Seasonal variability of lipophilic shellfish toxins in bivalves and waters, and abundance of dinophysis spp. in Jinhae Bay, Korea.
Marine toxins (Properties)
Marine toxins (Environmental aspects)
Bivalvia (Physiological aspects)
Kim, Ji Hoe
Lee, Ka Jeong
Kang, Yang Soon
Kim, Poong Ho
Song, Ki Cheol
Lee, Tae Seek
|Publication:||Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2010 National Shellfisheries Association, Inc. ISSN: 0730-8000|
|Issue:||Date: Dec, 2010 Source Volume: 29 Source Issue: 4|
|Topic:||Event Code: 310 Science & research|
|Geographic:||Geographic Scope: South Korea Geographic Code: 9SOUT South Korea|
ABSTRACT Lipophilic shellfish toxins (LSTs) produced by
dinoflagellates, including Dinophysis spp., can accumulate in bivalves.
The seasonal variability of LSTs in bivalves and waters, and the
abundance of Dinophysis spp. were investigated in Jinhae Bay, Republic
of Korea, in 2007. Diarrhetic shellfish poisoning (DSP) such as okadaic
acid and dinophysistoxin-1, and other lipophilic toxins such as
pectenotoxin-2 (PTX2) and yessotoxin were analyzed by liquid
chromatography-tandem mass spectrometry (LC-MS/MS). The toxin
concentration in size-fractionated plankton material (20-100 [micro]m in
size) concentrated from a specific volume of water was converted into
the toxin contents per unit volume of water. Among the genus Dinophysis,
only D. aeuminata was observed. The seasonal variability of okadaic
acid, dinophysistoxin-1, and PTX2 per unit volume of water paralleled
that of the abundance of D. aeuminata. The cellular content and profile
of toxins in D. acuminata varied, but PTX2 was mostly the dominant
toxin. The toxins in the hepatopancreas of mussels (Mytilus
galloprovineialis) were found from spring to autumn, and relatively high
levels of toxins were detected in summer (June to August). Variation of
the toxins in the hepatopancreas of mussels was remarkably consistent
with both abundance of D. aeuminata and toxin concentration in the
water. Our results suggest that LC-MS/MS analysis of toxin in plankton
concentrates has the possibility of becoming an alternative to or
complement of the conventional microscopic method in toxic phytoplankton
KEY WORDS: diarrhetic shellfish toxin, DSP, okadaic acid, dinophysistoxin, pectenotoxin, yessotoxin, Dinophysis acuminata, LC-MS/MS
Contamination of bivalves with lipophilic shellfish toxins (LSTs) including diarrhetic shellfish poisoning (DSP) and other lipophilic toxins is a worldwide problem for public health and fishery industries (FAO 2004, FAO/IOC/WHO 2004). DSP toxins such as okadaic acid (OA) and dinophysistoxins (DTXs), and lipophilic toxins such as pectenotoxins (PTXs) are known to bc produced by dinoflagellate species of the genera Dinophysis and Prorocentrum (Lee et al. 1989, Andersen et al. 1996, Mackenzie et al. 2005, Kamiyama & Suzuki 2008, Suzuki et al. 2009, Vale et al. 2009). Protoceratium reticulatum, Lingulodinium polyedrum, and Gonyaulax spinifera have also been described as producers of yessotoxins (YTXs) that are lipophilic toxins (Paz et al. 2007, Suzuki et al. 2007, Paz et al. 2008). These toxins produced by dinoflagellates can accumulate in filter-feeding bivalves such as mussels, oysters and clams. The concentration of toxins in a bivalve depends on the concentration of the causative organisms in the water column and the toxin content per cell. Monitoring of the causative phytoplankton in water is helpful for forecasting toxic outbreaks before they are concentrated in bivalves. However, discrepancies between the occurrence of Dinophysis spp. in the water column and the toxicity of shellfish have been reported from a number of countries (Hoshiai et al. 1997, Dahl & Johannessen 2001, Marcaillou et al. 2005).
