Environ. Sci. Technol. 2005, 39, 1492-1505
Use of Transplanted Zebra Mussels (Dreissena polymorpha) To Assess the Bioavailability of Microcontaminants in Flemish Surface Waters L I E V E N B E R V O E T S , * ,† J U D I T H V O E T S , † ADRIAN COVACI,‡ SHAOGANG CHU,‡ DIAB QADAH,† ROEL SMOLDERS,† PAUL SCHEPENS,† AND RONNY BLUST† Department of Biology, Ecophysiology, Biochemistry and Toxicology Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, and Department of Pharmaceutical Sciences, Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium
Zebra mussels (Dreissena polymorpha) were translocated in cages to 56 water bodies in Flanders (Belgium) during summer 2001. After six weeks, concentrations of polychlorinated biphenyls (PCBs), hexachlorobenzene (HCB), p,p′DDE, and trace metals were measured in the transplanted mussels. It was investigated whether total dissolved water and sediment pollutant levels or bioaccumulation factors (BAFs) and biota-sediment accumulation factors (BSAFs) were predictive for mussel tissue levels. The sample sites covered a broad range both in terms of the type and concentration of the pollutants, and this was reflected in large differences in tissue concentrations of all pollutants among the sites. The highest pollutant levels in mussels were among the highest reported in the literature. For Cd and Zn levels up to 33 and 1994 µg/g dry wt. respectively were found. The lowest levels were comparable to those from uncontaminated sites in Europe and the U.S. For Cd and Zn respectively 51 and 75% of the variation in tissue levels was described. For both metals, dissolved and particulate metal contributed to the variation in accumulation. For other pollutants, relationships between tissue concentration and water or sediment concentration were weak or nonsignificant. Then the measured environmental factors (dissolved calcium, pH, oxygen, organic carbon and clay content in the sediment) were taken into account applying multiple regression analysis, and no increase in the described variation of pollutant accumulation was observed. The BAF and BSAF for all pollutants varied up to 1000-fold even after TOC-normalization. Clear negative relationships were found between BAFs/ BSAFs and environmental levels. However, even at constant environmental concentrations a 10- to 100-fold variation in BAFs/BSAFs was observed. This study illustrated the need for biological monitoring since neither environmental * Corresponding author phone: ++32 3 265 33 49; fax: ++32 3 265 34 97; e-mail:
[email protected]. † Department of Biology, Ecophysiology, Biochemistry and Toxicology Group. ‡ Department of Pharmaceutical Sciences, Toxicological Center. 1492
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levels nor BAFs/BSAFs predict bioaccumulation under natural conditions.
Introduction Organic micropollutants and metals are continuously introduced in the aquatic environment by anthropogenic activities. In recent decades several of these compounds have been recognized as hazardous because of their ability to accumulate in the aquatic food chain and their toxic effects to organisms. Therefore many of them, such as metals, biocides, and polychlorinated biphenyls (PCBs) have been identified as priority pollutants by regulatory agencies (13). The assessment of pollutants in the environment by regulatory agencies, including the Flemish Environmental Agency, involves physical and chemical measurements in water and sediments, ecotoxicity tests conducted under controlled conditions, and, to a lesser extent, field observations on impacted indigenous populations or individuals (46). The bioavailability of dissolved and sediment-bound chemicals is determined by the physical, chemical and biological characteristics of the sediment and the overlying and interstitial water. Moreover, due to changes in pollution load and environmental conditions, water or sediment samples that are representative of temporal fluctuations cannot easily be obtained. The chemical partitioning between different sediment fractions is very important in determining the levels of trace metals (7-11) and organic pollutants (1214) available to aquatic organisms. As a consequence, neither physical and chemical measurements nor laboratory toxicity tests will give a complete or correct picture of the pollution status of a water body. Consequently, reliable