Management of common off-flavors

in pond-raised channel catfish

(Ictalurus punctatus)

Kevin K. Schrader, Ph.D.

U.S. Dept. of Agriculture, Agricultural Research
Service, Natural Products Utilization Research Unit
University, Mississippi 38677 USA
 CATFISH
PRODUCTION in USA

2010 : Total processor

sales =
U.S. $577 million

2010 : 214 million kg

catfish processed

Over 50% of U.S. catfish production occurs in

Mississippi (2011: 43,500 acres or 176 km2)
 Preharvest off-flavor
problems cost producers
U.S. $10-60 million
annually due to delayed
harvest

Losses due to:

• Additional feed costs
• Delay in stocking a new crop
• Diseases
• Bird depredation
• Water quality deterioration
Catfish are flavor tested prior to harvest:

1) 1-2 fish sampled two weeks prior to harvest

2) Additional fish (2-7) sampled day before or day of
harvest
3) At processing plant, 1-2
fish are sampled from live-
haul truck
4) Additional 7-8 fish may
be sampled
From: SRAC Publication #431 by Dr. Martine van der Ploeg
Preharvest off-flavor problems in pond-raised
channel catfish:

1) environmentally-derived sources
(e.g., microbial metabolites = earthy, musty;
grass/wild onion clippings = onion;
dead catfish = rotten, sulfury, sewage)

2) anthropogenic sources
(e.g., diesel, metallic)
Common environmental off-flavor metabolites:

1) “Musty”

2-Methylisoborneol or MIB
CH 3 CH3 CH3 CH 3

OH HO
CH 3 CH3

CH 3 CH3

Produced by the cyanobacteria (blue-green algae) species

Planktothrix perornata (Oscillatoria perornata)
Planktothrix perornata
Common environmental off-flavor metabolites:

2) “Earthy” CH 3 CH3

Geosmin or trans-1,10-
dimethyl-trans-9-decalol OH
CH 3
OH
CH3

A B

More prevalent in east Mississippi CH3 CH 3

and west Alabama catfish
ponds than ponds in Miss. Delta

(Anabaena spiralis; OH
CH3
OH
CH 3

other Anabaena spp.?) C D

Human sensory detection threshold (µg/kg)

Average consumer Flavor tester

MIB 0.7
0.1-0.2

Geosmin 8.4? 0.25-0.5

Microbial sources of geosmin and MIB

Cyanobacteria
Actinomycetes (Streptomyces, Nocardia)
Fungi (Penicllium spp.)
Myxobacteria (Myxococcus xanthus)

In recirculating aquaculture
systems, actinomycetes are
primary contributors
to off-flavors.
Geosmin-producing actinomycetes
Management Approaches
 Harvestaround off-flavor episodes---
farmers wait for odor-producing
cyanobacteria to disappear

 Prevention of off-flavor episodes: 1)

manipulation of nutrient availability; 2)
biomanipulation (use of planktivorous fish
such as tilapia Oreochromis aureus)
Management Approaches
 Depuration of off-flavors from fish flesh:

1) move crop of fish to pond free of off-flavor

compounds

2) application of algaecides
Management Approach: Depuration
after application of algaecides

 Reduce the abundance of P. perornata,

thereby reducing MIB levels in pond water

 MIBwill be eliminated from fish flesh by

passive diffusion across gills and
metabolism
Application of algaecides

 Only USEPA-approved algaecides:

1) diuron; 2) copper-based products (e.g.,
copper sulfate, chelated copper
products); and 3) sodium carbonate
peroxyhydrate (SCP) formulated products

 Each has several negative attributes

Algaecides
 Diuron (Karmex® DF, Karmex ® XP, Direx ®
4L); 10 µg/L application rates; 9
applications per pond per year
 Copper sulfate pentahydrate [0.25-2.0
mg/L]; chelated copper products
(Cutrine+, K-Tea) [0.2-0.5 mg Cu/L]

 Sodiumcarbonate peroxyhydrate (SCP)

products (Pak™ 27, GreenClean Pro)
Negative attributes of copper-based
products and diuron
 Low degree of selective toxicity towards
undesirable cyanobacteria
 High persistence in the environment