The identification and enumeration of toxic phytoplankton by conventional microscopy requires substantial expert knowledge and human effort because all species of Dinophysis are similar in morphological features in the genus, and exhibit intraspecific morphological variability. These characteristics of this genus may lead to misidentification (Zingone et al. 1998, Culverhouse et al. 2003). Furthermore, the cellular toxin concentration of the dinoflagellates and the ability to produce toxins can vary among species of the same genus and among strains of the same species (Lee et al. 1989, Andersen et al. 1996, Mackenzie et al. 2005, Paz et al. 2007, Suzuki et al. 2007, Suzuki et al. 2009, Vale et al. 2009). Seasonal variability in toxin content per cell was also reported in the same species at the same location throughout the year (Mackenzie et al. 2005, Pizarro et al. 2009). Nevertheless, toxic and nontoxic strains of a phytoplankton species cannot be distinguished by microscopy.
Analysis of toxins in plankton material concentrated from a specific volume of water may take account of the variability of cellular toxin content in causative organisms. Recently, several research groups successfully quantified the toxin contents in handpicked cells of Dinophysis species by liquid chromatographytandem mass spectrometry (LC-MS/MS) (Puente et al. 2004, Fernandez et al. 2006, Suzuki et al. 2009, Pizarro et al. 2009). Only 10s to 100s of single-cell isolates of Dinophysis spp. were enough for confirmation of toxins. This LC-MS/MS technique may be challenging to apply as an alternative or complementary approach to monitoring toxic phytoplankton, although it is not possible to enumerate and/or identify the exact causative organism.
DSP incidents have never been officially reported in Korea, but low levels of OA and its derivatives have been found in the hepatopancreas of Korean bivalves (Kim et al. 2008), and the dinoflagellates Dinophysis spp. and Prorocentrum spp. have also been reported in Korean coastal waters (Lee et al. 1993, Kwak et al. 2001, Shin et al. 2005). However, there are currently no published data available to describe the relationship between the occurrence of causative organisms and related toxin contents in shellfish on the Korean coast.
In this study we investigated spatial and temporal variation of lipophilic shellfish toxins such as OA, DTXI, PTX2, and YTX in bivalves and size-fractionated plankton by LC-MS/ MS, and Dinophysis species abundance in Jinhae Bay on the south coast of the Republic of Korea in 2007. We also discuss the possibilities of applying the LC-MS/MS method for toxin analysis in plankton concentrate as an alternative or as a complement to the conventional microscopic method in routine toxic phytoplankton monitoring.
MATERIALS AND METHODS
High-performance liquid chromatography (HPLC)-grade solvents (acetonitrile,
methanol) and an analytical-grade solvent (methanol) were obtained from Merck (Darmstadt, Germany). Analytical-grade reagents (formic acid, ammonium formate) were purchased from Fluka (Buchs, Germany) or Sigma (St. Louis, MO). Distilled water was passed through a Milli-Q water purification system (Millipore, Bedford, MA) and was used for the preparation of HPLC mobile phases.
Standard solutions of OA (14.3 [micro]g/mL), PTX2 (8.6 [micro]g/mL), and YTX (5.3 [micro]g/mL) were obtained in methanol from the Institute for Marine Biosciences of National Research Council (Halifax, Nova Scotia). DTX1 was purchased from Wako Chemicals (Osaka, Japan) and dissolved in HPLC-grade methanol. Multistandard solutions for calibration and quantification of toxins were prepared in HPLC-grade methanol at concentrations of 10, 25, 50, and 100 ng/mL.
[FIGURE 1 OMITTED]
Field Sample Collection
Samples were collected twice a month at 2 stations (Geoje and Masan) in Jinhae Bay on the south coast of the Republic of Korea in 2007 (Fig. 1). Water samples were collected using a 5-L Niskin bottle at a depth of 2 m because of greater biomass in the surface water. The water collected in the Niskin bottle was mixed and used for Dinophysis cell counts and size fractionation of plankton material. Water temperature and salinity were measured at the same depth using a YSI 556 multiprobe system (YSI Inc., Yellow Springs, OH). Water samples for phytoplankton enumeration were collected in 1-L polypropylene bottles, and 10 mL Lugol's solution was added to each sample (American Public Health Association, American Water Works Association, and Water Environment Federation 1998). Plankton concentrate for toxin analysis in the water was obtained after size fractionation by sequential filtration. Twenty liters of sample was passed through a 100-1am mesh nylon sieve to eliminate large particles, and then plankton material was collected using a 20-[micro]m mesh nylon sieve. Plankton concentrate was resuspended in 20 mL filtered seawater in a 50-mL Falcon polypropylene conical tube (Becton, Franklin Lakes, NJ), and kept frozen at -20[degrees]C until used for the extraction of the toxins. Bivalve samples, cultured mussels (Mytilus galloprovincialis) and oysters (Crassostrea gigas), were collected at 2-3 m in depth from a hanging rope culture. The hepatopancreas from at least 12 individuals was combined and used for the toxin analysis.
