flow rate in m3/d divided by the
active surface area of the media (m2).
(2j/2k) Bed height—no flow, BH0, and bed
height—operating, BHop: These parameters are
needed for fluidized bed systems.
(2m/2n) Submergence (%) and rotational speed, v:
These parameters are needed for rotating biological
contactors systems.
(2o/2p/2q) Biofilter, make-up, reuse, rearing unit,
and discharge flows, Qfilter, Qmu, Qreuse, Qru, and Qout:
384 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388
These parameters are needed for computation of basic
performance numbers and their accuracy is critical in
biofilter studies. The flow measurements should be
accurate to 1%. This may be more of a problem with
larger systems because of the lack of straight pipe runs
necessary for the use of relatively inexpensive flow
meters. In small-scale systems, a simple volumetric
technique (bucket and stopwatch) may be adequate.
Because of the wide variety of systems used in
different aquaculture systems, flow measurement and
sampling sites must be clearly shown on the process
flowsheet (for example, see Fig. 1).
(3e) Cumulative feed burden, CFB: CFB is a
measure of intensity of the overall system. This
parameter is equal to:
mg feed=d
CFB ¼
liters of make-up flow=d
The units of CFB are mg feed/L or parts per million on
a weight basis. This parameter considers a control
volume surrounding the entire production system (the
largest box in Fig. 1). Internal processes and chemical
reactions are not considered.
(3f) Cumulative oxygen consumption, COC: The
COC is the total amount of oxygen consumed within
the rearing units (mg/L) and has been widely used to
describe the intensity in single-pass or serial reuse
systems. This parameter will depend on water
temperature, species, size, feed inputs, feeding
frequency, and general level of activity. While the
COC can be estimated by measuring the influent and
effluent DOs (DOin  DOout), accurate estimates of
COC requires measurement over the whole day on a
regular or continuous basis (Niimi, 1978; Steffensen,
1989) or can be estimated from the feeding rate and
oxygen feed ratio (Colt and Orwicz, 1991):
FR
OFR
COC ¼ PF
1440Qru
where PF is the peaking factor (1.2–1.4 for daily
maximum COC); FR the feeding rate (kg/d);
OFR the mg oxygen consumed/kg of feed (about
200,000 mg/kg feed for fingering and larger fish
and typical dry diet); Qru is the rearing unit flow (lpm).
(3g) Cumulative loading, CL: This is more a
conventional intensity parameter and is used in the
design of salmon and trout production systems.
(3h/3i/3j) Total ammonia nitrogen, TAN, nitrite-
nitrogen, NO2-N, and nitrate-nitrogen, NO3-N: All
nitrogen compounds should be reported in terms of
nitrogen (molecular weight = 14.01 g/mol). The fol-
lowing terminology should be used for ammonia
compounds:
Total ammonia nitrogen (TAN) Sum of NH3-N + NH4+-N
expressed in mg/L
Ionized ammonia (IA) NH4+-N expressed in mg/L
Un-ionized ammonia (UIA) NH3-N expressed in mg/L or
mg/L
Nitrite-N NO2-N expressed in mg/L
Nitrate-N NO3-N expressed in mg/L
At the current time, there are no recognized
standard methods for the computation of UIA. The
measure of solution pH at laboratory temperature may
introduce a 25–50% error in the estimates of UIA for
coldwater applications (J. Colt, unpublished).
Most ammonia tests measure the concentration of
TAN, although gas-sensing electrodes can directly
measure the concentration of UIA. Generally, the gas-
sensing electrodes are used to measure TAN by adding
a strong base to convert all ammonia components into
UIA before measurement.
The most commonly used method for measuring
TAN is the phenate method (Clesceri et al., 1998;
www.standardmethods.org). For TAN < 0.1–0.2 mg/L
the phenate method is subject to high and variable
blanks and difficulties at low concentrations. Within the
linear absorbance range of the phenate method, it may
be possible to increase the accuracy of the ammonia
removal estimate by zeroing out the spectrophotometer
with the influent water sample rather than a blank. This
will allow the difference in TAN between the two
samples to be read directly instead of subtracting two
separate readings. The use of this method has not been
documented in the published literature. More accurate
methods are available for the measurement of TAN
levels in the low mg/L concentrations (Holmes et al.,
1999).
