Aquaculture environment interactions

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AQUACULTURE ENVIRONMENT INTERACTIONS

Aquacult Environ Interact

Vol. 5: 107–116, 2014

doi: 10.3354/aei00100

Published online May 8

INTRODUCTION

Taste-and-odour compounds (TOCs) can reduce

the palatability of freshwater fish produced in aqua-

culture systems, especially when water recirculation

is applied (Robertson & Lawton 2003, Smith et al.

2008). The most commonly identified TOCs are

geosmin (earthy flavour) and 2-methylisoborneol

(MIB; mildewed flavour), and both of these off-

flavours have been shown to concentrate 200- to 400-

fold in fish flesh, relative to the ambient concentra-

tion (Howgate 2004). TOCs are produced by several

© The authors 2014. Open Access under Creative Commons by

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restricted. Authors and original publication must be credited. 

Publisher: Inter-Research · www.int-res.com

*Corresponding author: [e-mail]

Geosmin off-flavour in pond-raised fish in southern

Bangladesh and occurrence of potential 

off-flavour producing organisms

Mikael A. Petersen

1

, Md. Ariful Alam

2

, Md. Mizanur Rahman

2

, Md. Lokman Ali

2

, 

Sultan Mahmud

2

, Louise Schlüter

3

, Niels O. G. Jørgensen

4,

*

1

Department of Food Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

2

Faculty of Fisheries, Patuakhali Science and Technology University, Dumki, Patuakhali 8602, Bangladesh

3

Environment and Toxicology, DHI Group, Agern Allé 5, 2970 Hørsholm, Denmark

4

Genetics and Microbiology, Department of Plant and Environmental Sciences, University of Copenhagen, 

Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

ABSTRACT: Pangas Pangasianodon hypophthalmus and tilapia Oreochromis niloticus were culti-

vated for 6 mo in earthen ponds in Bangladesh to examine occurrence of the off-flavour geosmin

in water and fish and to test procedures for reduction of off-flavour. In the ponds (~1 m depth and

area of 400 m

2

), the average geosmin concentration was 3.9 ng l

−1

(range 0.2 to 20 ng l

−1

). No

effects of season or water treatment (sand filtration or probiotic microbes) were found. The content

of geosmin in the fish was 21 ng kg

−1

(range: 0.0 to 91 ng kg

−1

) for pangas and 17 ng kg

−1

(range:

0.0 to 68 ng kg

−1

) for tilapia. Water treatment reduced the geosmin content by 56 to 74% in pan-

gas, but no effect was found in tilapia. Likewise, depuration for 

≥12 h in groundwater lowered the

geosmin content in pangas (by 65 to 90%) but not in tilapia. Sensory analysis indicated a positive

effect of both water treatment and depuration, and the fish were graded ‘no or mild flavour’ after

such treatment, compared to ‘strong off-flavour’ in controls. Abundance analyses of known off-

flavour producing microorganisms (streptomycete bacteria and cyanobacteria) showed a high

density of streptomycetes (0.5 to 13% of the bacterial population), while cyanobacteria made up a

maximum of 9.3% of the phytoplankton biomass or were absent. This first study on off-flavours in

pangas and tilapia in Bangladeshi ponds indicates that geosmin was not a major off-flavour in the

fish, but improvement of sensory quality by water treatment and depuration suggests that other,

unidentified off-flavours were present in the fish.

KEY WORDS:  Off-flavour · Aquaculture · Pangasianodon hypophthalmus · Oreochromis niloticus ·

Streptomycetes · Cyanobacteria · Geosmin · GC-MS · Sensory analysis

O

PEN

PEN

 

A

CCESS

CCESS

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Aquacult Environ Interact 5: 107–116, 2014

species of cyanobacteria (also known as blue-green

algae) and by non-photosynthetic Streptomyces bac-

teria (Gram-positive, filamentous bacteria), but recent

research indicates that other bacteria, e.g. species

within the order Myxococcales, may also produce

off-flavours in some aquaculture systems (Zaitlin &

Watson 2006, Auffret et al. 2013).

TOCs impact taste and flavour of fish in both

warm- and cold-water aquaculture systems. In warm

water, tainting by geosmin and MIB was observed in

barramundi Lates calcarifer from freshwater lakes in

northern Australia (Percival et al. 2008), in catfish

Ictalurus punctatus from ponds in Mississippi and

Alabama, USA (Schrader & Dennis 2005), and in

tilapia Oreochromis niloticus reared in pond cages in

Thailand (Gutierrez et al. 2013). In cold-water sys-

tems, tainting of rainbow trout Oncorhynchus mykiss

by geosmin and MIB was detected in outdoor tanks

with recirculation in Denmark (Petersen et al. 2011)

and in arctic charr Salvelinus alpinus from an indoor

system with water recirculation in West Virginia,

USA (Houle et al. 2011). TOCs reduce market value

of fish from aquaculture due to consumer reluctance

to buy food with an earthy or mildewed taste, but the

actual reduction in market value of fish due to taint-

ing is difficult to determine. For American catfish,

TOCs have been estimated to reduce the sale by

30% (Engle et al. 1995).

In Bangladesh, occurrence of off-flavour in pan-

gasiid catfish has been reported for fish reared in

ponds (Khan et al. 2009), but the extent of off-flavour

problems in pond-raised fish in Bangladesh has not

been scientifically documented. Various treatments

of fish meat to reduce off-flavours are practised in

Bangladesh and Indian households, e.g. dipping and

washing in tamarind pulp, lemon juice, lemon grass,

salt solution, or a suspension of banana leaf ash, and

some of these treatments appear to improve the sen-

sory quality of the fish (Mohsin et al. 1999).