Enumeration of the Toxic Phytoplankton Species
The Lugol-fixed samples were concentrated by sedimentation for 2 days and then siphoning off the overlying water. The remaining seawater was allowed to settle for more than 24 h and concentrated to a final volume of 20 mL in graduated tubes. LST-producing phytoplankton species were identified using a Nikon Eclipse 80i microscope (Nikon Co., Tokyo, Japan). Numerical abundance of toxin-producing phytoplankton cells was determined by counting in duplicate in a Sedgwick-Rafter counting chamber using a concentrated water sample (0.2 mL) (UNESCO 1974, American Public Health Association, American Water Works Association, and Water Environment Federation 1998). The plankton cell counts were expressed per unit volume of water. The lower limit of plankton detection was 100 cells/L in this study. Although the modified Utermohl method that requires only 2 h settling time is even more preferable for counting phytoplankton (Paxinos & Mitchell 2000), the standard method that requires at least 24 h of settling was used in this study because the method is relatively simple.
Sample Treatment for Toxin Analysis
For toxin analysis of the size-fractionated plankton material, we used the solid phase extraction method of Suzuki et al. (1997, 1998) with minor modifications. Frozen plankton samples in seawater were thawed at room temperature and sonicated for 2 min in a water bath ultrasonicator (Branson ultrasonic cleaner; Branson Ultrasonics Co., Danbury, CT), and filtered through Toyo No. 5A filter paper (Toyo Roshi Kaisha Ltd, Tokyo, Japan). Two milliliters of 200 mM phosphate buffer (pH, 5.8) was added to 20 mL of the filtrate, then loaded onto a Sep-Pak C18 plus cartridge column (Waters, Milford, MA), which had been previously conditioned with 5 mL methanol and 20 mM phosphate buffer (pH, 5.8). The cartridge was washed with 10 mL distilled water to remove the seawater salt, and the toxins were eluted with 5 mL methanol. The methanol eluates were evaporated, and residues were dissolved in 200 [micro]L methanol. An aliquot of this solution was directly analyzed by LC-MS/MS. The dissected hepatopancreas of shellfish were homogenized with 9x volume of 90% methanol and the extracts were centrifuged at 3,000g for 5 min. An aliquot of supernatant was passed through a 0.45-[micro]m Minisart RC4 filter (Sartorius-Stedim, Goettingen, Germany) for direct injection into the LC-MS/MS.
LC-MS/MS analysis of toxins was carried out according to a previous method (Suzuki et al. 2005, Kim et al. 2008) with a slight modification by using selective reaction monitoring (SRM) instead of selected ion monitoring. The HPLC unit consisted of a Surveyor MS Pump plus and Surveyor AS plus (Thermo Electron, Finnigan; San Jose, CA). The separation was performed on a Luna C18(2) column (150 x 2.0 mm, 5 [micro]m; Phenomenex, Torrance, CA) preceded by a guard column cartridge (4.0 x 2.0 mm; Phenomenex). The column temperature was maintained at 35[degrees]C. Eluent A was water and B was acetonitrile-water (95:5), both containing 2 mM ammonium formate and 50 mM formic acid (Quilliam 2003). Linear gradient elution from 20-100% B was performed for 5 min and then held at 100% B for 10 min, followed by re-equilibration with 20% B (5 min). The flow rate was 0.2 mL/min and the injection volume was 10 [micro]L. The sample tray temperature in the Surveyor AS plus was set at 7[degrees]C. A triple-quadrupole mass spectrometer (Thermo Electron, Finnigan, TSQ Quantum Discovery Max, San Jose, CA) was used for mass detection. The eluate from the column was introduced into an electrospray ionization interface without splitting. All analyses were carried out in negative ion electrospray ionization, with the spray voltage set at 4.5 kV. The heated capillary temperature was set at 200[degrees]C. Nitrogen sheath gas was set at 37 arbitrary units and auxiliary gas at 10 arbitrary units. SRM LC-MS/MS analysis for toxins was carried out using [[M-H].sup.-] (OA, DTX1, YTX) or [[M + HCOOH-H].sup.-] (PTX2) as the target parent ions in Q1 and particular fragment ions of each toxin in Q3. The detailed SRM conditions are shown in Table 1.