(3o) Equilibrium pH, pHe: This is the pH of a
water sample in equilibrium with atmospheric carbon
dioxide (Colt and Orwicz, 1991) and depends
primarily on alkalinity and to a lesser degree on
water temperature. The change in pH upon aeration
(pHinitial  pHe) is a measure of the amount of carbon
 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388
dioxide in solution:
pHinitial  pHe ¼ positive;
CO2 supersaturated with atmosphere
pHinitial  pHe ¼ 0;
CO2 in equilibrium with atmosphere
pHinitial  pHe ¼ negative;
CO2 undersaturated with atmosphere
(5a) Time of acclimation before the start of data
collection: Compared to heterotrophic bacteria, the
autotrophic bacteria responsible for ammonia oxida-
tion grows very slowly. It may take up to 60 d for a
new filter to approach steady-state conditions, es-
pecially with some plastic media in seawater. In
general, the response of biofilters during the acclima-
tion phase is not very important, except that this phase
should be as short as possible.
(5i/5p) Percent TAN removal, PTR and volumetric
TAN conversion rate, VTR: These are two of the most
important biofilter performance parameters. They are
estimated by measuring influent and effluent TAN
across the biofilter (between points (c) and (d) in
Fig. 1). It is assumed that this parameter is a measure
of the steady-state performance of the biofilter. The
VTR does not require an estimate of the total
active surface area of media and can be used directly
for system design. These reported ammonia removal
rates should be characterized by a mean and standard
deviation. A PTR or VTR estimate based on a
single influent and effluent concentration may be
biased because of a lag between actual response
and measured response (Niimi, 1978; Steffensen,
1989).
Three statistical problems have been identified
when reporting PTR and VTR values. They include:
(1) the statistical definition of steady-state TAN
removal, (2) the randomization of treatments, and (3)
the proper replication of experimental units during
experimental design.
2.1. Definition of steady-state
Few biofilter studies have statistically defined what
is meant by ‘‘steady-state’’. Generally, steady-state is
385
achieved when the TAN in the effluent stabilizes and
a horizontal straight line can be drawn by eye
through observed points on a TAN versus time
graph. In a review of 14 biofilter articles published
in the last 15 years, only five definitions of steady-
state were found in the Material and Methods
sections:
 Biofilters were considered fully acclimated when a
steady-state culture was established, capable of
keeping TAN and NO2-N levels below 0.7 mg/L in
each system, under conditions of 8.64 g/d mass
ammonia loading for 7 consecutive days (Sandu
et al., 2002).
 Ammonium chloride was added to both systems at
the concentration of 2 mg/L and sea water was
recirculated until ammonia-N and nitrite-N were
reduced to 0.2 mg/L; it took 7 d (Menasveta et al.,
2001).
 The system reached steady-state when the TAN
removal rate equaled the rate of addition, which
could be indicated by a stable TAN concentration
(Zhu and Chen, 2001).
 The ammonia removal rate was monitored until a
quasi steady-state condition for nitrifying popula-
tions were established after approximately 8 weeks
(Greiner and Timmons, 1998).
 Continuous feeding was maintained until a steady-
state culture, as evidence by a stable TAN
concentration for each reactor at a fixed TAN
feeding rate, was established. This required about 5
weeks of operation (Zhu and Chen, 2002).
No statistical tests were used to determine ‘‘steady-
state’’ in any of the studies reviewed. One approach
would be to perform a linear regression on TANout
versus time and test to see if the slope of this line is
statistical different from 0 at a stated probability level.
The time for development of ‘‘steady-state’’ condi-
tions may also be different for ammonia and nitrite
oxidation or other measures of biofilter performance.
Additional work is needed to statistical define the
‘‘steady-state’’ condition in biofilter studies.
