CHEMISTRY/BIOCHEMISTRY

Storage Temperature Effect on Histamine

Formation in Big Eye Tuna and Skipjack
CLIA C.G. SILVA, DUARTE J.B. DA PONTE, and MARIA L.N. ENES DAPKEVICIUS
ABSTRACT
Notable histamine formation (30 mg/100g) was detected in
big eye tuna (Thunnus obesus) and skipjack (Katsuwonus
pelamis) captured in Azorean waters and stored for 1, 3 and
6 days at 22, 10 and 4C, respectively. Higher levels (p0.05)
of histamine were produced by skipjack reflecting its higher
histidine content. Measurements of pH or volatile basic nitrogen were not adequate for estimating the extent of histamine-related health hazard. Counts of histamine-forming
bacteria increased during storage at 4 and 10C, and histamine formation was suppressed at 4C. Storage temperature and histidine content were the main factors controlling
histamine levels in tuna.
Key Words: histamine, tuna, fish, biogenic amines

INTRODUCTION
TUNA AND OTHER SCOMBROID FISHES HAVE BEEN IMPLICATED IN
outbreaks of histamine poisoning (Arnold and Brown, 1978; Taylor,
1986). Histamine is produced mainly by bacteria with histidine decarboxylase activity. Usually, the content of free histidine in tuna
fish muscle is very high, therefore there is a risk that toxic levels of
histamine can be formed, especially under unfavorable storage conditions (Eitenmiller et al., 1982). The stability of histamine to heat
may explain the levels often reported in canned tuna (Frank and Yoshinaga, 1984). The European Community proposed that the average
content of histamine in fish should not exceed 10 mg/100g and no
sample may contain 20 mg/100g (Luten et al., 1992). The U.S.
Food & Drug Administration established that levels 5 mg/100g in
tuna constitute a potential health hazard based on data collected from
outbreaks (FDA, 1996).
Many studies have been reported on the effects of storage temperature on histamine formation in fish, but results very often have
been ambiguous (Edmunds and Eitenmiller, 1975; Salguro and
Mackie, 1979; Yamanaka et al., 1984; Frank and Yoshinaga, 1987;
Yamanaka and Matsumoto, 1989; Veciana-Nogus et al., 1990;
Wendakoon et al., 1990, Wei et al., 1990, Rodriguez-Jerez, 1994).
This may be due to differences in type and level of bacterial flora in
the fish. Histamine-producing bacterial species and strains vary considerably in amounts of histamine formation, and the type of spoilage bacteria present depends on the aquatic environment (Omura et
al., 1978; Yoshinaga and Frank, 1982; Lpez-Sabater et al., 1994;
Lpez-Sabater et al., 1996a). Thus, the tuna fish caught in the Azorean
waters should have a specific microflora with a specific temperature-related histamine production.
Our objective was to study the effects of temperature storage on
histamine production in big-eye tuna and skipjack caught in Azorean waters in order to assess the effectiveness of the refrigeration system (circulation of refrigerated seawater) that has been introduced
by tuna fisheries in the region. Histamine was measured and compared with the concentration of free histidine, pH and volatile basic
nitrogen (VBN) during storage at 22, 10 and 4C. The growth of

Authors da Ponte and Enes Dapkevicius are affiliated with the Depto. de Cincias
Agrrias, Universidade dos Aores, 9700 Angra do Herosmo, Portugal. Author Silvas
present address: School of Animal & Microbial Sciences, The Univ. of Reading,
Whiteknights, PO Box 228, Reading RG6 6AJ, UK.

644

JOURNAL OF FOOD SCIENCEVolume 63, No. 4, 1998

aerobic mesophilic microorganisms and histamine-forming bacteria

was also measured.