information on the bioavailability of pollutants can only be obtained by in situ biomonitoring (15-17). Bivalves have been widely used since the late 1980s to monitor the quality of freshwater ecosystems. In Europe the zebra mussel Dreissena polymorpha has been frequently used for this purpose (18-22). Zebra mussels fulfill the requirements of a good test organism for accumulation studies. They are easy to collect and to handle and available in large numbers. Further they have a relatively long life span, are sedentary and resistant to various types of pollution without suffering mortality. Finally their high filtration rate favors the bioaccumulation of both organic and inorganic contaminants and they accumulate pollutants both from water and particulate material (15, 18, 20, 23, 24). In a previous study levels of microcontaminants in transplanted zebra mussels were compared to levels in indigenous mussels (25). The transplanted zebra mussels accumulated similar levels of microcontaminants to indigenous mussels after only 6 weeks. The aim of the present study was to assess the bioavailability of trace metals, polychlorinated biphenyls (PCBs), p,pdichlorodiphenyldichloroethylene (p,p′-DDE) and hexachlorobenzene (HCB) to zebra mussels in different surface waters in Flanders. We therefore related accumulated levels of micropollutants in mussels to dissolved levels and/or levels in the sediment. Bioaccumulation factors (BAF) were calculated for the measured metals and organic pollutants and their usefulness in predicting the bioaccumulation of micropollutants in zebra mussels was evaluated. The bioconcentration factor (BCF) 10.1021/es049048t CCC: $30.25
2005 American Chemical Society Published on Web 01/27/2005
FIGURE 1. Map of the sampling sites for collecting and transplanting zebra mussel, REF: site of collection. or bioaccumulation factor (BAF) are used as criteria by regulatory agencies to assess the potential of micropollutants to bioaccumulate and as a consequence to have environmental impact (3).
Materials And Methods Test Organism and Field Exposure. Zebra mussels (Dreissena polymorpha) were collected in June 2001 at the drinking water reservoir of the Antwerp Drinking Water Company (AWW, Duffel) and selected by length (17-20 mm). This site is further called the Reference site. Since this site is used for drinking water purposes it is considered as uncontaminated. Random groups of twenty-five individuals were placed in polyethylene cages, consisting of two attached pond baskets that allowed free circulation of water. The dimensions of the resulting cages were 11 × 11 × 22 cm with a mesh size of 2 × 4 mm. Cages were anchored by attaching a stone and secured to the bank with a rope and a peg. All cages were free floating at a depth of 30 cm below the water surface. The cages were deployed in situ at the beginning of July 2001 and mussels were collected after 6 weeks of exposure and transferred to the laboratory for analyses. At sites where the cages were lost, resident mussels were collected, if present. Study Area and Sample Processing. At 56 sites through Flanders, including the reference site, three to six cages containing 25 mussels each were deployed in situ. The selected sites included canals (14 sites), lakes (14 sites), and running waters (28 sites). Figure 1 shows the location of the sampling sites including the site where the original mussels were collected (henceforth referred to as the ‘Reference site’). In the laboratory the soft body parts of the mussels were dissected from the shell, byssus threads were removed and tissue was rinsed with deionized water. The pooled mussels from the individual cages were homogenized using an UltraTurrax T8 homogenizer (Ika Labortechnik, Staufen, Germany), resulting in three to six replicate samples from each site. Each sample was divided in two subsamples: one for metal analysis and one for analysis of organic pollutants. Samples were quickly frozen in liquid nitrogen and stored at -80 °C until analyzed. Metal Analysis. Samples for metal analysis were placed in acid washed polypropylene preweighted vials and dried for 24 h at 60 °C. Subsequently, the biological material was digested in a microwave oven, adding a mixture (5:1) of nitric acid (70%) and hydrogen peroxide (30%) following the protocol as described by Blust et al. (26). Digested samples were frozen at -20 °C until further analysis. Silver, aluminum,