Calculation of Toxin Concentrations in Water Columns and Plankton Cells
The concentrations of OA, DTX1, PTX2, and YTX per unit volume of seawater (10 L) were calculated from toxin concentrations in size-fractionated plankton material from 20 L water. In addition, the concentration of OA, DTX1, or PTX2 per cell of Dinophysis sp. was only calculated when the plankton was counted in the water sample and the toxins were detected from size-fractionated plankton material in the same water column. The toxin content per cell was calculated by dividing the measured amount of toxin of the size-fractionated plankton sample with the corresponding cell density.
RESULTS AND DISCUSSION
The Jinhae Bay region has a temperate climate. A seasonal cycle in seawater temperature was observed at both stations. The water temperature ranged between a minimum of 6.9[degrees]C in January and a maximum of 28.2[degrees]C in August. Salinity values displayed larger variations from 27.78-33.56 psu at the Geoje station (Fig. 2A) and from 25.63-33.07 psu at the Masan station (Fig. 3A). Salinity also showed remarkable seasonal variations. The lowest salinities at each station were recorded in September because of the monsoon, and the highest salinities were noted during winter months.
Among the genus Dinophysis, only D. acuminata was identified during this study. D. acuminata was sporadically detected in the range of 200-1000 cells/L from March to September (Fig. 2B and Fig. 3B). D. acuminata was found within a wide range of water temperature, between 9.4[degrees]C and 26.0[degrees]C. The occurrence and abundance of D. acuminata differed between the sampling stations. Our results are consistent with those of other studies indicating that D. acuminata was observed in the water temperature range of 4-27[degrees]C in Jinhae Bay (Kwak et al. 2001).
The seasonal variability of OA, DTX1, and PTX2 per unit volume of seawater (Fig. 2C, D and Fig. 3C, D) paralleled that of the abundance of D. acuminata at both stations. The toxins in size-fractionated plankton were detected from spring to early autumn, and the relatively high toxin levels per unit volume of the water were found in summer (July to August). When the cell counts of D. acuminata reached 1000 cells/L (Fig. 3B) in September 2007, toxin concentrations per unit volume of water were 7.9 ng/10 L for OA, 8.0 ng/10 L for DTX1, and 81.0 ng/10 L for PTX2, but were not detectable for YTX (Fig. 3C, D). Although the same abundance of D. acuminata cells (200 cells/ L) was observed 3 times, concentration of toxins in the water varied: 0.8-1.7 ng/10 L for OA, 0.0-1.1 ng/10 L for DTX1, and 0.0-37.0 ng/10 L for PTX2. The peak concentrations of OA, DTX1, and PTX2 in seawater were consistent with the abundance of D. acuminata, but low levels of the toxins were detected even during periods when cells were below detection levels. No YTX producers, such as P. reticulatum, were found during the study, and very low concentrations of YTX per unit volume of seawater were detected. These results are thought to occur because our detection system for toxins was very sensitive whereas that for phytoplankton species (100 cells/L) was not. Thus, our results suggest that LC-MS/MS analysis of toxins in plankton concentrates has the ability to be a very sensitive alternative, or a complement, to the conventional microscopic method in routine toxic phytoplankton monitoring.
