Anal. Chem. 1996,67, 225-228
Chemiluminescence Detection of Red Tide Phytoplankton Chattonella marina Tsai-yun Lee, Naohim Ootoh,t Etsuo Niki, Kenji Yokoyama,* Mikio Tsu#lki,@ Toshiftmi Takeuchi)' and 1-0 Karube*
Research Center for Advanced Science and Technology, University of Tokyo, 4-6- I Komah, Meguru-ku, Tokyo 153,Japan resulted in about 20 billion yen (=$190 million) lost during the A sensitive method was developed for monitoring the cell years 1970-1988.2 concentration of red tide phytoplankton Chattonella Till now, many approaches for red tide detection have been marina, which frequently caused fish death via an obscure mechanism. 2-Methyl-6-(p-methoxyphenyl)-3,7- proposed, including remote sensing by multispectral scanner from dihydroimidazo[1,2-alpyrazin-3-one (MCIA), a Cypyair~raft.~ Although these methods may be useful for sketching ridina luciferin analog, which emits light strongly at 465 the distribution map of red tides, none of them could offer the nm in the presence of superoxide, was applied as a sensitivity that is urgently needed for early detection of red tides chemiluminescenceprobe. The MCIA-dependent chemito prevent the damage of aquacultures. luminescence of room-cultured axenic C. marina was Recently, Shimada et al. found that Chattonella antiqua causes efficiently suppressed by adding superoxide dismutase superoxide dismutase (SOD)-inhibitablecytochrome c red~ction,~ (20 units mL-l) to the phytoplankton suspension but not and Oda et al. proved the production of the spin adduct of by filtering out the plankton cells, indicating the chemisuperoxide anion (02-) OMPO-OOH) in a suspension of Chatluminescence was due to the superoxide released from tonellu marina by spin-trappingassay: suggestingthat Chattonella C.marina. Several species of other tested phytoplankspp. release 02- into water. Although superoxide can be generton, such as Heterosigma akashiwo, Skeletonema ated during photosynthesis in chloroplasts,7chloroplast thylakoids costatum, Chaetoceros sociale, and Porphyridium showed little permeability to this negatively charged 02- in cruentrum did not induce this chemiluminescence experiments where spinach was used! Superoxideis harmful to whereas Chattonella antiqua showed characteristic living organisms due to its own toxicity; more than that, a more similar to C. marina, suggesting that this method has harmful radical, OH', could be formed from superoxide through specificity for the Chattonella genus. As compared with a Fenton-type Haber-Weiss reaction in the presence of Fe3+ion the cell density of C. marina during the red tide seasons, in seawater. Although many of the researchers have suggested the detection limit of 200 cells mL-' based on this these free radicals are the agents of the fish kills during C. marina luminescent method is considered to be applicablefor C. red tides, the progress of the investigation in this field is still very marina detection in the early stage of its red tides. Red tide is a phenomenon caused by small algae or phytoplankton where seawater becomes red. Both toxic and nontoxic red tides have occurred throughout recorded history, but in recent years, there has been a global increase in the number of these events due to coastal pollution and, probably, to other unclear factors, for example, long-distance transport of species across oceans.' Some kinds of red tides cause mass fish death and thus bring serious economic problems to the countries where people take fish as one of the important protein supplies. In Japan, one of the most troublesome red tides is caused by Chattonella spp., which have killed 30 million yellowtail in the Set0 Inland Sea and Resent address: Kao Corp., Kashima Research Labs., 20,Higashi Fukashiba, Kamisu-machi, Kashima-gun, Ibaraki, 314-02 Japan. + Resent address: Faculty of Material Science, Japan Advanced Insititute of Science and Technology Hokuriku, 15 Asahidai, Tatsunohchi-machi, Nomi-gun, Ishikawa, 92512. School of Life Science, Tokyo University of pharmacology, Horinouchi, Hachioji, Tokyo 192-03Japan. II Resent address: Faculty of Information Sciences, Hiroshima City University, Asaminami-h, Hiroshima 731-31Japan. (1) Anderson, D. M. In Red ti&: biology, environmental science and toxicology; Okaichi,T., Anderson. D. M., Nemoto, T., Eds.;Elsevier. New York, 1989 pp 11-16. +