 Public’s negative perception of the use of

herbicides in aquaculture
 Diuron use can take months to provide
“on-flavor” catfish
 2-Methylisoborneol (MIB)
Levels in Pond 2 during 2003

3500
solid arrow indicates
diuron application
3000

2500

2000

1500

1000 catfish
harvested
MIB (ng/L) in Pond Wa

500

7 28 36 42 49 57 66 70
8/4 8/25 9/2 9/8 9/15 9/23 10/2 10/6
Time (d)
Management of Musty Off-flavor
in Channel Catfish from
Commercial Ponds with Weekly
Applications of Copper Sulfate

Kevin K. Schrader, C.S. Tucker, T.R.

Hanson, P.D. Gerard, S.K. Kingsbury, and
A.M. Rimando. 2005. North American
Journal of Aquaculture 67:138-147.
Copper Sulfate: Main Disadvantages
 At higher application rates (> 0.5 mg Cu/L), loss
of selective toxicity towards cyanobacteria

 Toxicity of cupric ion is inversely proportional

to pH, alkalinity, hardness, and dissolved
organic matter  difficult to achieve consistent
results
 Little difference between phytotoxic and
ichthyotoxic concentrations of copper
Copper Sulfate Treatments
 At each location, 6 ponds were untreated
(controls) and 6 ponds received weekly
applications of copper sulfate pentahydrate (0.5
mg/L CuSO4•5H2O or 0.12 mg Cu/L)
 Copper sulfate crystals (Phelps Dodge Corp.)
placed in double-layered burlap bags were
suspended in front of paddlewheel aerators
 Water current from aerators helped distribute
copper throughout the pond
 Applications occurred from June until water
temperatures dropped below 20oC
Results: Comparison of Fish Flavor

 In general, 55% reduction in MIB off-flavored

catfish

 Results were less dramatic than those observed

by Tucker et al. (2001)
Recommendations
 Use in ponds scheduled for harvest in summer or
autumn, especially those with chronic history of
musty off-flavor episodes
 Should commence before water temperature is
≥20oC (P. perornata more prevalent)
 Weekly treatments must be maintained
 Proper placement of burlap bags on outflow side of
aerators
 May be more practical for smaller commercial farms
(i.e., fewer ponds to manage)
SCP-based Products (e.g., Pak ™ 27)
 2Na2CO3 •3H2O2 = 85%
Na2CO3 = 12.7% Na2SiO3 = 1.4%

 2Na2CO3 • 3H2O2 _ 2Na2CO3 + H2O2

 Previousefficacy testing yielded

inconsistent positive results
Sodium Carbonate Peroxyhydrate (SCP)
Rapid Bioassay Results
Test Org anism: P. perornata

Test Product LOEC a LCIC b

Diuron 1.0 1.0
SCP 1.0 100.0
a
LOEC = Lowest -observed -effect concentration (in µM)
b
LCIC = Lowest -complete -inhibition concentration (in µM)

(Schrader et al. 1998. Aquaculture 163:85)

Efficacy testing of SCP in limnocorrals
 Limnocorrals (fiberglass enclosures; 2.4 m dia. and
1.5 m in height) placed in catfish pond
 SCP test concentrations: 2.5, 5.0, and 10.0 mg/L
 Positive results were inconsistent:
At 5 and 10 mg/L, MIB
levels decreased after
2 days; increased at
5 days
Barley straw extracts: algaecides?