In large and industrial aquaculture systems, off-

flavours in fish may be reduced by depuration for few

to several days in clean water and without feed

before slaughtering (Robertson et al. 2005, Percival

et al. 2008). Depuration is an efficient procedure for

off-flavour reduction, but access to clean and off-

flavour-free water may be limited, and the fish lose

weight (and hereby value) during the treatment

(Burr et al. 2012).

In this study, the presence of geosmin and proce-

dures for its removal from water and fish were exam-

ined in tilapia Oreochromis niloticus and pangas

Pangasianodon hypophthalmus (also known as pan-

gasius), reared in local earthen ponds in southern

Bangladesh (Dumki Upazila). The 2 fish species con-

stitute a major source of animal protein to the local

population, and both species are of large economic

importance (Ahmed et al. 2012). When considering

the importance of the 2 fish species, the lack of stud-

ies on sources of the reported off-flavour problems

(see above) is surprising. In southeast Asia, the con-

tent of geosmin has been examined in tilapia in Thai

fish ponds (Gutierrez et al. 2013), but the content of

off-flavours in wild or farmed pangas has not previ-

ously been measured. Here, we determined the con-

tent of geosmin in water and fish (tilapia and pangas)

by a dynamic headspace extraction method and GC-

MS detection. MIB was targeted for inclusion in the

study, but the compound could not be quantified

with an acceptable accuracy. In addition, the taste

and flavour of the fish were characterized by sensory

analysis. Different procedures were applied to re 

-

duce potential off-flavours in water and fish, includ-

ing sand filtration and probiotic treatment of the

pond water during the production phase as well as

depuration of the fish in groundwater before slaugh-

tering. The presence of potential off-flavour produc-

ing organisms (cyanobacteria and streptomycetes)

in the water was examined by a chromatographic

method (for cyanobacteria) and a molecular ap 

-

proach (for streptomycetes).

MATERIALS AND METHODS

Set-up and sampling

Details on ponds, experimental set-up, growth

rates of fish and water quality are reported sepa-

rately (S. Mahmud et al. unpubl.). Briefly, 8 earthen

ponds of 309 to 497 m

2

area and with depth of 1.1 to

1.6 m were selected in Dumki Upazila, Patuakhali,

in southern Bangladesh (22° 21

′ 15″ N, 90° 23′21″ W).

Each pond received 50 tilapia Oreochromis niloticus

fry (5.2 to 5.3 g each) and 30 pangas Pangasianodon

hypophthalmus fry (62 to 75 g each) in late January

2012. The fish were fed artificial feed (Mega Fish

sinking feed pellets; www.spectragroup.com.bd) at

the rate of 30% of their body weight for first month,

20% for the next month and 3 to 10% for the last

4 mo. Planktonic matter in the ponds is another

important food source to the fish. After 6 mo, the fish

had grown to 182−199 g (tilapia) and 689−870 g (pan-

gas). At termination of the study (early August 2012),

selected fish were taken for depuration in cement

tanks with ~2 m

3

groundwater for up to 48 h. The

water was not exchanged and no agitation was

108

---

Petersen et al.: Geosmin in pond-raised fish

applied during the depuration. After slaughtering,

each of the fish were divided into 2 fillets. One fillet

was used for chemical analysis of content of geosmin

and MIB in the flesh, while the other fillet was used

for sensory analyses (see ‘Sensory profiling of fish’).

The fillets were kept frozen in individual plastic bags

at −20°C until analysis.

To test the effect of water treatment on the content

of off-flavour sources in the water, the following

treatments were performed in duplicates (2 ponds for

each treatment): continuous filtration of the water

through a sand filter, addition of probiotic material,

either PondPlus® by Novozymes (www.novozymes.

com) or AquaPhoto by ACI (www.aci-bd.com) at

weekly intervals according to the producer’s instruc-

tions, or no treatment (controls). The probiotic prod-

ucts are designed to improve remineralisation of

inorganic nutrients to promote growth of fish-benefi-

cial phytoplankton.

Samples for analysis of off-flavours in the water

were collected every second month. Volumes of

10 ml water were transferred in duplicates to 20 ml

glass vials and treated with 3 g NaCl before capping

with silicone-PTFE seals and were kept at 4°C until

analysis. To detect potential off-flavour producing

organisms in the water, volumes of 200 and 500 ml

water were filtered through 47 mm diameter 0.2 µm

pore size cellulose nitrate membrane filters (for

Streptomyces bacteria) or 47 mm diameter GA 100

GF/F filters (for cyanobacteria; www.advantecmfs.

com) (see below). The filters were folded and

wrapped individually in aluminium foil and kept at

−20°C until  analysis.

Dynamic headspace sampling of volatiles

 (including geosmin) in water and fish

Volatiles were extracted from water samples and

fish flesh by dynamic headspace sampling using pro-

cedures similar to those applied by Petersen et al.

(2011). Briefly, water samples were transferred to a

gas-washing bottle, which was immediately closed

with a purge head. The bottles were purged with

100 ml of N

2

min

−1

for 60 min in a water bath at 37°C,

simultaneous with collection of volatiles on Tenax TA

traps. To extract volatiles from the fish fillets, 10 g of

fish flesh was transferred to a gas-washing bottle

with 20 ml of water and 100 µl of internal standard

solution (5 mg 4-methyl-1-pentanol l

−1

). The flesh

was homogenized for 45 s at 13 500 rpm using an

Ultra Turrax homogenizer (www.ika.com) followed

by rinsing with 10 ml of water before immediate clos-

ing with a purge head. The bottles were purged with

100 ml of N

2

min

−1

for 60 min in a water bath at 50°C

with simultaneous collection of the volatiles on Tenax

TA traps. After headspace sampling, the Tenax traps

were dried at 100 ml min

−1

flow of dry N

2

for 10 min.