MATERIALS & METHODS

Storage conditions

Big-eye tuna (Thunnus obesus) and skipjack (Katsuwonus pelamis) weighing 1.42.5 kg, were obtained from a local tuna fishing
boat. The fishes caught in the Azorean waters (T22C) were kept
on board at 4C for four days in refrigerated sea-water tanks,
brought to the laboratory in isothermal boxes and kept on ice during
one day, before beginning the experiments. The fish muscles were
aseptically cut in random portions of about 15 g and placed in sterile
Petri dishes for chemical analysis. Portions (10g) were aseptically
cut and placed in sterile stomacher bags for bacterial analysis. Both
Petri dishes and stomacher bags containing the fish samples were
incubated at 4C 0.5C, 10C0.5C or 22C2C for various
periods of time. Each incubation was run in duplicate.
Histamine and histidine analysis

Histamine and histidine were determined in accordance with

methods of Gouygou et al. (1987) and Pozo and Saitua (1988), with
minor modifications. Fish muscle (5g) was homogenized with 30
mL of 10% trichloroacetic acid (TCA) using a Potter-Elvejham homogenizer (B. Braun, West Germany). The homogenate was centrifuged (1000g, 15 min at 4C) and the supernatant was separated.
TCA (10 mL, 10%) was mixed with the pellet and the mixture was
centrifuged again (1000g, 15 min, 4C). Both supernatants were
filtered (Whatman, 2.3 m) and the filtrate was stored at 4C until
derivatization.
The fluorophere was prepared by mixing 150 L of the filtrate
with 1.9 mL deionized water and 0.4 mL 1N NaOH, in a tube protected from light. OPA-reagent (100 L, prepared by dissolving 10
mg o-phtalaldehyde-OPA, SIGMA in 1 mL methanol) was added,
the solution was mixed, and left for 4 min. before adding 0.2 mL 3N
HCl. This solution (20 L) was directly injected into the chromatograph.
Analyses were performed with a Hitachi Liquid Chromatograph
(Hitachi, Ltd., Tokyo, Japan), consisting of a Model 655A-11 pump,
a syringe loading sample injector with a loop of 20 L, a Model
F1000 fluorescence spectrophotometer and a Spectra-Physics integrator (Model SP 4600). A Hibar Lichrosorb RP-18 reverse phase
column (5 m, 125 4 mm i.d., Merck, Poole, UK) was used and
fluorescence was monitored at 358 nm excitation and 447 nm emission wavelengths. The isocratic mobile phase consisted of acetonitrile: 25 mM KH2PO4 (1:1 v/v) at a flow rate of 0.7 mL/min. At
about 3.48 min and 4.56 min after injection, peaks of histidine (3.48
min) and histamine (4.56 min) appeared on the data processor.
Duplicate test samples were extracted, and each extract was analyzed at least three times.
Volatile basic nitrogen (VBN) and pH

Samples of fish muscle (5g) were homogenized with 10 mL 5%

TCA using a Potters homogenizer. Volatile basic nitrogen (VBN)
was determined by a modification of the micro-diffusion method of
Conway (Anonymous, 1988). Other samples (5g) of fish were homogenized with 5 mL distilled water and the pH measured with a
combined glass calomel electrode.

Microbiological analysis

At the end of each incubation period the samples were homogenized with Ringer solution (1:10 w/v) in a Stomacher homogenizer.
The homogenates were serially diluted and surface plated on culture
media. Plate count agar (Difco Laboratories) was used to enumerate
aerobic mesophilic microorganisms and microorganisms that grew
at 10C in the tuna samples. Duplicate plate counts were incubated
at 22C for 2 days and 10C for 6 days.
Nivens differential agar medium (Niven et al., 1981) modified
by addition of 4% agar, was used to enumerate histamine producing
bacteria in tuna samples. This medium (with 2% NaCl) was also
used to enumerate bacteria that could produce histamine at that salt
concentration (similar to the concentration in sea water). Both media were sterilized for 10 min. at 121C to avoid excess hydrolysis
of the agar at the low pH. Duplicates were incubated at 22C for 3
days and visually examined for purple colonies with a purple halo
on the yellow background.
Statistical analysis

Histamine levels in big eye tuna and skipjack were compared

and analyzed by unpaired two-tailed t-test. Significance of differences was defined at p 0.05. Linear correlation analysis was used
to examine the relationship between free histidine levels and histamine formation in both types of tuna.