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
On the other hand, if it is assumed that D. acuminata is solely responsible for all the toxins detected in the plankton concentrate, the toxin concentration per cell of the organism ranged from 0.4-0.9 pg/cell for OA, 0.0-0.8 pg/cell for DTX1, and 0.018.5 pg/cell for PTX2 (Table 2). Although spatial and temporal variability were observed in cellular toxin content and profiles of D. acuminata, in most cases PTX2 was the dominant toxin in this species. The toxins such as OA, DTXs, and PTXs have previously been identified in several species of Dinophysis, and PTX2 is found as the predominant toxin in wild or cultured D. acuminata (Mackenzie et al. 2005, Kamiyama & Suzuki 2008, Suzuki et al. 2009). The OA concentration per D. acuminata cell has been reported to range from trace to 2.8 pg/cell by LC-MS/ MS (Mackenzie et al. 2005, Adachi et al. 2008). DTX1 was found at concentrations of 0.1-2.4 pg/cell (Mackenzie et al. 2005) and 0.3-1.1 pg/cell (Suzuki et al. 2009) from wild D. acuminata in New Zealand and Japan, respectively. Kamiyama and Suzuki (2008) also reported that cultured D. acuminata contained 2.54.8 pg/cell of DTX1 and 14.7-14.8 pg/cell of PTX2, but there was no DTX1 in wild cells collected at the same location where the cultured D. acuminata strain was isolated. Thus, these results revealed that prediction of shellfish toxicity by only monitoring toxic phytoplankton is very difficult, because the cellular concentration and profile of toxins in D. acuminata varied temporally and between strains from different regions. The ratios of OA to DTX1 in the plankton concentrates, with the exception of a specimen collected March 26 at Geoje, were calculated in the range of 1.0-2.0, reflecting a slightly higher production of OA than DTX1 (Table 2). And the ratios of PTX2 to total OA (OA plus DTX1) ranged from 5.1-12.3. This means that PTX2 is the dominant compound in the plankton concentrates. Similar PTXs-to-OAs ratios (8.0-29.0) in D. acuminata cell concentrates was previously reported in New Zealand (Mackenzie et al. 2005).
Although the approximate distance between sampling stations was about 21 km in a straight line (Fig. 1), the levels and profiles of toxin in the hepatopancreas of mussels that were collected on the same date differed significantly between sampling stations. Roughly, toxins were detected in the hepatopancreas of mussels from February to October, but relatively high levels of toxins, with the exception of DTX1, were found in summer at both stations. The toxicity of the hepatopancreas of mussels collected at Masan was higher than those collected at Geoje. The highest levels in the hepatopancreas of mussels of OA (0.046 [micro]g/g at Geoje, 0.168 [micro]g/g at Masan), PTX2 (0.051 [micro]g/g at Geoje, 0.105 [micro]g/g at Masan), and YTX (0.081 [micro]g/g at Geoje, 0.011 [micro]g/g at Masan) were found in summer (June to August). DTX1 was also detected in summer, but the highest level at each station appeared in February (0.171 [micro]g/g at Masan) and March (0.028 [micro]g/g at Geoje). The concentration of the sum of OA, DTX1, and PTX2 in the hepatopancreas of some mussel samples exceeded the European standard (0.16 [micro]g/g, whole body) (European Commission 2002); however, the toxin concentration calculated in whole-body tissue was at a safe level for consumption. Similarly, large geographical variation in LST levels, even between adjacent cultivation areas, has previously been reported in European waters (Carmody et al. 1996, Lindahl et al. 2007). Geographical variation in toxicity indicates that causative organisms are not distributed uniformly in an area. In the hepatopancreas of oysters, OA and PTX2 were hardly detected, and low levels of YTX were rarely detected in spring, but DTX1 was once found at 0.015 [micro]g/g in July (Fig. 2F). A remarkable difference in toxin levels was observed between the bivalve species (mussels, M. galloprovincialis and oysters, C. gigas) at the same station (Fig. 2E, F). The concentrations of OA, DTX1, PTX2, and YTX in mussels were significantly higher than those in oysters. It was reported in a field study that the content of the OA group of toxins was at least 10 times higher in mussels (Mytilus sp.) compared with oysters (C. gigas) (Vale & Sampayo 2002, Madigan et al. 2006, Lindegarth et al. 2009). Therefore, oysters could accumulate less DSP and other lipophilic toxins than mussels. We have previously reported that OA and DTX1 were found in a few mussels collected in April and June, but PTX and YTX were not detected from any bivalve samples from the south coast of Korea (Kim et al. 2008). In this study, we report the first finding of PTX and YTX in Korean bivalves.