arsenic, cadmium, cobalt, chromium, copper, mercury, manganese, nickel, lead and zinc were measured in the samples with either an Inductively Coupled Plasma-Atomic Emission Spectrometer (ICP-AES, Varian Liberty Series II, Victoria, Australia) equipped with a microconcentric groove nebulizer (27) or an Inductively Coupled Plasma-Mass Spectrophotometer (ICP-MS, Varian UltraMass 700, Victoria, Australia). Concentrations of the metals in the tissues were calculated on a dry weight basis and expressed as µg/g. Analytical accuracy was determined using certified reference material of the Community Bureau of Reference of the European Commission (Geel, Belgium); standard for trace elements in river sediment; Certified Reference Material 320 (CRM 320) and mussel tissue (CRM 278). Recoveries were within 10% of the certified values. Organic Pollutants. Samples for organic pollutants analysis were placed in acid washed polypropylene preweighted vials and stored at -20 °C. All individual standards of PCBs and pesticides were obtained from Dr. Ehrenstorfer Laboratories (Augsburg, Germany). Acetone, n-hexane, dichloromethane and iso-octane were of pesticide grade (Merck, Darmstadt, Germany). Anhydrous sodium sulfate and silica gel (Merck) were used after heating overnight at 120 °C. An accelerated Soxhlet extractor B-811 (Buchi, Zurich, Switzerland) was used for the extraction of target compounds from tissues. The following organohalogenated pollutants were determined in mussel tissue: hexachlorobenzene (HCB), hexachlorocyclohexanes (R-, β-, and γ- HCH), p,p′-DDD, p,p′DDT, p,p′-DDE, and polychlorobiphenyls (∑ PCB ) 16 congeners identified according to IUPAC (International Union of Pure and Applied Chemistry) numbers: 28, 52, 99, 101, 105, 110, 118, 138, 153, 156, 170, 180, 183, 187, 194, and 199). Methods used for the determination of organic pollutants were previously validated and are briefly described below (28). The sample (usually between 1 and 4 g) was ground with 5-8 g anhydrous sodium sulfate until a fine powder was obtained and placed into the extraction thimble. The internal standards (PCB46, PCB143, -HCH) were added to the powder and then the sample was extracted with 100 mL n-hexane/ acetone (3/1, v/v) in hot Soxhlet extraction mode for 2 h. The lipid content of each sample was determined on an aliquot of the extract by evaporating the solvent at 105 °C for 12 h. The remaining extract was cleaned up by 8 g acidified silica (40% concentrated sulfuric acid, w/w) column. The analytes were eluted with 25 mL of hexane:dichloromethane (1:1, v/v). The eluate was concentrated and the volume was finally VOL. 39, NO. 6, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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reduced to 100 µL by a gentle nitrogen stream. A Gas Chromatograph with Electron Capture Detection (Hewlett-Packard 6890 GC-µECD) was equipped with a 25 m × 0.22 mm × 0.25 µm HT-8 capillary column. A 1 µL sample was injected in pulsed splitless mode with the spilt valve opened after 1 min. The injector and detector temperatures were 290 and 320°C, respectively. Helium was used as carrier gas (1 mL/min) and Ar/CH4 as makeup gas (40 mL/min). The temperature program of the GC oven started at 90 °C, hold for 1 min, and then with 15 °C/min to 180 °C, hold for 1 min, then with 3 °C/min to 250 °C and further by 15 °C/ min to 290 °C, hold for 6 min. Method limits of detection (LOD) for individual PCB congeners ranged between 0.03 and 0.80 ng/g wet weight (ww). HCB and p,p′-DDE, LOD were 0.23 and 0.63 ng/g wet weight, respectively. Recoveries of target compounds ranged between 72 and 80%. Quality assurance: Multilevel calibration curves were created for the quantification and good linearity (r2>0.999) was achieved for tested intervals that included the whole concentration range found in samples. The analyte identification was based on their relative retention times to the internal standard used for quantification. Peak area ratios (analyte response/internal standard response) were plotted against the concentration ratios (analyte concentration/ internal standard concentration). The method performance was assessed through rigorous internal quality control, which included daily check of calibration curves and regular analysis of procedural blanks and certified material