RESULTS & DISCUSSION

HISTAMINE CONTENT OF BIG-EYE TUNA AND SKIPJACK WAS NEGLIgible (0.1 mg/100g) at the start of incubation. Formation of histamine occurred very quickly at 22C, reaching health hazardous levels (50 mg/100g; FDA, 1982, 1996) in one day for skipjack and
two days for big-eye tuna (Fig. 1). The rise of histamine content was
delayed at refrigerated temperatures (10 and 4C, Fig. 1) but notable
amounts were detected after 3 days at 10C and 6 days at 4C, exceeding the limit of 5 mg/100g (FDA, 1996). During storage, histamine levels increased exponentially. In samples stored at 10C they
decreased after a maximum at day 9. These results confirmed the
effects of temperature on histamine formation as reported by Yamanaka et al. (1984), Frank and Yoshinaga (1987), Sato et al. (1995)
and Lpez-Sabater et al. (1996b).
Histamine levels in skipjack muscle were higher (p0.05) at all
storage temperatures (Fig. 1). Initially, skipjack contained 50% more
free histidine than big-eye which could explain the higher formation
of histamine in skipjack. However, the formation of histamine was
lower than the correspondent decrease of histidine during storage.
This difference was probably due to the deamination of histidine to
uruconate (Mackie and Fernandez-Salguro, 1977). It could also be

due to the histaminase activity in the spoilage bacteria (Buffoni,

1966), which may be responsible for the decline in histamine observed in both species after 12 days storage at 10C. Negative correlations (p0.05) were observed between histidine levels and histamine formation for all temperatures (except big eye tuna at 10C,
where negative correlation was significant only until 9 days storage). These results suggest that histamine production was dependent on storage temperature and also on free histidine levels. Therefore, skipjack would be more likely to cause histamine intoxication
due to its high histidine content.
Changes in pH and VBN levels in big-eye (Table 1) and skipjack
(Table 2) were compared. During initial storage, sample pH values
were considered normal for fish (6.0 to 6.3). Values above 6.3 were
only reached when samples had been stored for 3 days at 22C, 6
days at 10C or 12 days at 4C, by which time toxic levels of histamine were produced. The highest pH values were observed in samples stored at 10C during 12 days (8.4 in big-eye and 7.0 in skipjack).
Eitenmiller et al. (1982) reported low histidine decarboxylase activity
when histamine-forming bacteria were grown at pH 8.5. Thus, the observed increase in pH may have been responsible for the decrease of
histidine decarboxylase activity, which would explain the reduction of
histamine production after 12 days storage at 10C (Fig. 1).
Initial VBN values were higher in skipjack possibly indicating a
greater degree of spoilage. In both types of tuna stored at 22C, VBN
increased rapidly (Tables 1 and 2) which confirmed results of Lpez-Sabater et al. (1996b) that the decomposition of tuna at 20C
progressed faster than histamine formation. In contrast, at refrigerated temperatures, an important rise in VBN values was only observed
after 6 and 9 days in samples stored respectively at 10C and 4C.
These results confirmed reports that toxic histamine levels were found
in tuna stored at refrigerated temperatures before they developed
external signs of decomposition (by general appearance; e.g. odor,
texture, color of muscle) and would be rejected for human consumption (Sato et al., 1995; Lpez-Sabater et al., 1996b).
Counts of mesophilic aerobic microorganisms and histamineforming bacteria as related to storage temperature are summarized
(Fig. 2). At the start of incubation total aerobic counts were very low
(102 CFU/g), however they increased quickly at all incubation temperatures. After 3 days incubation at 22C, mesophilic aerobic microorganisms reached 108 CFU/g. This value was not reached until
10 days of incubation at 10C in either tuna. The growth of microorganisms at 10C was identical to that of mesophilic microorganisms
(Fig. 2). This was expected considering that the tuna were held 5
days at refrigeration temperatures before the beginning of the experiment. Lpez-Glvez et al. (1995) also had reported that the spoilage
flora of refrigerated tuna were dominated by psychrotrophs.