Analysis of volatiles by GC-MS

Volatiles were stripped from the traps in an auto-

matic 2-step thermal desorption unit (ATD 400;

Perkin Elmer). In the first step, the volatiles were des-

orbed by heating to 250°C at 60 ml He min

−1

for

15 min. The volatiles were focussed on a cold (5°C)

Tenax TA trap that subsequently was heated to

300°C for 4 min (second step). The 2-step procedure

allowed for a rapid transfer of volatiles to a GC-MS

(7890A GC system interfaced with a 5975C VL MSD

with a Triple-Axis detector; www.agilent.com) by a

heated (225°C) transfer line.

The volatiles were separated on a DB-Wax capil-

lary column (30 m 

× 0.25 mm internal diameter and

0.50 µm film thickness) at 1 ml He min

−1

and with a

temperature program of 40°C (10 min) and 8°C min

−1

until 240°C (5 min). The mass spectrometer was oper-

ated in electron ionization mode at 70 eV, and mass-

to-charge ratios between 15 and 300 were scanned.

Simultaneously, data were collected in selected ion

monitoring mode, monitoring mass 95 for MIB and

mass 112 for geosmin. Peak identity was confirmed

by probability-based matching of mass spectra

with those of a commercial database (Wiley275.L,

HP

product no. G1035A) and by comparison

with retention times of authentic standards. For

analysis of the acquired data, MSD Chemstation soft-

ware (v. E.02.00, Agilent Technologies) was used. For

absolute quantification of geosmin and MIB in fish

meat, amounts of 0.25, 1, and 4 µg kg

−1

were added

to meat from 4 selected fish during homogenization.

Correspondingly, absolute quantification of water

samples was carried out by adding 1, 5, 10, 50, and

100 ng l

−1

to samples of 10 ml water with 3 g NaCl.

Serial dilutions of the geosmin and MIB stocks (in

methanol; products G5908 and M3933, www. sigma-

aldrich.com) were performed in ethanol, but water

was used for the most dilute concentrations. An

acceptable linearity between peak area and concen-

tration was observed (R

2

= 0.91 to 0.93), and the

obtained graphs were used to generate peak area-to-

concentration calibration curves. MIB was identified

in most of the chromatograms but co-eluted with an

unknown compound. Therefore, no results for MIB in

water or fish are presented.

109

---

Aquacult Environ Interact 5: 107–116, 2014

Sensory profiling of fish

Sensory evaluation of the fish was performed at

Patuakhali Science and Technology University by a

panel of 10 people. The panel members were not

trained in off-flavour characterization but consisted

of typical local consumers of freshwater fish: 2 mem-

bers of the Fish Breeder Community Based Organi-

zation (CBO), 2 teachers and 2 students at Patuakhali

Science and Technology University, 2 business per-

sons, and 2 local fish farmers. The sensory partici-

pants did not receive training in identification of

geosmin or MIB before the tests but were asked to

categorize the fish flavour (Table 1). Fillets from the

fish were prepared in boiling water for 10 min,

divided in 10 pieces, and served randomly and with-

out identification to the panel members.

Identification of potential off-flavour producing

organisms

Densities of Streptomyces bacteria in the water

were determined by quantitative PCR (TaqMan pro-

cedure) targeting the 23S rRNA gene according to

Rintala & Nevalainen (2006) with the modifications

given by Lylloff et al. (2012). Genomic DNA was

extracted from the 47 mm membrane filters after cut-

ting each filter in 3 portions, using the PowerWater

®

DNA isolation kit by Mobio (www.mobio.com)

according to instructions by the producer. The PCR

reactions were performed in an Mx3000P Stratagene

system (www.genomics.agilent.com) using TaqMan

Universal PCR Master Mix (www.appliedbiosys-

tems.com). Densities of Streptomyces on the filters

were determined from calibration curves produced

by amplification of DNA from known densities of

spores in 2 species of Streptomyces. Possible interfer-

ence on the PCR reaction by compounds in the sam-

ples was checked by measuring the amplification

efficiency of a plasmid construct with and without

addition of DNA extracted from the water samples

(Lylloff et al. 2012).

Densities of cyanobacteria were estimated from the

composition and concentration of specific pigments

in the phytoplankton according to Schlüter et al.

(2004). The GF/F filters were transferred to vials with

95% acetone with internal standard (vitamin E). The

samples were vortexed, sonicated on ice, extracted at

4°C for 20 h, and mixed again. The extracts were fil-

tered through 0.2 µm Teflon syringe filters into HPLC

vials and placed in the cooling rack (4°C) of the

HPLC system together with a parallel set of vials with

injection buffer (28 mM aqueous tetrabutyl ammo-

nium acetate [TBA] at pH 6.5 and methanol, in a

90:10 ratio). The extracts were injected by program-

ming the auto-injector to make a sandwich injection

of buffer and sample in the ratio 5:2. The total injec-

tion volume was 500 µl. For detection of the pig-

ments, a Shimadzu LC-10A HPLC system was used

following the method by Van Heukelem & Thomas

(2001) (with slight modifications as given below). The

HPLC system consisted of a LC-10ADVP pump, an

SPD-M10A VP photodiode array detector, an SCL-

10ADVP System controller with Lab Solution soft-

ware, an auto sampler (set at 4°C), a column oven

(CTO-10ASVP), and a degasser. The column was an

Eclipse XDB C8, 4.6 mm × 150 mm (Agilent Technol -

ogies) operated at 60°C. The solvents were (A) meth -

anol and 28 mM aqueous TBA at pH 6.4 in a 70:30

ratio and (B) 100% methanol. The following time

programming was used: 0 min: 95% A, 5% B; 27 min:

5% A, 95% B; 34 min: 5% A, 95% B; 35 min: 0% A,

100% B; 38 min: 0% A, 100% B; 39.5 min: 95% A,

5% B; 50 min: stop. The flow rate was 1.1 ml min

−1

.