CRM 349 (PCBs in cod liver oil). For the certified material, the standard deviation of PCB congeners no. 28, 52, 101, 118, 153, and 180 at the concentration of 22.5; 62.0; 164.0; 142.0; 317.0 and 73.0 ng/g were 2.0; 0.6; 3.0; 2.2; 13.1; and 2.6 ng/g, respectively. Relationship with Environmental Levels. Most of the sites are sampled monthly by the Flemish Environmental Agency (FEA) for water analysis and 26 sites were sampled in 2001 for sediment analysis. Metals measured by the FEA in water and sediments were As, Cd, Cu, Cr, Hg, Ni, Pb and Zn. Organic pollutants measured in the sediments were seven PCB congeners (no. 28, 52, 101, 118, 138, 153, and 180), HCB and p,p′-DDE. Dissolved metal concentrations and the concentrations of micropollutants in the sediment were provided by the FEA (website:http://www.vmm.be/geoview/ geoinfo/fysico-chemie). Additionally, contaminant levels in sediment of five sites were analyzed by ourselves. To evaluate whether it was possible to predict the bioavailability of micropollutants to aquatic organisms, concentrations of pollutants measured in the mussels were related to levels in the sediment and/or water. For 32 sites data were available to relate 9 organic pollutants and 8 metals in mussels to levels in water and/or sediment. To take into account differences in bioavailabilty of pollutants in sediments, some environmental agencies, including the FEA, normalize the measured concentrations of micropollutants to a “standard sediment” with a clay content of 10% and a organic carbon content of 5% (29, 30). Organic carbon content was measured with a TOC-analyzer Stro¨hlein C-MAT 5500 (Stro¨hlein Instruments, Viersen, Germany). Clay content was measured using laser diffraction (Malvern Mastersizer S., Hoersholm, Denmark). To account for these factors, for metals the following formula is applied (29, 30), taking into account the measured clay content and the total organic carbon.
LN ) Lm*(A + (B*10) + (C*5)/(A + (B*x) + (C*y) For the organic pollutants the used formula is
LN ) Lm*(5/y) where LN: normalized concentration of a pollutant, Lm: 1494
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measured pollutant concentration in the sediment, A,B,C: constants dependent on the metal (29, 30), x: clay content of the sample (%), y: organic carbon content of the sample (%). As with the measured raw concentrations, the relationships between normalized sediment concentrations and tissue levels were also analyzed. For both metals and organics, bioaccumulation factors (BAFs) and biota-sediment accumulation factors (BSAFs) were calculated. The BAF/BSAF is defined as the ratio of tissue concentration to dissolved or sediment concentration respectively (31). The BAFs/BSAFs were calculated in two different ways. In the first case (BAFa and BSAFa) we took the ratio of the measured tissue concentration to the measured water or sediment concentration. In the second case TOCnormalized BSAFs were calculated (BSAFb). For the organic micropollutants the BSAFb was calculated as BSAFb ) Cl/Csoc and for the metals BSAFb ) Cdw/Csoc, where Cl is the lipid normalized concentration in the mussel, Csoc the concentration in the sediment normalized for organic carbon content, and Cdw the metal concentration in the mussel on a dry weight basis (32-34). The relationships between BAF/BSAF values and environmental concentrations were analyzed for eight metals (As, Cd, Cr, Cu, Hg, Ni, Pb and Zn) and for 9 organics (PCB nr: 28, 52, 101, 118, 153, 138, 180; HCB and p,p-DDE). Statistics. Analysis of variance (ANOVA, with post-hoc Duncan’s multiple range test) and linear and nonlinear (multiple) regressions were used to analyze the data. All data were tested for homogeneity of variance by the log-anova test and for normality by the Kolmogorov-Smirnov test for goodness of fit. Significance levels of tests are indicated by asterisks according to the following probability ranges: * p e 0.05; ** p e 0.01; *** p e 0.001. Statistical methods used are outlined in Sokal and Rohlf (35). In the few cases where the concentration of a pollutant in one or more of the replicates was below the limit of detection (LOD), a value of LOD/2 was used for the calculation of the means and for the summation of the congeners (36, 37). All statistical analyses were performed using the software package STATISTICA (StatSoft Inc., Tulsa, OK, USA).