0003-2700/95/0367-0225$9.00/0 Q 1994 American Chemical Society
slow. In this study, a flow-type chemiluminescence analyzer was constructed for the detection of superoxide released from C. marilta. The chemiluminescent probe used was 2-methyl+@ methoxyphenyl)-3,7-dihydroimidazo[1,2-alpyrazin-3-one (MCLA), a Cypyridina luciferin analog, a superoxidespecific reagent that was originally synthesized by Nishida et al. and has been applied for estimating the superoxide-generation ability in systems such (2) Okaichi, T. In Red tides: bioloa, environmental science and toxicology; Okaichi,T., Anderson, D. M., Nemoto,T., Eds.; Elsevier: New York, 1989 pp 137-142. (3) Matswura, S.;Yokota, M. In Red fides: biology, environmental science and toxicology; Okaichi, T., Anderson, D. M., Nemoto, T., Eds.; Elsevier: New York, 1989; pp 193-196. (4) Yentsch, C. M. In Red tides: biologv, environmental science and toxicology: Okaichi, T., Anderson, D. M., Nemoto, T., Eds.; Elsevier New York, 1989 pp 221-224. (5) Shimada, M.; Shmono, R; Mwakami, T. H.; Yoshmatsu, S.;Ono, C. In Red tides: bioloa, environmental science and toxicology: Okaichi, T., Anderson, D. M., Nemoto, T., Eds.; Elsevier: New York, 1989 pp 443446. (6) Oda, T.;Ishimatsu, A; Shimada, M.; Takeshida, S.;Muramatsu, T. Mar. Biol. 1992,112,505-509. (7)Halliwell, B.; Gutteridge J. M. C. Free Radicals in Biology and Medicine; Oxford University Press: London, 1985; Chapter 5. (8) Takahashi, M.; Asada, K PIanf Cell Phydol. 1982, 23 (S),1457-1461.
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995 225
as human granulocytes and monocytes? Because this is the first time for use of MCLA in a flow-type analysis, its concentration and pH effects were investigated to obtain sufficient detection sensitivity. In order to confirm whether the superoxidereleasing ability is common to phytoplankton or not, other species such as C. antiqua, Heterosigma akashiwo, Skeletonema costatum, and Chaetoceros sociale were also analyzed.
IQI
MATERIALS AND METHODS
Plankton Culture. ChaftoneZZa marina N I B 3 and NIES14, C. antiqua NIESl and NIESll4, Heterosigma akashiwo NIESG, Skeletonema costatum "324, and Chaetocerossociale N E 3 7 7 were provided by the National Institute for EnvironmentalStudies, Environment Agency, Japan. Porphyridum cruentrum R-1 was obtained from the IAM Culture Collection, Institute of Molecular and Cellular Biosciences, University of Tokyo. These plankton were cultured at 25 "C, under fluorescent light sources of 40 p E m-2 s-l with a light-dark period of 12 and 12 h. Erd-Schreiber modified (ESM) medium was applied for cultures in which soil extract was excluded from the original medium composition. Namely, 120 mg of NaN03, 5 mg of bHP04, 100 pg of vitamin B1,lO pg of vitamin B12,l pg of biotin, 260 pg of Fe-EDTA, 330 pg of Mn-EDTA, and 1 g of tris(hydroxymethy1)aminomethane were dissolved in 1 L of seawater, and the pH was adjusted to 8.0. All the plankton were harvested on the second day after transfer to a new culture medium in order to keep the pH and medium composition from obvious change, which likely affected the intensity of chemiluminescence. C. marina N I B 3 was used as the plankton sample, unless otherwise mentioned. Reagents. MCLA was dissolved in distilled water passed through Milli-Q PLUS (Millipore, Bedford, MA), and the MCLA solution was stored in 1.0 mL aliquots at -80 "C until needed. For chemiluminescence analyses, dilution was performed with carbonate buffer at pH 9.7 except experiments on the effects