The analysis was calibrated with pigment standards

from DHI Lab Products. The internal standard was

detected at 222 nm, while the phytoplankton pig-

ments were detected at 450 nm. Peak identities were

routinely confirmed by on-line PDA analysis.

Pigment concentrations were subsequently loaded

into the CHEMTAX 1.95 program to calculate chloro-

phyll  a biomass of individual phytoplankton groups

according to Mackey et al. (1996) and Higgins et al.

(2011). Pigment ratios used as input values for

CHEMTAX calculations were from Schlüter et al.

(2006).

RESULTS AND DISCUSSION

Geosmin in water

Occurrence of geosmin in the water from the 4

types of ponds (sand filtration, PondPlus or Aqua 

-

Photo enrichment, and untreated controls) were

110

Off-flavour level

Grade

Numeric score

Strong 

A

1.0 to 2.5

Medium

B

2.5 to 5.0

Mild 

C

5.0 to 7.5

No off-flavour

D

7.5 to 10.0

Table 1. Categories for sensory analysis of fish

---

Petersen et al.: Geosmin in pond-raised fish

rather similar during the 6 mo period with bimonthly

sampling of duplicate ponds. Mean concentrations of

geosmin (in ng l

−

) in the pond water were 4.5 (range:

1.0 to 14.6) with sand filtration, 3.4 (range: 0.3 to 20.0)

for enrichment with PondPlus, 3.7 (range: 0.2 to 16.1)

for enrichment with AquaPhoto, and 4.1 (range 0.6 to

8.1) for the controls. Mean concentration of geosmin

in all water samples was 3.9 ng l

−1

, and only 3 of 56

water samples had geosmin concentrations > 10 ng

l

−1

. No significant effects of water treatment or sea-

sonality were observed (p > 0.05; t-test).

Occurrence of geosmin in the pond water is low

relative to concentrations measured in other fish

ponds in warm water, e.g. catfish ponds in Louisiana,

USA (4 to 246 ng l

−1

; Hurlburt et al. 2009), and barra-

mundi ponds in Queensland, Australia (range <1 to

14 370 ng l

−1

, typical level of 1000 ng l

−1

; Jones et al.

2013). Geosmin concentrations in the Patuakhali

ponds more resembled levels measured in cold

stream water (< 20°C), e.g. in Denmark, where levels

of geosmin in inlet water of traditional fishponds

were <10 ng l

−1

(Klausen et al. 2005). The low level of

geosmin, as well as the lack of effects of the different

treatments, suggests that geosmin-producing organ-

isms were not abundant in the water, as discussed

below.

Geosmin in fish

Mean concentration of geosmin in meat from all

the analysed fish was 21 ng kg

−1

for pangas Pan-

gasianodon hypophthalmus (range 0 to 91 ng kg

−1

)

and 17 ng kg

−1

for tilapia Oreochromis niloticus

(range: 0 to 68 ng kg

−1

). Statistical analyses showed

that there was a systematic effect of water treatment

and depuration on the content of geosmin in the fish.

An initial ANOVA test demonstrated significant

interaction between both fish species and effect of

water treatment and between fish species and the

duration of depuration. Therefore, data from pangas

and tilapia are treated separately. For pangas, treat-

ment of the pond water reduced the level of geosmin

by 56 to 74% relative to the content in fish from the

control ponds (Table 2). The effect of the 3 treatment

types (PondPlus, AquaPhoto, and sand filter) was not

significantly different. In contrast, for tilapia, no sig-

nificant effect of any of the water treatments on the

content of geosmin was observed. In fish from the

control ponds, a lower content of geosmin was found

in tilapia (11 ng kg

−1

) than in pangas (54 ng kg

−1

), but

this difference was not observed in the other water

treatments (Table 2).

Depuration of the fish for 12 to 48 h had a signifi-

cant effect in pangas, in which the geosmin content

was reduced to 65 to 90% of the level in the control

fish (Table 3). No effects of duration of the depura-

tion time (12 to 48 h) were found. For tilapia, the

depuration did not affect the content of geosmin in

the flesh.

The concentration of geosmin in tilapia and pangas

in the present ponds is low compared to geosmin

content in tilapia in other studies, e.g. fish from cages

in Thailand. Gutierrez et al. (2013) measured 0.49 to

3.51 µg geosmin kg

−1

wet weight in a seasonal study

of tilapia cultured in pond cages in northern Thai-

land. The relatively large content (10- to 70-fold

above the level in tilapia in the Patuakhali ponds)

may reflect the higher concentrations of geosmin in

the pond water (0.41 to 2.33 µg l

−1

, or 100- to 600-fold

above the level in the Patuakhali ponds). In addition

to geosmin, high levels of MIB were measured in

both fish and water in tilapia in the Thai ponds.

The geosmin content in tilapia and pangas was also

low relative to barramundi from cages in freshwater

in tropical Australia. Jones et al. (2013) reported con-

centrations of 0.74 to 4.47 µg geosmin kg

−1

wet

weight (13- to 82-fold above the levels in Patuakhali

fish). The larger geosmin content in the barramundi

111

Water treatment

Pangas

n

Tilapia

n

Control 54 A

6

11 A

6

(no water treatment)

PondPlus

24 B

5

24 A

6

Sand filter

18 B

6

23 A

6

AquaPhoto

14 B

6

12 A

5

Table 2. Mean concentrations (least squares, from ANOVA)

of geosmin (ng kg

−1

wet weight) in meat from fish from

ponds with different water treatments. For each species,

treatments not marked with the same letter (A or B) are

 significantly different (p < 0.05). n: number of analyzed fish

Depuration time

Pangas

n

Tilapia

n

Control (no depuration)

54 A

6

11 A

6

12 h 

5 B

6

8 A

6

24 h 

12 B

3

39 A

2

36 h 

19 B

6

13 A

5

48 h 

16 B

5

15 A

2

Table 3. Mean concentrations (least squares, from ANOVA)

of geosmin (ng kg

−1

wet weight) in meat from fish from

ponds with different depuration times. For each species,

depuration times not marked with the same letter are

 significantly different (p < 0.05). n: number of analyzed fish

---

Aquacult Environ Interact 5: 107–116, 2014

may reflect the high geosmin concentration in the

water (<1 to 14.4 µg l

−1

, or up to 3700-fold higher than

in the Bangladeshi ponds). No data for geosmin con-

tent in pangas are available in the literature.