Fig. 1Changes in histamine(Hm) and histidine (His) contents of big-eye tuna (A) and skipjack (B) during storage as related to temperature.

Volume 63, No. 4, 1998JOURNAL OF FOOD SCIENCE 645

Histamine Formation in Tuna . . .

Table 1Changes in pH and VBN in big-eye tuna during storage as
related to temperature
Storage time

pH

VBN (mg/100g)

Table 2Changes in pH and VBN in skipjack during storage as related to temperature

Storage time

pH

VBN (mg/100g)

(days)

4C

10C

22C

4C

10C

22C

(days)

4C

10C

22C

4C

10C

22C

0
1
2
3
6
9
12

6.0

6.0

20

20

6.0

30

30
38

30

21
24
29
36

24
31
61
151

0
1
2
3
6
9
12

6.0
6.2

6.2
6.6
7.4
8.4

20
33
34
39

6.0

6.2
6.3
6.2
6.6

6.0
6.1
6.2
6.8

6.2
6.3
6.2
6.5

6.2
6.6
6.7
7.0

27
34
38
47

31
50
52
97

Histamine-forming bacteria were very low and undetectable at

the beginning of the assay (Fig. 2), which confirmed with the negligible histamine content initially present. However, during subsequent incubation, counts of histamine-forming bacteria increased
rapidly, especially at 22C. At the early stage of incubation, counts
of histamine-forming bacteria increased faster in skipjack than in
big-eye, confirming the faster histamine production in skipjack. The
growth of histamine-forming bacteria showed good agreement with
the formation of histamine in both tuna, in early storage at 22C and
10C. Refrigeration at 4C did not prevent development of microorganisms with histidine decarboxylase activity.
The histamine producing bacteria grew well in both media containing 0.5% and 2% NaCl (Fig. 2). This was probably because they
had adapted to the aquatic environment where the fishes were caught
and the storage on board in refrigerated sea water. Although storage
in refrigerated sea water is widely used on board fishing vessels, the
increase of spoilage bacteria and uptake of salt in fish are limitations
of this system (Heen, 1981). The refrigerated sea water system used

6.1
6.4

47
64

on board did not prevent, and may promote microflora in tuna fish
consisting of a large number of histamine-producing bacteria that
grow at refrigerated temperatures in the presence of NaCl.

CONCLUSIONS
THE AMOUNT OF HISTAMINE FORMED DURING STORAGE OF TUNA
captured in Azorean waters increased gradually, reaching toxic levels after 1 day at 22C, 3 days at 10C and 6 days at 4C. In skipjack
higher levels of histamine were produced than in big-eye, probably
due to initial higher levels of free histidine. VBN or pH did not reflect the degree of spoilage in tuna fish stored at refrigerated temperatures. It is essential to determine histamine content in order to control potential toxic effects. The growth of histamine-forming bacteria showed good agreement with the formation of histamine in both
species, at early stages of incubation. Histamine-forming bacteria
can grow at 4C but with suppressed histamine formation. A low
refrigeration temperature during storage is critical for reducing the
histamine.

Fig. 2Time-related changes in counts of microorganisms growing in Plate Count Agar (PCA) from big-eye tuna (A) and skipjack (B); and
in counts of histamine-forming bacteria cultured in Nivens differential agar medium in the presence of 0.5% and 2% NaCl in big-eye tuna
(C) and skipjack (D) as related to temperature.