of pH. The concentration of MCIA was calculated by using the value of -6 = 9600 (mol L-l)-l cm-l. Bovine liver superoxide dismutase (SOD) and catalase were purchased from Sigma Chemical Co., St, Louis, MO. All other reagents were of analyticalreagent grade and were used as received. ChemiluminescenceAnalysis. The schematic diagram of the flow-type chemiluminescence analyzer is shown in Figure la. MCIA solution was cooled by an ice bath, and the temperature of plankton suspension was usually controlled by using a thermostat at 25 "C except in the experiments on the effects of temperature. Before and after the luminescence measurement, distilled water flowed through the analyzing system to wash it. For the luminescence measurement, MCLA solution and phytoplankton suspension were simultaneouslypumped into the spiral flow cell via the mixer (Figure lb) (refer to the description of Imailo), where MCLA and phytoplankton suspension were mixed by a rotary flow (Figure IC). Much higher luminescence was obtained from the mixture that passed through the rotary-flow mixer than through a simple Y-type or T-type mixer. Chemiluminescence emitted from the mixture of MCIA and algal suspension was measured with a combination of photomultiplier tube (9) Nishida, IC;Kimura, H.;Nakano, M.;G0to.T. Clin. a i m . Acta 1989,179. 177- 182. (10) Imai, K. In Bioluminescence and Chemiluminescence,Basic andL3petimental; Imai, IC, Ed.; Hirokaw Tokyo, 1989; pp 257-276.
226 Analytical Chemistry, Vol. 67, No. 7, January 7, 7995
D 1.0 mm i. d. 0.5 m m i. d.
-m,
Figure 1. (a) Schematic diagram of the flow type chemiluminescence analyzer: A, MCLA solution; B, ice-bath; C, phytoplankton suspension; D, thermostat; E, peristaltic pump; F, rotary-flow mixer; G,spiral flow cell; H, photomultiplier; I, photocounter; J, personal computer. (b) Three-dimensional schematic diagram of the rotaryflow mixer. (c) The rotary flow in the mixer.
(R 1332, Hamamatsu photonics Co., Ltd., Shizuoka, Japan) and a photon counter (C 5410, Hamamatsu). RESULTS AND DISCUSSION
QuantitativeDetermination of MCIA-Dependent Chemiluminescence. Simultaneous flow of MCLA and the suspension of C. marina caused a marked luminescence, and a stationary and maximal luminescence was observed after the spiral flow cell was filled with the mixture (Figure 2). The internal diameter in every part of the analyzing system was all wider than 0.5 mm, much greater than the size of the tested phytoplankton. As confirmed by light microscopy, even the highest pumping speed (1.6 mL min-l) did not rupture the very delicate cells of C. marina. Excluding 90% of the plankton cells of C. marina by 4 gentle cotton filter in the inlet of plankton suspension caused only a 10% decrease in luminescence, indicating that the detected MCLAdependent chemiluminescence was due to some species in the suspension of C. marina but not due to the direct interaction between MCLA and the plankton cells themselves. The remarkable chemiluminescence was suppressed by the addition of 20 units mL-l SOD to the C. marina suspension to the level of the blank ESM medium where C. marina was not included (Figure 2). This result suggests the contribution of superoxide to this chemiluminescence. ESM medium itself also induced the luminescence of MCLA, which was somewhat reduced by SOD addition. Even deionized water and MilliQ water caused this blank luminescence, indicating that the chemicals in the ESM medium were not the main agents of this background luminescence.
,
h
C. marina + MCLA
ESM medium + MCLA
+SOD .....
,_
(ESM medium + 20 U/ml SOD ) + MCLA
,.'
y ..' 0
9.5
9
10.5
10
11
pH of MCLA I
I
Figure 4. pH effect of MCLA solution. MCLA was dissolved in 0.2 M carbonate buffer: C. marina concentration 17000 cells/" [MCLA] = 0.4 GM.
- 7001
I
I
0
0.4
0
40
0.8
1.2
80 120 [luminol] (pM)
1.6
160
Flgure 3. Concentration effects of MCLA (0)and luminol (W): C. marina concentration. 15000 cells/mL.