The higher content of geosmin in pangas than in

tilapia (54 vs. 11 ng kg

−1

) from the control ponds was

unexpected since similar concentrations of geosmin

were found in the 2 fish species (12 to 24 ng kg

−1

)

from all ponds with water treatments. We have no

explanation for the greater geosmin content in pan-

gas from the control ponds. The ponds in this study

are individual, earthen, rain-fed ponds, and specula-

tively, environmental conditions in the 2 adjacent

pangas control ponds were similar, e.g. with respect

to general water quality, vegetation, shading, and

influence from livestock and households, but might

have differed from the other ponds. Feeding habits of

the fish may also have influenced the geosmin con-

tent, although the mechanisms are not clear. Tilapia

is a ‘column feeder’ fish, but it can also feed on ben-

thos and ingest mud, while pangas tend to prefer

artificial feed in the water column if feed is available

(data not shown). Sediment in lakes has previously

been found to harbour higher concentrations of geo -

smin than the water above (Nielsen et al. 2006).

However, the occurrence of geosmin in bottom mate-

rial of the ponds is unknown, and more research is

needed to determine possible relations between

feeding habits, feed sources, and levels of off-flavour

compounds in fish.

Effect of depuration on geosmin content in fish

The positive effect in pangas of depuration for 12

to 48 h in groundwater (reduction of the geosmin

content to about one-third of the original value), but

not for tilapia, shows that even a short depuration

period can purge geosmin from pangas. Tilapia had

a low content of geosmin before depuration, and

this might explain the lack of effect from the depu-

ration. Unfortunately, the geosmin content of the

groundwater used for the depuration is unknown,

and the groundwater might have had a variable

geosmin content, affecting the geosmin content of

the fish. For cold-water fish, such as rainbow trout,

depuration periods of ~1 wk are typically practised

(Petersen et al. 2011) and are supported by experi-

mental studies of off-flavour depuration in this spe-

cies (Howgate 2004). Possibly, the high water tem-

perature (~30°C) in this study increased the release

of geosmin in pangas, e.g. due to a higher metabolic

activity.

Sensory analysis of off-flavour

For pangas, grading of the off-flavour level by the

panel members largely reflected the geosmin content

determined by the chemical analysis. Fish from the

control ponds had the highest grading (strong off-

flavour) relative to PondPlus (medium off-flavour),

sand filter (mild off-flavour), and AquaPhoto (no off-

flavour) treatments (Table 4). For tilapia, the grading

pattern was similar, except that the treatments with

sand filter and AquaPhoto were similar (mild off-

flavour) (Table 5). Thus, AquaPhoto treatment and

sand filtration were most efficient in reducing the off-

flavour content of the fish, followed by PondPlus.

Depuration of pangas and tilapia in tanks with

groundwater significantly reduced the perceived off-

flavour relative to control fish. In pangas, all fish

were categorized as flavour-free (grade D) after 12 h

depuration, although a depuration time of 

≥24 h gave

a slightly higher numeric score (Table 6). For tilapia,

all fish were given a score of mild off-flavour (grade

C) after 12 to 48 h of depuration. No data on geosmin

concentrations in the groundwater are available, but

the decline in geosmin content in the fish suggests a

lower content than in the pond water.

The coincidence between a lower geosmin content

and ‘less off-flavour’ grading after depuration, as

112

Water treatment

Average grade

Off-flavour level

Control

2.4 ± 0.2

Strong (A)

PondPlus

4.8 ± 0.8

Medium (B)

Sand filter

7.1 ± 1.1

Mild (C)

AquaPhoto

7.8 ± 0.1

No off-flavour (D)

Table 4. Off-flavour profiling of pangas from ponds with dif-

ferent treatment. Numeric scores of grades (see Table 1) are

mean ± SE; n = 48 (12 from each pond treatment). The grad-

ing was statistically different for each water treatment type 

(p < 0.05; ANOVA)

Water treatment

Average grade

Off-flavour level

Control

2.7 ± 0.0

Strong (A)

PondPlus

4.9 ± 0.1

Medium (B)

Sand filter

6.2 ± 0.3

Mild (C)

AquaPhoto

6.5 ± 0.2

Mild (C)

Table 5. Off-flavour profiling of tilapia from ponds with dif-

ferent treatment. Numeric scores of grades (see Table 1)  are

mean ± SE; n = 48 (12 from each pond treatment). The grad-

ing was statistically different for each water treatment type

(p < 0.05; ANOVA) except between the sand filter and 

AquaPhoto treatments

---

Petersen et al.: Geosmin in pond-raised fish

observed for pangas but not for tilapia (the geosmin

content was unchanged after depuration in tilapia),

might point to geosmin as a dominant component in

the perceived off-flavour. However, in sensory stud-

ies of channel catfish and rainbow trout, the human

threshold for geosmin detection was determined as

~250 ng kg

−1

fish (Grimm et al. 2004, Robertson et al.