2.2. Randomization of treatments
When biofilters are tested at a number of
environmental conditions (e.g., 30, 25, 20 15, 10,
386 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388
and 5 8C), both the order of the treatments and location
must be randomized. It is not an acceptable
experimental design to study the effects of tempera-
ture in a uniform manner (30, 25, and 20 8C, etc.),
although it would certainly reduce the acclimation
time when temperatures are changed. It is not
desirable to have all the 30 8C treatments grouped
together (e.g., next to the window), although equip-
ment constraints may make this necessary. Randomi-
zation of treatment locations and levels can be based
random number tables, random number generators, or
use of slips of paper in a hat. Regardless of the
technique used, it may be necessary to repeat the
process several times to find an acceptable ‘‘random’’
selection. Additional information on randomization
can be found in Cochran and Cox (1957) and standard
statistical texts.
2.3. Replication of experimental design
When replicate experimental units are available,
the location of replicates for a single treatment
should not be grouped in the same location (for
example, all four replicates for the BOD/TAN = 0.5
should not be located closest to the window). It may
be difficult to randomized biofilter location for
experiments involving temperature when a water
bath is used to maintain temperature. This is a
common problem in agricultural trials and requires
the use of a blocked experimental design (Cochran
and Cox, 1957).
It is important to understand what is and what is
not a true replicate. Assume that three biofilter are
operated with different influent TAN concentrations
for 8 weeks until the effluent TAN remains ‘‘stable’’.
Then, the biofilter is operated for 4 weeks and the
mean weekly PTR and VTR are computed from daily
TAN values. The 4 weekly PTRs and VTRs are not
true replicates, but repeated measures and cannot be
used in conventional analysis of variance. Com-
monly, it is found that a series of repeated
measurements are autocorrelated (the value of
parameter at t + 1 depends on the value at t). True
replication for this example would require 12
independent systems operated for an 8-week accli-
mation period, followed by 1-week of observations.
It is also possible to replicate the experiment in time
by repeating the experiment four times (36 weeks
total duration). Ideally, the biofilter would be cleaned
at the start of each experiment and acclimated in the
same manner and both concentration and location
randomized. Seasonal changes in temperature, light,
and personnel can have significant negative impacts
on this approach.
If there are questions about the uniformity of
conditions between replicate systems, a uniformity
trial should be conducted (a single treatment level
assigned to all replicates). For most researchers
working on biofilters, consultation with a statistical
consultant prior to the start of experimental work is
highly recommended. No amount of statistical
analysis can save a poorly designed experiment.
Biofilter experiments take a major expenditure of time
and resources; some statistical short-cuts may be
necessary to complete experimental work within a
reasonable time and budget.
The publication of unreplicated experimental work
is becoming difficult in peer-reviewed journals.
Statistical analysis generally requires replicated
experiments (one exception will be discussed below).
Experiments can be replicated by use of a number of
similar units or replicated by repeating the experi-
ments in time. For new systems, it may be necessary to
conduct preliminary experiments to estimate varia-
bility. This initial estimate of variability can be used to
determine the number of replicates needed to test for
the required difference in means (Quin and Keough,
2002).
With well-behaved data, it is possible to make
statistical tests on unreplicated data. This involves
fitting regression lines to the data points (for example,
impact two different BOD/TAN ratios on TAN
removal kinetics) and checking for statistical differ-
ences between the slopes and intercepts of different
regression lines. Some aquaculture journals as a
matter of publication policy will not accept unrepli-
cated studies except for special experimental designs.
The publication of un-replicated biofilter studies
should be restricted to evaluation of large systems,
where the construction and operation of multiple
systems can be very expensive. Depending on the
objectives of these studies, 4–16 months of data may
be needed.
(5j) Filter system ratio, FSR: The FSR is equal to
the amount of TAN removed within the filter per day
divided by the total amount of TAN removed in the
 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388
entire system by nitrification:
1440Qfilter ðTANin  TANout Þ
FSR ¼
Total nitrification in system ðmg=dÞ
The total nitrification in the system considers the
ammonia removed by: (1) bacteria attached to the
inside of pipes, fittings, and other unit processes, (2)
suspended bacteria in the water column, and (3)
bacteria attached to the filter media. Although limited
information is available, FSR values vary from 50 to
90% in laboratory systems (Malone and Beecher,
2000; Hargrove et al., 1995).