Singlet oxygen (l02) is also known to react with MCLA with high reaction rate;" however, it seems little or none was released from C. marina, as confirmed by the addition of its efficient quencher, sodium azide. No detectable inhibition was caused by catalase addition, indicating that H202, a product of the dismutation reaction of 0 2 - , did not contribute to this luminescence. This result is reasonable because only a very low concentration of H202 could be formed from the dismutation of 02- at pH 8 in the ESM medium according to calculation by the reaction rate equations. Concentration and pH Effects of the MCIA Solution. The relation between chemiluminescence intensity and MCLA concentration is shown in Figure 3. The higher the concentrationof MCLA used, the stronger the chemiluminescence obtained. In the case of 15 000 cells C. marina mL-l, the luminescence was almost linearly related to the concentration of MCLA from 0.1 to 1.6pM, which coincides with the results in the xanthiie/xanthine oxidase system.ll hminol, a widely used chemiluminescent probe, emitted much less light than MCLA even if its concentration was lWfold greater than MCLk The most effective parameter for this chemiluminescence detection was the pH value of the MCLA solution. At low pH, chemiluminescencewas very low, which resulted in a compara(11) Suziki,N.;Ogawa, K; Yoda, B.;Nomoto, T.;Inaba, H.;Goto, T.Nippon s u h n Gukkaishi 1991,57,1711-1715.
tively low signal/noise (S/N) value. The chemiluminescence increased rapidly as the pH exceeded 9.2 and reached to its maximum as the pH was raised to 9.7 (Figure 4). This luminescence intensity at pH 9.7 was 2 orders of magnitude greater than the background, and the lower concentration of C. marina therefore became detectable. There was a suggested mechanism for Cypyridina luciferin (L) anion and oxygen, involving a nonchain reaction between the luciferin and superoxide that gives a hydroperoxide anion through a radical pair intermediate92
LH
- L- -[L"O,-] - L-00- 0 2
products
+ hv
MCLA, being an analog of Cvpvridinu luciferin, is proposed to react with superoxide by a mechanism similar to the above. Until recently, since analysis of systems for superoxide produced from stimulated macrophages or xanthine/xanthine oxidase system are carried out in batch cells, the pH of the MCLA solution itself has not been studied. Our results, however, suggest the importance of the pH of the MCLA solution, which strongly affects the light yield of chemiluminescence in a flow analyzing system where the pH of the sample itself cannot be widely changed.
O
H
'
MCLA
Incubation Temperature of Chattonella murinu. The activity of phytoplankton, as with other organisms, varies with temperature. Temperaturesthose lower than 20 "C or higher than 25 "C reduced the MCLAdependent chemiluminescence of C. marina; the luminescence maximum was observed in the region from 20 to 25 "C (Figure 5). This result coincides with the multiplication rate for C. marina in this temperature range. The temperature of the suspension itself did not affect the luminescence intensity of MCLA in this range, agreeing with the blank (12) Goto, T.Pure Appl. Chem. 1968,17, 421-441.
Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
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- 2001
1
?
5
15 20 25 30 35 INCUBATION TEMPERATURE ("C)
40
Figure 1. Incubation temperature effect of C. marina on MCLAdependent chemuluminescence: C. marina concentration 8600 cells/ mL; [MCLA] = 0.8 pM.