2005, Robin et al. 2006), although a slightly lower

threshold of ~100 ng kg

−1

fish was found for rainbow

trout by Petersen et al. (2011). In the Patuakhali

ponds, the maximum geosmin content was 68 and

91 ng kg

−1

for tilapia and pangas, respectively. As -

suming that the detection threshold of geosmin is

identical for catfish, trout, pangas, and tilapia, our

results indicate that compounds other than geosmin

caused the off-flavour in the Patuakhali fish. MIB is

frequently re ported as a major off-flavour compound

in fish (Tucker 2000), but our analyses suggest that

this compound occurred at similar or lower concen-

trations in fish in this study. Unfortunately, MIB co-

eluted with an unknown compound and could not be

properly quantified.

The positive effect of off-flavour reduction after

only 12 h of depuration is surprising, e.g. when com-

pared to depuration of rainbow trout Onchorhynchus

mykiss for several days to obtain a content below

the human threshold level (Robertson et al. 2005).

Although the initial off-flavour (geosmin) content

was higher in the rainbow trouts, the warmer water

in Bangladesh (~30°C, or 15°C above the tempera-

ture used during the depuration of the rainbow trout)

probably increased the off-flavour removal due to a

high metabolic rate. Supporting a temperature effect,

Jones et al. (2013) also observed an efficient reduc-

tion of geosmin in barramundi in water at 26°C after

24 h.

The sensory panel in this study did not receive

training in identification of specific off-flavours or in

description of sensory properties, and thus, compar-

ison of off-flavour grading by this panel and trained

panels in other studies is problematic. Yet, despite

the lack of training, grading by the panel agreed

well with the different levels of geo -

smin in the fish (although geosmin

did not appear to be a dominant off-

flavour). An advantage of the local

panel is that its members can identify

flavour preferences by Bangladeshi

consumers, implying that their grad-

ing is valuable to fish farmers in the

region.

Potential producers of off-flavours

Since streptomycete bacteria and cyanobacteria

are recognized as notorious off-flavour producers in

freshwater, including aquaculture systems (Zaitlin &

Watson 2006), abundances of these 2 groups of

microorganisms were determined in the ponds. The

density of Streptomyces bacteria in the pond water

varied from 0.21 × 10

9

l

−1

to 4.2 × 10

9

l

−1

(Fig. 1). Large

seasonal and pond-to-pond variations, even between

duplicate ponds, occurred. A general trend, however,

was presence of higher densities in March than in

May and July (p < 0.05; t-test). Except for the high

density in AquaPhoto Pond #1 at March 25, water

treatment by sand filtration or probiotics resulted in

lower densities of streptomycetes, relative to the con-

trols, in March, but this trend was not found in May

and July. The density of all bacteria in the pond

113

Fish

No 

12 h

24 h

36 h

48 h

species

depuration

Pangas

2.4 ± 0.1 A

7.9 ± 0.2 B

9.2 ± 0.2 C

9.0 ± 0.2 C

8.7 ± 0.2 C

Tilapia

2.4 ± 0.2 A

6.7 ± 0.3 B

6.9 ± 0.2 B

7.1 ± 0.2 B

6.7 ± 0.3 B

Table 6. Off-flavour profiling of fish without and with depuration for varying

time periods. Mean numeric scores of grades (see Table 1) ± SE are shown; n =

40 (4 fish of each species from each treatment). Treatments not marked with 

the same letter (A, B and C) are significantly different (p < 0.05; ANOVA)

Pond

Sand filter 1 Sand filter 2 PondPlus 1 PondPlus 2AquaPhoto 1AquaPhoto 2

Control 1 Contro

l 2

D

e

n

s

it

y o

f s

tre

p

to

m

yc

e

te

s

 (10   c

e

lls

 l

-1

9

)

0

1

2

3

4

5

March 25

May 25 

July 25 

Fig. 1. Abundance of Streptomyces in the different ponds

during the growth period. Mean values of triplicate samples 

(on filter divided into 3 portions) ± 1 SD are shown

---

Aquacult Environ Interact 5: 107–116, 2014

water was not determined, but the abundance of bac-

teria in 4 similar fish ponds in the area was deter-

mined in December 2011 as 23 × 10

9

to 32 × 10

9

cells

l

−1

(data not shown). Assuming that these densities

are also representative of the 8 studied ponds, strep-

tomycete bacteria made up 0.45 to 13.3% (mean of

3.4%) of all bacteria.

Occurrence of Streptomyces in aquatic environ-

ments is relatively non-documented, but they are as -

sumed potential contributors of off-flavours (Klausen

et al. 2005). Using cultivation methods, densities of

10

2

to 10

5

streptomycetes l

−1

were found in various

freshwater environments (Lanciotti et al. 2003, Lee et

al. 2011), while fluorescence in situ hybridization,

targeting rRNA, indicated up to 2 × 10

9

cells l

−1

in a

water reservoir (Nielsen et al. 2006). In contrast,

using the present PCR approach, Lylloff et al. (2012)

only found up to 46 × 10

3

streptomycetes l

−1

in vari-

ous Australian freshwaters. Relative to these studies,

the present abundance of Streptomyces in the Patu-

akhali ponds appears relatively high. The low con-

centrations of geosmin in the pond water suggest

that streptomycetes, although abundant, were not

significant producers of geosmin in the water during

the studied period.

Cyanobacteria made up a relatively small portion

of the plankton biomass during the study, ranging

from 0.7% (May 25) to 1.9% (July 25) of the phyto-

plankton biomass (based on chlorophyll a) (Fig. 2).

The highest abundance (9.3% of the biomass)

occurred in AquaPhoto Pond #2 on 25 March. Treat-

ment of the water (sand filtration or probiotics)

reduced the abundance of cyanobacteria by 69% rel-

ative to the controls (p < 0.02; Mann-Whitney rank

sum test of all samples). The phytoplankton commu-

nity was dominated by chlorophytes (green algae),

followed by diatoms and cryptophytes, making up

53−76%, 13−23%, and 7.5−22%, respectively, of the

mean biomass from March to July.