 Commercial products claim the following:

“natural extract of barley straw”
“concentrated to work faster than barley
straw”
“goes to work on contact”

 Not
USEPA-approved for use in
aquaculture
Barley straw extracts
Rapid Bioassay Results
Test Org anism: P. perornata

Test Product LOEC a LCIC b

Microbe -Lift >5.0% >5.0%
Savio >5.0% >5.0%
a
LOEC = Lowest -observed -effect concentration
b
LCIC = Lowest -complete -inhibition concentration

Label-recommended application rate for Microbe-Lift is 0.0066% (v/v)

and for Savio is 0.0078% (v/v)
(Schrader. 2005. J. Aquat. Plant Manage. 43:100)
 USDA, Agricultural
Research Service,
Natural Products
Utilization Research Unit
University, MS 38677 USA
Thad Cochran National Center for Natural Products Research

Research Mission: Control of Undesirable

Microbes and Off-flavors in Aquaculture with
Natural Products
Approach
 Selectiveremoval of P. perornata
amongst other cyanobacteria, green
algae, and diatoms in catfish ponds

 Use a rapid bioassay to screen a large

number of natural compounds and
extracts (plants, marine algae and
cyanobacteria)
Development of Bioassay
 Must be rapid; permit the evaluation of
compounds (and extracts) at different
concentrations
 Must allow a determination of selective toxicity,
i.e., compound kills or prevents the growth of P.
perornata but not green algae (Selenastrum
capricornutum)
 Must be inexpensive and reproducible
Solution:

96-Well microplate bioassay

Schrader et al. 1997. Weed Technology 11:767
Compounds and Extracts Screened
 Pure natural compounds (hundreds)
(e.g., fatty acids, phenolics, quinones)

 Crude extracts (thousands)

(e.g., from aquatic and terrestrial plants,
marine organisms, marine algae and
cyanobacteria)
Promising compounds and extracts
 Compounds: complete growth inhibition at ≤ 1
mg/L (LCIC)
 Extracts: complete growth inhibition at ≤ 10
mg/L (LCIC)
 Selectivity of at least one order of magnitude
 Extracts undergo bioassay-guided fractionation
 Mini-scale up assay using a 6-well plate
 Promising compounds undergo efficacy testing
Compounds Tested in Ponds
 trans-Ferulic
acid: algistatic; half-life in
pond of 4 hours

 Menadione sodium bisulfite = derivative of

vitamin K3 and the active ingredient in
SeaKleen®
 Evaluation of Natural Product
SeaKleen®
 Produced by Garnett, Inc., Georgia, USA
 Used mainly to treat ballast water from ships
 Water-soluble
 Bioassay results: IC50 of 1.3 mg/L for P.
perornata compared to IC50 of 39.8 mg/L for
S. capricornutum
 Efficacy Testing of SeaKleen ®

 Limnocorrals were used

 Results found 1.3 mg/L to be effective
 Dissipation half-life was < 1 day
 Not economically practical for catfish
aquaculture due to cost: U.S. $41/kg and
higher application rate than diuron (10 µg/L)

(Schrader et al. 2004. N. Amer. J. Aquacult. 66:20)

 Efficacy Testing of Promising
Natural Compounds
 9,10-Anthraquinone: found in plant tannin
extracts from teak wood and in ryegrass (Lolium
perenne)
 Main ingredient in a commercial bird repellent
registered as a biopesticide with USEPA
 Problem: Not water soluble; not very soluble in
ethanol
Efficacy Tested in Summer of 1998

Result: Anthraquinone was not effective in controlling P. perornata in

the manner that it was applied, i.e., formulation problem
OO9,10-Anthraquinone
Novel Anthraquinone Derivative
 Provided by Dr. Dhammika Nannayakkara,
(National Center for Natural Products
Research, University of Mississippi)
 Modified anthraquinone to make it water-
soluble (natural-product derivative)
 Compound is patented (U.S. #6,949,250);
one cooperative agreement established
for primary development
 O

N
H
H3PO4

2-[Methylamino-N-(1’-methylethyl)]-
9,10-anthraquinone monophosphate = Anthraquinone-59
Synthesis of Anthraquinone-59

O O

CH2 Cl
1. H2 N N
H
H3PO 4
2. H 3PO 4
O O
 Comparison of Quinone Derivative
Comparison of 96 -h IC50 ( ? M)