Special care must be taken to prevent nitrification
from occurring in storage reservoirs and feed lines
during filter performance tests. The refrigeration of
feed solutions will help retard the growth of nitrifying
bacteria, but fresh feed solutions must be prepared
every 3–5 d depending on temperature. Nitrifying
bacteria grow extremely well on some plastic tubing
(e.g., Tygon) so they must be carefully cleaned every
3–4 d to prevent significant loss of ammonia.
Under cases where suspended nitrifying bacteria
are not significant, it may be possible to estimate FSR
values by removing the media from the filter and
observing the resulting nitrification rate (Hargrove
et al., 1995) or running a blank system (a reactor
without media).
Information on FSR is more important in small
systems, because the ratio of filter:non-biofilter TAN
removal may not scale linearly as the size of the
system is increased. A low value of FSR for a
laboratory system will increase the uncertainty of the
estimation of the performance of larger systems. In
full-scale production systems, the value of FSR is not
as important for design as the ratio of the filter:non-
biofilter TAN removal is fixed by the design. Another
system of the same size (and operation) should have a
similar FSR value.
If significant nitrification from suspended bacteria
is occurring, some type of general mass balance
approach will be needed to estimate FSR. In high
intensity conventional pond and heterotrophic reactor
systems, uptake of ammonia by suspended bacteria,
algae, and other microorganisms may be the primary
ammonia removal mechanism.
(5q/5r) Apparent volumetric nitrite conversion
rate, VNRA and volumetric nitrite conversion rate
387
(VNR): The apparent volumetric nitrite conversion
rate is a function of the influent and effluent nitrite
value:
1440Qfilter ½ðNO 
2 -Nout Þ  ðNO2 -Nin Þ
VNRA ¼
Vmedia
The actual volumetric nitrite conversion rate must
consider the total amount of TAN converted to nitrite
(Malone and Beecher, 2000) and is equal to:
VNR ¼ VTR þ VNRA
(5s) Surface TAN conversion rate, STR: This is
equal to the amount of TAN (mg) removed per day
divided by the total active surface area of the media
contained in the biofilter. As previously discussed, the
computation of the total active surface area of the
media is difficult.
(5t) Volumetric oxygen consumption rate, VOCRtot:
This is a measure of the total oxygen consumption (mg/
d) divided by the volume of media in the biofilter. The
VOCRtot includes the oxygen consumed by both the
nitrifying and the heterotrophic bacteria contained in
the biofilter.
(5u) Volumetric oxygen consumption rate for
nitrifying bacteria, VOCRnit: In theory, 4.571 mg O2
is necessary to convert 1 mg of TAN into nitrate
(NO3). From this, the VOCRnit can be related to the
VTR alone (assuming that the conversion from TAN to
NO2 limits the conversion from NO2 to NO3):
1440Qbiofilter DDO
VOCRnit ¼
Vbiofilter
1440Qbiofilter ½ð4:571Þ D TAN
¼
Vbiofilter
¼ ð4:571 g O2 =g TANÞVTR
A form of this equation was presented (Golz et al.,
1999) for floating bead filters, relating VTR and VNR
to VOCRnit:
VOCRnit ¼ ð3:47 VTR þ 1:09 VNRÞ
0:92
The factor 0.92 corrects for oxygen assimilation
during bacterial growth. Additional experimental
work is needed to assess the accuracy of this equation
for other biofilters and operating conditions.
(5v/5w) Volumetric oxygen consumption rate for
heterotrophic bacteria, VOCRhet: This parameter is
computed by subtracting the nitrifying component
388 J. Colt et al. / Aquacultural Engineering 34 (2006) 377–388
(VOCRnit) from the total oxygen consumption
(VOCRtot).
(5w) Oxygen consumption ratio, OCR: This
parameter is equal to the oxygen consumption by
nitrifying bacteria divided by the total oxygen
consumption across the biofilter:
VOCRnit
OCR ¼
VOCTtot
(5aa/5bb/5cc) Ammonia removal efficiency, ARE,
ammonia removal efficiency-system, AREsys, and
oxygen utilization efficiency, OUE: The ARE and
OUE are the daily TAN removed and oxygen
consumed in the biofilter divided by the energy
consumed. If a single pump is used for the biofilter and
gas transfer systems, the allocation of the power to the
biofilter must be clearly stated.