The pigment analyses indicate that cyanobacteria

had a low abundance among phytoplankton in the

ponds. Microscopy analysis of phytoplankton in

some fish ponds in the region, e.g. in the main PSTU

campus fish pond, showed that the phytoplankton in

May and June 2013 was dominated by Oscillatoria,

Microcystis,  Anabaena, and other cyanobacterial

species (data not shown). This contrasts our obser -

vations of phytoplankton populations determined by

pigment analysis. Here, cyanobacteria were identi-

fied by the pigments myxoxanthophyll, echinenone,

and canthaxanthin. Myxoxanthophyll and echinenone

have been found in selected species of Oscillatoria

and Microcystis (Schlüter et al. 2006), while Ana -

baena spp. may contain all 3 pigments (Schlüter et

al. 2004). These 3 cyano-pigments were only present

in some of the samples and in relatively small con-

centrations, as reflected in Fig. 2. Zeaxanthin is

another cyanobacterial pigment that was detected in

all samples in relatively low concentrations. How-

ever, zeaxanthin is also present in chlorophytes and

has limited value as diagnostic pigment in the pres-

ent study, where chlorophytes dominated.

Pheophytin  a and pheophorbide a (degradation

products of chlorophyll a; not included in the CHEM-

TAX calculations) were present in considerable

amounts in some samples and constituted up to 27%

of chlorophyll a (average of 14% of chlorophyll a in

all samples). These degradation products indicate a

considerable breakdown of phytoplankton in the

ponds.

114

May 25

Chlorophyll 

a 

(µg l   ) 

-1

0

25

50

200

July 25

0

10

20

30

40

100

March 25

0

50

100

150

200

350

400

Dinoflagellates 

Diatoms 

Chlorophytes 

Cryptophytes 

Cyanobacteria 

Sand filter 1 Sand filter 2 PondPlus 1 PondPlus 2 AquaPhoto 1AquaPhot

o 2

Control 1 Control 2

Fig. 2. Concentrations of chlorophyll a and composition of

major groups of phytoplankton in the ponds determined by

pigment and CHEMTAX analyses. For each sampling, result 

from 1 filter is shown

---

Petersen et al.: Geosmin in pond-raised fish

Cyanobacteria are assumed dominant producers of

off-flavours in open, outdoor aquaculture systems

due to presence of sunlight and inorganic nutrients

(Tucker 2000, Schrader & Dennis 2005). In the Patu-

akhali ponds, the low content of both cyanobacteria

and geosmin suggests that cyanobacteria were not

major producers of off-flavours in the water. How-

ever, more information on species of cyanobacteria

and their potential production of off-flavours in the

ponds is needed to draw conclusions, e.g. on the risk

of off-flavour episodes due to cyanobacteria.

CONCLUSION

The off-flavour geosmin in pangas and tilapia from

the Bangladeshi ponds was below the human detec-

tion threshold based on other studies of off-flavours

in fish, and geosmin probably did not contribute to

tainting of the fish. The chemical analyses demon-

strated that treatment of the pond water (via sand fil-

tration or probiotic microbes) and depuration for at

least 12 h reduced the geosmin content in pangas but

not in tilapia. Sensory studies confirmed a positive

effect of both water treatment and depuration on the

off-flavour content of the fish, especially for pangas.

This improvement of the sensory quality, despite the

fact that the geosmin content appeared to be consis-

tently below the human threshold, suggests that off-

flavour compounds other than geosmin were present

in water and fish. Studies are underway to demon-

strate whether phytoplankton, vegetation, or anthro-

pogenic sources may contribute to the off-flavour

content in water and fish in Bangladeshi ponds.

Acknowledgements.  We thank the Regional Fisheries &

Livestock Development Component − Barisal TSU, Danish

International Development Assistance (Danida)/Govern-

ment of Bangladesh, Barisal, Bangladesh, for providing a

grant for this study. Technician Ulla Rasmussen, University

of Copenhagen, is acknowledged for skillful PCR analyses

of streptomycetes.

LITERATURE CITED

Ahmed N, Young J, Dey M, Muir J (2012) From production

to consumption:  a case study of tilapia marketing sys-

tems in Bangladesh. Aquacult Int 20: 51−70

Auffret M, Yergeau É, Pilote A, Proulx É and others (2013)

Impact of water quality on the bacterial populations and

off-flavours in recirculating aquaculture systems. FEMS

Microbiol Ecol 84: 235−247

Burr GS, Wolters WR, Schrader KK, Summerfelt ST (2012)

Impact of depuration of earthy-musty off-flavors on fillet

quality of Atlantic salmon, Salmo salar, cultured in a re -

circulating aquaculture system. Aquacult Eng 50: 28−36

Engle CR, Pounds GL, van der Ploeg M (1995) The cost of

off-flavor. J World Aquacult Soc 26: 297−306

Grimm CC, Lloyd SW, Zimba PV (2004) Instrumental versus

sensory detection of off-flavors in farm-raised channel

catfish. Aquaculture 236: 309−319

Gutierrez R, Sompong U, Prarom W, Iwami N, Itayma T,

Nomura N, Sugiura N (2013) Off-flavour in Nile tilapia

(Oreochromis niloticus) cultured in an integrated pond-

cage culture system. Maejo Int J Sci Tech 7(Spec Issue): 

1−13

Higgins H, Wright S, Schlüter L (2011) Quantitative inter-

pretation of chemotaxonomic pigment data. In: 

 Roy S

(ed) Characterization, chemotaxonomy and applications

in oceanography. UNESCO, Paris, p 257−313

Houle S, Schrader KK, Le Francois NR, Comeau Y and oth-

ers (2011) Geosmin causes off-flavour in arctic charr in

recirculating aquaculture systems. Aquacult Res 42: 

360−365

Howgate P (2004) Tainting of farmed fish by geosmin and

2-methyl-iso-borneol:  a review of sensory aspects and of

uptake/depuration. Aquaculture 234: 155−181

Hurlburt BK, Brashear SS, Lloyd SW, Grimm CC, Thomson

JL, Zimba PV (2009) Impact of weather on off-flavour

episodes at a Louisiana commercial catfish farm.