646

JOURNAL OF FOOD SCIENCEVolume 63, No. 4, 1998

REFERENCES
Anonymous 1988. Pescado. Determinao do teor de azoto bsico voltil total
(A.B.V.T.). Mtodo de Conway. Norma Portuguesa NP 2930. Instituto Portugus de
Qualidade.
Arnold, S.H. and Brown, W.D. 1978. Histamine (?) toxicity from fish products. Adv.
Food Res. 24: 113-154.
Buffoni, F. 1966. Histaminase and related amine oxidases. Pharmacol. Rev. 18(4):
1163-1199.
Edmunds, W.J. and Eitenmiller, R.R. 1975. Effect of storage time and temperature on
histamine content and histidine decarboxylase activity of aquatic species. J. Food
Sci. 40: 516-519.
Eitenmiller, R.R., Orr, J.H., and Wallis, W.W. 1982. Histamine formation in fish: Microbiological and biochemical conditions. In Chemistry & Biochemistry of Marine
Fish Products, R.E. Martin, G.J. Flick, C.E. Hebard, and D.R. Ward (Ed.), p.39-50.
The Avi Publishing Company, Inc., Westport, CT.
FDA. 1982. Defect action levels for histamine in tuna, availability of guide. Fed. Reg.
47(173): 40487.
FDA. 1996. Decomposition and histamine in raw, frozen tuna and mahi-mahi, canned
tuna; and related species. Compliance Policy Guides 7108.240, Sec. 540.525.
Frank, H.A. and Yoshinaga, D.H. 1984. Histamine formation in tuna. In Seafood Toxins, E.P. Ragelis (Ed.), p.443-451. ACS Symposium Series, N262, American Chemical Society, Washington, DC.
Frank, H.A. and Yoshinaga, D.H. 1987. Table for estimating histamine formation in
skipjack tuna, Katsuwonus pelamis, at low nonfreezing temperatures. Mar. Fish. Rev.
49(4): 67-70.
Gouygou, J.P., Sinquin, C., and Durand, P. 1987. High pressure liquid chromatography
determination of histamine in fish. J. Food Sci. 52: 925-927.
Heen, E. 1981. Developments in chilling and freezing of fish. In Advances in the refrigerated treatment of fish, International Institute of Refrigeration (Ed.), 41-46.
Proceedings of the Meetings of Commissions C2, D1, D2 and D3, Boston.
Lpez-Glvez, D., de la Hoz, L. and Ordez, J.A. 1995. Effect of carbon dioxide and
oxygen enriched atmospheres on microbiological and chemical changes in refrigerated tuna (Thunnus alalunga) steaks. J. Agric. Food Chem. 43(2): 483-490.
Lpez-Sabater, E.I., Rodrguez-Jerez, J.J., Hernndez-Herrero, M., and Mora-Ventura, M.A.T. 1996a. Incidence of histamine-forming bacteria and histamine content in
scombroid fish species from retail markets in the Barcelona area. Int. J. Food Microbiol. 28: 411-418.
Lpez-Sabater, E.I., Rodrguez-Jerez, J.J., Hernndez-Herrero, M., Roig-Sagus, A.X.,
and Mora-Ventura, M.A.T. 1996b. Sensory quality and histamine formation during
controlled decomposition of tuna (Thunnus thynnus). J. Food Prot. 59(2): 167-174.
Lpez-Sabater, E.I., Rodrguez-Jerez, J.J., Roig-Sagus, A.X., and Mora-Ventura,
M.A.T. 1994. Bacteriological quality of tuna fish (Thunnus thynnus) destined for
canning: Effect of tuna handling on presence of histidine decarboxylase bacteria
and histamine level. J. Food Prot. 57(4): 318-323.
Luten, J.B., Bouquet, W., Seuren, L.A.J., Burggraaf, M.M., Riekwel-Booy, G., Durand, P., Etienne, M., Gouyou, J.P., Landrein, A., Leclerq, M., and Guinet, R. 1992.
Biogenic amines in fishery products: Standardization methods within EC. In Qual-