test of ESM medium. The intensity of MCLAdependent chemiluminescence therefore only reflects the superoxide-producing ability in each sample. However, we could not get a conclusion of the rank of this producing ability for each individual cell because a part of the cells in the samples incubated at 10, 15, and 35 "C seemed to have lost part (for 15 and 35 "C) or all (for 10 "C) of their activity during the incubation process and could not recover even when the incubation temperature was held to 20 "C for 1 h. Similar sensitivity to temperature was also observed in maintenance of the C. marina culture. At temperatures lower than 10 "C, the suspension of C. marina became more transparent and its color changed from yellowish brown to yellow. As confirmed with light microscopy, all the cells had been broken down. In addition, the absorption peaks of chlorophylls and carotenoids, which were not clear in the absorption spectrum of the C. marina suspension, became sharp and strong in this 10 "C incubated sample, indicating that these pigments had been released from the broken cells to the culture medium. Since superoxideis very reactive to the carbon-carbon double bond, the highly unsaturated carotenoids thus could easily consume this nucleophilic anion. Superoxide dismutase may also contribute to the superoxide disappearing process. As the algal cells were broken down, superoxide dismutase could be released from the plankton cells and promote the dismutation of superoxide. Since C. antiqua, which appeared before or after the red tides of C. marina, also showed a similar temperature effect on its growth rate (u) and final concentrationafter %day culture,13this strong temperature dependence of Chattonella might be the reason why their red tides only occur in summer. The comparatively higher growth rate and MCLAdependent chemiluminescence of C. marina at 20-25 "C suggest a relatively high probability of superoxiderelease in the summer where outbreaks of the red tides of C. marina occur and cause mass fish death. Calibration and Specificity. The typical calibration curve of C. marina is shown in Figure 6, where the plankton suspensions were incubated at 25 O C and with 0.8 pM MCLA at pH 9.7. Although greater errors appeared among lower cell concentrations, the chemiluminescencedue to a concentrationof 200 cells mL-l was detectable. (13) Nakamura, Y.;Watanabe, M. M. /. Oceanog. SOC./fin. 1983,39,110114. (14) Montani,S.;Tokuyasu, M.; Okaichi,T. In Red fides: biobgy, environmental science and toxicology; Okaichi, T., Anderson, D. M., Nemoto, T., Eds.; Elsevier. New York, 1989 pp 197-200. 228 Analytical Chemistry, Vol. 67, No. 1, January 1, 1995
0
2000 4000 6000 CELL CONCENTRATION (cells/ml)
8000
Figure 6. Typical calibration curve of C. marina (0).Other tested red tide phytoplankton, such as H. akashiwo (W), did not show a detectable MCLA-dependent chemiluminescence in the same detection condition: [MCLA] = 0.8pM; pH of MCLA, 9.7; 25 "C. Each value is the mean f S D for triplicate determinations.
In the Set0 Inland Sea, a density of 10 cells/ml appeared in several samples in early July and 5400 celldm1 was observed in its most dense patch on 22 July in the red tide outbreak of 1983.14 The detection limit of 200 celldm1 based on our method thus strongly suggests an applicable early detection of C. marina by utilizing its MCLA-dependent chemiluminescence. Heterosigma akashiwo, which belongs to the same class as C. marina, Raphytophyta, did not show as detectable a chemiluminescence ( F i r e 6) as C. marina. Other tested species in different classes, Skeletonema costutum, Chaetoceros sociale @acillariophyta) and Porphyridium cruentrum (Rhodophyta), also did not show this chemiluminescence even though their cell concentrations were high as 20 OOO cells mL-l. Although superoxide can be generated in the chloroplasts, nondetectable luminescence induced by other tested species suggests that the detected superoxide is generated in some sites other than the chloroplasts of these photosynthetic organisms or that the scavenging system in Chattonella is different from other species. Both the tested strains of C. marina, NIES3 and NIES14, and another fish-killing species of Chattonella genus, C. antiqua (two strains were investigated, NIESl and NIES114), all showed this MCLAdependent chemiluminescence, indicating that both the red tides of C. marina and C. antiqua could be detected by this method . In conclusion, the described MCLAdependent chemiluminescent method could provide a simple monitoring for C. marina with sensitivity higher than that obtained by other investigators. Furthermore, since this method is based on the superoxide released from phytoplankton, application to other phytoplankton that have the same characteristics as C. marina could therefore also be expected. ACKNOWLEDQYENT
The authors thank Prof. Y. Fukuyo and K. Ikebukuro for their help in collecting information, and IC Hayashi for helpful advice in the fabrication of the rotary-flow mixer. Received for review May 18, 1994. Accepted October 4, 1994.a AC940503L @Abstract published in Advance ACS Abstracts, November 15,1994.