A remarkably consistent tendency was observed between concentrations of OA, DTX1, and PTX2 in the hepatopancreas of mussels and the abundance of D. acuminata or the toxin concentrations per unit volume of seawater. With some exceptions, the concentrations of PTX2 in seawater were 10-20 times higher than those of OA, but the concentration of PTX2 in the hepatopancreas of mussels was much lower than that of OA. Because the blue mussels (M. edulis) and greenshell mussels (Perna canaliculus) have been reported to convert PTX-2 to PTX2 secoacid (Miles et al. 2004, Suzuki et al. 2001), this result may indicate that the PTX2 in D. acuminata was converted to PTX2 secoacid in the hepatopancreas of the mussels. However, the relation between the toxin concentrations per unit volume of seawater and shellfish toxicity is not always consistent. Such a discrepancy may be related to the very high spatial and temporal variability of Dinophysis spp. distributions and/or quality of total available food for shellfish, such as chlorophyll and seston (Reguera et al. 2003, International Council for the Exploration of the Sea 2005). Dahl and Johannessen (2001) found a positive relationship between the concentration of DSP toxins in blue mussels (M. edulis) and increasing ratios of D. acuta (measured in cells per liter) to chlorophyll a. The relative amount of toxins (in relation to the total seston volume) available in the food has been known to be as an important factor affecting the assimilation of toxins (Morono et al. 2001). When the paralytic shellfish poisoning toxins accumulation per mussel (M. galloprovincialis) was analyzed, volume-specific toxin concentration was the most important factor in the accumulation of toxins (Morono et al. 2001), and this can be generalized to DSP toxins as supported by the field observations of Sampayo et al. (1990). The use of the ratio between toxin concentration and the amount of food may be desirable for more accurate prediction of DSP events.
It was demonstrated that LC-MS/MS analysis of toxins in plankton concentrates is possibly an alternative or a complement to conventional microscopic methods in routine toxic phytoplankton monitoring for prediction of shellfish toxicity. Furthermore, the toxin analysis techniques may be useful for a more accurate estimate of toxin content in the seawater, because this technique may take into account the variation of cellular toxin content in causative organisms.
We thank the 2 anonymous reviewers for their many valuable comments that greatly improved the article. This work was funded by a grant from the National Fisheries Research and Development Institute, Korea (RP-2010-FS-010).
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JI HOE KIM, (1) * KA JEONG LEE, (2) TOSHIYUKI SUZUKI, (3) YANG SOON KANG, (4) POONG HO KIM, (2) KI CHEOL SONG (2) AND TAE SEEK LEE (1)
(1) Southeast Sea Fisheries Research Institute, National Fisheries Research & Development Institute, 361, Yeongun, Sanyang, Tongyeong, Gyeongnam 650-943, Republic of Korea; (2) Food Safety Research Division, National Fisheries Research & Development Institute, 408-1, Sirang, Gijang, Busan 619-705, Republic of Korea; (3) Biochemistry and Food Technology Division, National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fukuura, Kanazawa, Yokohama 236-8648, Japan; (4) Southwest Sea Fisheries Research Institute, National Fisheries Research & Development Institute, 347, Anpo, Hwayang, Yeosu, Jeonnam 556-823, Republic of Korea
* Corresponding author. E-mail: email@example.com
TABLE 1. Selective reaction monitoring tandem mass spectrometry conditions of each toxin. Ions Toxins Q1 (parent ion) Q3 (fragment ion) CE (eV) Okadaic acid 803.5 255.4 -48 Dinophysistoxin-1 817.5 255.4 -48 Pectenotoxin-2 903.5 137.0 -60 Yessotoxin 1141.5 1061.5 -40 CE, collision energy. TABLE 2. Calculated cellular toxin content of Dinophysis acuminata cells in Jinhae Bay, Korea in 2007. Sampling Station and Collection Date Geoje Masan Toxins (pg/cell) March 26 July 2 June 4 July 18 September 3 Okadaic acid (OA) 0.5 0.9 0.4 0.9 0.8 Dinophysistoxin-1 0.0 0.6 0.2 0.6 0.8 (DTX 1) Pectenotoxin-2 0.0 18.5 5.6 10.9 8.1 (PTX2) OA/DTX1 N/A 1.5 2.0 1.5 1.0 PTX2/OAs N/A 12.3 9.3 7.3 5.1 (OA + DTX 1) N/A, not applicable.
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