Aquacult Res 40: 566−574

Jones B, Fuller S, Carton AG (2013) Earthy-muddy tainting

of cultured barramundi linked to geosmin in tropical

northern Australia. Aquacult Environ Interact 3: 117−124

Khan S, Hossain MS, Haque MM (2009) Effects of feed 

-

ing schedule on growth, production and economics of

pangasiid catfish (Pangas hypophthalmus) and silver carp

(Hypophthalmichthys molitrix) polyculture. J Bangla desh

Agricult Univ 7: 175−181

Klausen C, Nicolaisen MH, Strobel BW, Warnecke F,

Nielsen JL, Jørgensen NOG (2005) Abundance of acti-

nobacteria and production of geosmin and 2-methyl 

-

isoborneol in Danish streams and fish ponds. FEMS

Microbiol Ecol 52: 265−278

Lanciotti E, Santini C, Lupi E, Burrini D (2003) Actino-

mycetes, cyanobacteria and algae causing tastes and

odours in water of the River Arno used for the water sup-

ply of Florence. J Water Supply Res Technol 52: 489−500

Lee GC, Kim YS, Kim MJ, Oh SA and others (2011) Pres-

ence, molecular characteristics and geosmin producing

ability of Actinomycetes isolated from South Korean ter-

restrial and aquatic environments. Water Sci Technol 63: 

2745−2751

Lylloff JE, Mogensen MH, Burford MA, Schlüter L, Jør-

gensen NOG (2012) Detection of aquatic streptomycetes

by quantitative PCR for prediction of taste-and-odour

episodes in water reservoirs. J Water Supply Res Technol

61: 272−282

Mackey MD, Mackey DJ, Higgins HW, Wright SW (1996)

CHEMTAX — a program for estimating class abun-

dances from chemical markers: 

 application to HPLC

measurements of phytoplankton. Mar Ecol Prog Ser 144: 

265−283

Mohsin M, Bakar J, Selamat J (1999) The effects on colour,

texture and sensory attributes achieved by washing

black tilapia flesh with a banana leaf ash solution. Int J

Food Sci Technol 34: 359−363

Nielsen JL, Klausen C, Nielsen PH, Burford MA, Jørgensen

NOG (2006) Detection of activity among uncultured Acti-

nobacteria  in a drinking water reservoir. FEMS Micro-

biol Ecol 55: 432−438

115

®

®

®

®

®

®

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®

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®

®

®

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®

---

Aquacult Environ Interact 5: 107–116, 2014

Percival S, Drabsch P, Glencross B (2008) Determining fac-

tors affecting muddy-flavour taint in farmed barramundi,

Lates calcarifer. Aquaculture 284: 136−143

Petersen MA, Hyldig G, Strobel BW, Henriksen NH, Jør-

gensen NOG (2011) Chemical and sensory quantification

of geosmin and 2-methylisoborneol in rainbow trout

(Oncorhynchus mykiss) from recirculated aquacultures

in relation to concentrations in basin water. J Agricult

Food Chem 59: 12561−12568

Rintala H, Nevalainen A (2006) Quantitative measurement

of streptomycetes using real-time PCR. J Environ Monit

8: 745−749

Robertson RF, Lawton LA (2003) Off-flavor problems and a

potential solution within the UK trout industry. ACS

Symp Ser 848: 55−68

Robertson RF, Jauncey K, Beveridge MCM, Lawton LA

(2005) Depuration rates and the sensory threshold con-

centration of geosmin responsible for earthy-musty taint

in rainbow trout, Onchorhynchus mykiss. Aquaculture

245: 89−99

Robin J, Cravedi JP, Hillenweck A, Deshayes C, Vallod D

(2006) Off flavor characterization and origin in French

trout farming. Aquaculture 260: 128−138

Schlüter L, Garde K, Kaas H (2004) Detection of the toxic

cyanobacteria  Nodularia spumigena by means of a

4-keto-myxoxanthophyll-like pigment in the Baltic Sea.

Mar Ecol Prog Ser 275: 69−78

Schlüter L, Lauridsen TL, Jørgensen T, Krogh G (2006) Iden-

tification and quantification of phytoplankton groups in

lakes using new pigment ratios — a comparison between

pigment analysis by HPLC and microscopy. Freshw Biol

51: 1474−1485

Schrader

KK, Dennis

ME

(2005) Cyanobacteria and

earthy/musty compounds found in commercial catfish

(Ictalurus punctatus) ponds in the Mississippi Delta and

Mississippi-Alabama Blackland Prairie. Water Res 39: 

2807−2814

Smith JL, Boyer GL, Zimba PV (2008) A review of cyanobac-

terial odorous and bioactive metabolites: 

 impacts and

management alternatives in aquaculture. Aquaculture

280: 5−20

Tucker CS (2000) Off-flavor problems in aquaculture. Rev

Fish Sci 8: 45−88 

Van Heukelem L, Thomas C (2001) Computer assisted high-

performance liquid chromatography method develop-

ment with applications to the isolation and analysis of

phytoplankton pigments. J Chromatogr A 910: 31−49

Zaitlin B, Watson SB (2006) Actinomycetes in relation to

taste and odour in drinking water: 

 myths, tenets and

truths. Water Res 40: 1741−1753

116

Editorial responsibility: Megan La Peyre, 

Baton Rouge, Louisiana, USA

Submitted: October 14, 2013; Accepted: March 20, 2014

Proofs received from author(s): April 16, 2014

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