(6a/6b) Volumetric feed capacity, VFC: This
parameter describes the amount feed/d a volume of
biofilter can support and is an overall measure of
system efficiency and intensity. Because feed input
integrates feeding rate (and therefore animal size) and
biomass, it is likely that volumetric biomass capacity
(VBC) will depend more on animal size than the
volumetric feed capacity (VFC).
Acknowledgements
We would like to thank Tim Pfeiffer, Dallas
Weaver, Barnaby Watten, and Tom Losordo for review
of earlier drafts of this article.
References
ASTM, 1982. Standards of Metric Practice. ASTM E 380-82.
American Society for Testing and Materials, Philadelphia, PA.
Clesceri, L.S., Greenburg, A.E., Eaton, A.D. (Eds.), 1998. Standard
Methods for the Examination of Water and Wastewater. 20th ed.
APHA, Washington, DC.
Cochran, W.G., Cox, G.M., 1957. Experimental Design, 2nd ed.
John Wiley & Sons, New York.
Colt, J., Orwicz, K., 1991. Modeling production capacity of aquatic
culture systems under freshwater conditions. Aquacult. Eng. 10,
1–29.
EIFAC, 1986. Report of Working Group on Terminology, Format
and Units of Measurements as Related to Flow-through and
Recirculation Systems. European Inland Fisheries Advisory
Commission, EIFAC Tech. Paper 49. Food and Agricultural
Organization of the United Nations, Rome.
Golz, W.J., Rusch, K.A., Malone, R.F., 1999. Modeling the major
limitations on nitrification in floating-bead filters. Aquacult.
Eng. 20, 43–61.
Greiner, A.D., Timmons, M.B., 1998. Evaluation of the nitrification
rates of microbead and trickling filters in an intensive recirculat-
ing tilapia production facility. Aquacult. Eng. 18, 189–200.
Hargrove, L.L., Westerman, P.W., Losordo, T.M., 1995. Nitrification
in three-stage and single-stage floating bead biofilters in a
laboratory-scale recirculating aquaculture system. Aquacult.
Eng. 15, 67–80.
Holmes, R.M., Aminot, A., Kérouel, R., Hooker, B.A., Peterson,
B.J., 1999. A simple and precise method for measuring ammo-
nium in marine and freshwater ecosystems. Can. J. Fish. Aquat.
Sci. 56, 1801–1808.
Malone, R.F., Beecher, L.E., 2000. Use of floating bead filters to
recondition recirculating waters in warmwater aquaculture pro-
duction systems. Aquacult. Eng. 22, 57–73.
Menasveta, P., Panritdam, T., Sihanonth, P., Powtongsook, S.,
Chuntape, B., Lee, P., 2001. Design and function of a closed,
recirculating seawater system with denitrification for the
culture of black tiger shrimp broodstock. Aquacult. Eng. 25,
35–49.
Niimi, A.J., 1978. Lag adjustment between estimated and actual
physiological responses conducted in flow-through systems. J.
Fish. Res. Board Can. 35, 1265–1269.
Quin, G.P., Keough, M.J., 2002. Experimental Design and Data
Analysis for Biologists. Cambridge University Press, Cam-
bridge, UK.
Rusten, B., Neu, K.E., 1999. Down to size: moving-bed biofilm
reactors move into the small-flow treatment arena. Water
Environ. Tech. (January), 27–33.
Sandu, S.I., Boardman, G.D., Watten, B.J., Brazil, B.L., 2002.
Factors influencing the nitrification efficiency of fluidized
bed filters with a plastic bead medium. Aquacult. Eng. 26,
41–59.
Steffensen, J.F., 1989. Some errors in respirometry of aquatic
breathers: how to avoid and correct for them. Fish Phys.
Biochem. 6, 49–59.
Zhu, S., Chen, S., 2001. Impact of Reynolds number on nitrification
biofilm kinetics. Aquacult. Eng. 24, 213–229.
Zhu, S., Chen, S., 2002. The impact of temperature on nitrification
rate in fixed film biofilters. Aquacult. Eng. 26, 221–237.