ity Assurance in the Fish Industry, H. Huss, M. Jakobsen and J. Liston (Ed.), p.427439. Proceedings of an International Conference, Elsevier Science Publishers.
Mackie, I.M. and Fernandez-Salguro, J. 1977. Histidine metabolism in fish. Urocanic
acid in mackerel (Scomber scombrus). J. Sci. Food Agric. 28: 935-940.
Niven, C.F., Jeffrey, M.B., and Corlett, Jr. D.A. 1981. Differential plating medium for
quantitative detection of histamine-producing bacteria. Appl. Environ. Microbiol.
41: 321-322.
Omura, Y., Price, R.J., and Olcott, H.S. 1978. Histamine-forming bacteria isolated
from spoiled skipjack tuna and jack mackerel. J. Food Sci. 43: 1779-1781.
Pozo, R.G. and Saitua, E.S. 1988. Determinacin de histamina en pescado y sus produtos derivados por HPLC con deteccin flurimtrica. Alimentaria, Octubre: 27-29.
Rodrguez-Jerez, J.J., Lpez-Sabater, E.I., Hernndez-Herrero, M., and Mora-Ventura, M.A.T. 1994. Histamine, putrescine and cadaverine formation in Spanish semipreserved anchovies as affected by time/temperature. J. Food Sci. 59(5): 993-997.
Salguro, J.F. and Mackie, I.M. 1979. Histidine metabolism in mackerel (Scomber
scombrus) Studies on histidine decarboxylase activity and histamine formation during storage of flesh and liver under sterile and nonsterile conditions. J. Food Technol. 14: 131-139.
Sato, T., Okuzumi, M., and Fujii, T. 1995. Evaluation of polyamines of common mackerel during storage as indicators of decomposition. J. Food Hyg. Soc. Japan. 36(6):
743-747.
Taylor, S.L. 1986. Histamine food poisoning: toxicology and clinical aspects. CRC
Crit. Rev. Toxicol. 17(2): 91-128.
Taylor, S.L., Stratton, J.E., and Nordlee, J A. 1989. Histamine poisoning (scombroid
fish poisoning): an allergy intoxication. Clinic. Toxicol. 27: 225-240.
Veciana-Nogus, M.T., Vidal-Carou, M.C., and Marin-Font, A. 1990. Histamine and
tyramine during storage and spoilage of anchovie, Engraulis encrasicholus: Relationships with other fish spoilage indicators. J. Food Sci. 55: 1192-1193, 1195.
Wei, C.I., Chen, C.M., Koburger, J.A., Otwell, W.S., and Marshall, M.R. 1990. Bacterial growth and histamine production on vacuum packaged tuna. J. Food Sci. 55: 5963.
Wendakoon, C.N., Murata, M., and Sakaguchi, M. 1990. Comparison of nonvolatile
amine formation between the dark and white muscles of mackerel during storage.
Nippon Suisan Gakkaishi, 56(5): 809-818.
Yamanaka, H. and Matsumoto, M. 1989. Simultaneous determination of polyamines
in red meat fishes by high performance liquid chromatography and evaluation of
freshness. J. Food Hyg. Soc. Japan. 30: 396-400.
Yamanaka, H., Shiomi, K., Kikuchi, T., and Okuzumi, M. 1984. Changes in histamine
contents in red meat fish during storage at different temperatures. Bull. Jap. Soc.
Sci. Fish. 50(4): 695-701.
Yoshinaga, D.H. and Frank, H.A. 1982. Histamine-producing bacteria in decomposing
skipjack tuna. Appl. Environ. Microbiol. 44(2): 447-452.
Ms received 8/12/97; revised 11/25/97; accepted 1/26/98.
We acknowledge the financial support of Secretaria Regional de Agricultura e Pescas, Azores. We
also gratefully acknowledge Dr. M.J.R. Nout (Wageningen Agricultural University) and Dr. J.H.
Houben (Utrecht University) for critically reading the manuscript.

Volume 63, No. 4, 1998JOURNAL OF FOOD SCIENCE 647