Results Survival of the Mussels. Survival rates in the cages, dissolved oxygen, pH and calcium levels in the water and organic carbon and clay content in the sediment are presented in Table 1. At six sites all cages disappeared but resident mussels could be collected. At some others sites one or two of the cages were lost or dried up due to a decreased water level. Survival ranged from 0 to 100% with six sites where none of the exposed mussels survived. The median survival was 66.7% (25-percentile: 29.7%; 75-percentile 88.9%). When only canals and lakes are considered the median survival was 94.3% (25percentile: 59.0%; 75-percentile 98.2%). The decreased survival at some sites could not be attributed to decreased oxygen levels alone, since only a weak (though still significant) relationship between mean oxygen levels (N)12) and survival was found (r2)0.162; p)0.004, n)49). At some sites where mortality was high, packing of sediment particles in the cages was observed, although this also cannot have been the only cause of mortality. Concentrations of Organic Pollutants and Metals in Zebra Mussel. Concentrations of HCHs (three isomers), p,p′DDD and p,p′-DDT were below the limit of detection at all sites and were therefore not included in the analysis. The mean concentrations of PCBs and Organic Chloride Pesticides (OCPs) in zebra mussels at the different sites, including the reference site, are given in Table 2. Most individual PCB congeners were detectable in mussel tissue at all sites. Only congeners no. 28, 156 and 194 were below the detection limit
TABLE 1: Location and Mussel Survival, along with Selected Water and Sediment Characteristics for the Different Sampling Sitesa,b Site
Name and place
Type
Location
pH
Ca mg/L
O2 in %
Clay %
TOC %
Survival %
# cages
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 REF
Aa-voort Zarrebeek Nieuw Bedelf Kanaal Schipdonk Groenstraatbeek Watersportbaan Oude Durme Vondelbeek Meer van Weerde 1 Meer van Weerde 2 Willebroeksevaart Nekker Put van Niel Zennegat Kapittelbeek Muisbroek E-10 plas Netekanaal Kleine Nete K. Dessel-Schoten Nijlense Beek Grote Beek Dijle Valckelaereloop Edegemse beek Fort 4 Fort 5 Fort 6 Fort 7 Fort 8 K. Herentals-Bocholt K. Dessel-Schoten 2 K. Mol-Dessel Oude Dijkloop 1 Oude Dijkloop 2 Oude Dijkloop 3 Oude Dijkloop 4 Oude Dijkloop 5 Bollaak Bankloop Kneutersloop Molse Nete 1 Scheppelijke Nete 1 Scheppelijke Nete 2 Scheppelijke Nete 3 Molse Nete 2 Voorste Nete Desselse Nete Zwarte Nete K. Herentals-Bocholt K. Beverlo 1 K. Beverlo 2 Dommel Eindergatloop Z-Willemsvaart AWW