Test Organism
Compound P. perornata S. capricornutum Selectivity
Cutrine-Plus 1.78 1.86 1.04
Diuron 0.10 0.13 1.30
Diquat 0.036 5.00 138.9
Anthraquinone -59 0.006 5.62 936.7
Efficacy Testing of Anthraquinone-59:
Methods
 Dose-response and single concentration
studies
 Limnocorrals; six replicates per
concentration and six controls; 125 µg/L
 Phytoplankton identification/enumeration
 MIB levels (SPME-GC-MS)
 Level of anthraquinone derivative (HPLC-
MS)
 Effect of Anthraquinone-59 on the Abundance
of Oscillatoria perornata

4000
Control
125 ppb Quinone a
Derivative
3000
a
a
=n6)

a
2000 a
a
a

1000
Limnocorrals (

b b b b
b
0
Filaments/mL of P

0 1 2 3 4 5 6 7

Time (d)
 Effect of Anthraquinone-59 on 2-Methylisoborneol
Levels in Pond Water

2500
Control
125 ppb Quinone a
Derivative
2000
a

a
1500 a
a a

1000

b a

500 b a

b
b
2-Methylisoborneo

0
Pond Water withi

0 1 2 3 4 5 6 7

Time (d)
 Effect of Anthraquinone-59 on the Abundance
of Green Algae

14000
Control
12000 125 ppb Quinone
Derivative a

10000
=n6)

8000

a
6000
a
4000
Limnocorrals (

a b b
2000 a b
a
a
a a
0
Natural Units/mL

0 1 2 3 4 5 6 7

Time (d)
 Effect of Anthraquinone-59 on the Abundance
of Diatoms

70000
Control a
60000 125 ppb Quinone
Derivative

50000
a
=n6)

40000

30000

a
20000
Limnocorrals (

10000
a
aa b b
aa b b
0
Natural Units/mL

0 1 2 3 4 5 6 7

Time (d)
 Third Efficacy Study of Anthraquinone-59:
Determination of Dissipation Rate and Half-life
in Catfish Pond Water

100

90
Anthraquinone-59
80

70
=n6)

60

50

40

30
in Pond Water

20
within Limnocorrals (

10
Half-life = 19 hours
0
Anthraquinone-5

0 10 20 30 40 50 60 70 80 90 100

Time (h)
Pond Efficacy Testing of
Anthraquinone-59: Methods
 Used 0.1 ha earthern, levee-type pond
 Stocking rate of 615 kg of channel catfish
 Pond volume determined by salt method
(Provine. 1976. Prog. Fish-Cult. 38:135)
 Two applications of 125 µg/L anthraquinone-59
 Continuous aeration after first application;
1 hour aeration after second application
(Schrader et al. 2007. N. Amer. J. Aquacult. 69:360)
Pond Efficacy Testing of
Anthraquinone-59: Methods
 Phytoplankton identification/enumeration
 Zooplankton identification/enumeration
 Measurement of major phytoplankton divisions
(chl a; bbe cuvette fluorometer)
 MIB levels (SPME-GC-MS)
 Three catfish caught at days 0, 7, and 14; fillets
analyzed for MIB and geosmin levels
(microwave distillation method; SPME-GC-MS)
 Effect of Anthraquinone-59 on MIB Levels and O. perornata Abundance
7000 7000
6500 6500
* = Application of Anthraquinone-59
6000 6000
5500 5500
5000 5000
4500 4500
4000 4000

(filaments/mL)
3500 3500
3000 3000
2500 2500
2000 2000

O. perornata Abundance of
1500 1500
MIB Levels (ng/L)

1000 1000
500 500
0 * * 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Time (d)
MIB and Geosmin Levels in Catfish Fillets

Sampling time (d) MIBa,b (µg/kg) Geosmina,b (µg/kg)

0 12.962z±1.868 0.129z±0.023
7 7.354y±0.935 0.055y±0.009
14 1.292x±0.982 0.026y±0.012
a
Mean (n = 3) ± standard deviation
b
Numbers in a column followed by the same letter are not significantly different
(P > 0.10)
Development Issues
 Patent 

 Commercial development 

 Cost-effective method for synthesis ?

 Scale-up?

 USEPA approval for use in aquaculture??

Muchas Gracias
Dr. Laurence Massaut
Cámara Nacional de Acuacultura,
Guayaquil, Ecuador
Questions?