River River Canal Canal Canal Canal Lake River Lake Lake Canal Lake Lake Lake River Lake Lake Canal River Canal River River River River River Lake Lake Lake Lake Lake Canal Canal Canal River River River River River River River River River River River River River River River River Canal Canal Canal River River Canal Lake
Oostkerke Zarren Lombardsijde Oostkerke Oedelem Ghent Hamme Dendermonde Weerde Weerde Vilvoorde Mechelen Niel Mechelen Dworp Ekeren Schoten Nijlen Lier Schoten Nijlen Deurne Haacht Putte Antwerp Mortsel Antwerp Antwerp Antwerp Antwerp Dessel Turnhout Mol Beerse Beerse Gierle Lille Poederlee Zandhoven Olen Olen Balen Balen Mol Mol Mol Dessel Dessel Retie Kaulille Leopolsburg Lommel Neerpelt Neerpelt Rekem Duffel
7.8 7.6 8.2 nd nd 8.2 7.6 7.7 7.3 7.6 7.1 8.5 8.2 8.1 7.9 7.2 7.2 8.2 7.0 7.2 6.6 6.9 7.6 7.2 7.2 7.8 8.1 7.9 8.0 7.5 7.6 7.3 7.6 6.9 7.4 7.3 7.6 7.3 7.0 7.5 7.2 7.3 7.1 7.0 7.1 7.3 7.3 7.3 7.0 7.2 7.9 7.2 6.9 7.0 7.4 8.2
147 93 156 nd nd 61 35 103 72 118 63 nd 65 65 69 75 33 47 52 52 71 2697 115 61 nd 82 73 71 80 83 32 43 59 44 57 68 68 67 70 nd nd 37 56 60 60 45 54 38 34 48 43 60 40 68 57 49
46.3 47.0 78.8 50.0 62.6 87.5 nd 24.5 97.2 94.5 46.8 110 99.6 72.0 76.8 nd 97.2 94.7 59.4 93.5 60.9 61.9 58.2 39.9 67.7 86.8 63.0 56.5 60.9 38.2 67.1 84.5 95.1 52.0 66.8 81.3 65.2 61.3 76.0 52.7 53.8 57.7 85.9 78.1 67.9 77.7 77.2 74.2 70.1 70.8 95.1 93.4 60.2 56.9 77.8 90.1
18.0 2.10 3.70 nd nd nd nd nd 7.81 6.94 9.50 nd nd nd 0.85 nd nd nd 5.00 0.50 5.00 nd nd nd nd 4.70 2.63 3.70 nd nd 3.60 0.50 nd nd nd nd nd 1.40 2.41 2.49 2.81 1.50 1.05 1.00 16.0 0.97 0.89 1.40 1.20 6.70 0.00 9.90 1.20 0.31 17.9 nd
4.65 1.00 0.88 nd nd nd nd nd 1.00 4.80 5.00 nd nd nd 0.16 nd nd nd 0.96 1.10 2.10 nd nd nd nd nd nd nd 1.98 4.79 1.50 1.10 nd nd nd nd nd 0.52 1.63 0.88 0.43 0.57 1.90 2.80 21.0 2.90 0.97 0.76 0.81 2.70 3.20 6.20 0.97 0.00 12.0 nd
35.6 84.4 35.6 0.0 0.0 R R 0.0 97.8 R R 75.8 76.2 95.8 88.9 R 98.7 36.7 40.0 R 15.6 0.0 0.0 0.0 26.7 100 100 100 100 95.6 77.8 96.5 88.6 70.0 76.7 26.7 66.6 86.8 83.3 66.7 56.7 48.9 44.4 51.1 17.8 66.7 97.8 28.9 84.4 42.3 93.0 96.7 62.2 50.0 29.7 R
2 3 3 3 3 0 0 3 3 0 0 3 3 3 3 0 3 3 3 0 3 3 1 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 3 3 2 3 3 3 -
a
The number of cages recovered are indicated.
b
R: resident mussels sampled; nd: not determined; TOC: Total Organic Carbon.
at a considerable number of sites. The sum of all PCBs at the different sites ranged from 8.6 to 168 ng/g wet wt. For most running waters PCB levels in mussels were rather low whereas the highest levels were measured in mussels from lakes and canals. Mean HCB in mussel tissue ranged from below the detection limit to 5.2 ng/g wet wt. At 19 sites, including rivers, canals and lakes, the HCB concentration was also below the detection limit. Finally, p,p′-DDE was detectable in mussels from all sites, with mean values ranging from 0.51 to 8.3 ng/g wet wt. The average concentrations of metals in zebra mussel at the different sites are given in Table 3. With the exception of Ag and Hg, all other metals were detectable in zebra mussel
from all sites. At some sites very high levels of Cd, Cu, Ni, Pb and Zn were measured in zebra mussels. Concentrations of Organic Pollutants and Metals in the Environment. The average levels of the seven PCB congeners and their sum, HCB and p,p′-DDE, measured in the sediment are summarized in Table 4. At 32% of the sampling sites all the considered organic pollutants were below detection limit. Levels of HCB were below detection limit at 19 of the 31 sites and ranged up to 2 ng/g dry wt. Levels of p,p′-DDE were below detection limit at 24 sites and ranged up to 2 ng/g dry wt. VOL. 39, NO. 6, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Mean Concentrations of Organic Contaminants in Zebra Mussel in ng/g Wet wt from Waterbodies in Flanders in 2001-2002 Site
Lipidin %
PCB28
PCB52
PCB99
PCB101
PCB105
PCB110
PCB118
PCB138
PCB153
PCB156
PCB170
PCB180
PCB183
PCB187
PCB194
PCB199
∑PCB
p,p′-DDE
HCB
1 2 3 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 26 27 28 29 30 31 32 33 34 35 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 55 REF
1.01 0.89 0.86 3.14 2.34 1.65 0.66 0.68 0.88 1.56 1.36 1.00 1.10 1.10 0.73 0.55 1.07 0.72 0.75 1.00 1.29 1.17 0.83 0.68 0.69 1.04 1.09 1.17 0.57 1.11 1.00 0.90 0.97 0.91 0.78 0.80 1.09 0.70 1.11 0.68 1.05 0.65 0.86 0.90 0.82 0.87 1.23
< 0.17 < 0.17 0.21( 0.11 0.81 1.1 1.7 ( 0.14 5.8 11 0.23 ( 0.02 < 0.17 1.1 ( 0.08 < 0.17 < 0.17 < 0.17 0.26 ( 0.03 < 0.17 0.79 ( 0.04 0.80 0.26 ( 0.02 0.36 ( 0.01 0.36 ( 0.10 0.28 ( 0.01 0.61 ( 0.02 0.57 ( 0.05 0.48 ( 0.05 0.64 ( 0.02 < 0.17 < 0.17 < 0.17 < 0.17 < 0.17 0.34 0.80 < 0.17 0.89 0.83 2.6 0.83 < 0.17 0.22 ( 0.15 < 0.17 0.72 ( 0.06 0.54 ( 0.02 0.69 ( 0.05 0.96 ( 0.19 1.1 ( 0.04 0.31 ( 0.03
< 0.20 0.32 ( 0.15 0.59 ( 0.12 1.7 4.4 3.4 ( 0.22 10 11 0.73 ( 0.02 0.55 ( 0.02 3.2 ( 0.23 0.65 ( 0.07 0.48 ( 0.05 0.33 ( 0.01 1.2 ( 0.14 0.56 ( 0.15 2.2 ( 0.09 1.0 1.3 ( 0.13 1.5 ( 0.11 1.5 ( 0.35 1.0 ( 0.08 2.2 ( 0.08 2.0 ( 0.09 1.7 ( 0.22 1.6 ( 0.09 1.5 0.95 ( 0.02 0.87 ( 0.11 0.17 ( 0.14 < 0.20 0.61 0.96 0.63 ( 0.11 1.0 0.97 2.9 2.0 < 0.20 0.30 ( 0.20 0.27 ( 0.13 2.6 ( 0.11 1.4 ( 0.05 1.9 ( 0.13 0.28 ( 0.20 2.2 ( 0.09 0.68 ( 0.02
0.42 ( 0.14 0.36 ( 0.05 0.40 ( 0.11 1.0 2.3 2.5 ( 0.20 7.4 3.7 0.41 ( 0.02 0.29 ( 0.01 2.3 ( 0.14 0.50 ( 0.04 0.22 ( 0.03 0.34 ( 0.06 0.88 ( 0.06 0.42 ( 0.06 1.9 ( 0.10 0.65 0.63 ( 0.08 0.78 ( 0.05 0.86 ( 0.22 0.71 ( 0.04 1.3 ( 0.17 1.4 ( 0.08 1.1 ( 0.08 1.4 ( 0.06 1.6 1.1 ( 0.03 0.68 ( 0.00 0.47 ( 0.01 0.31 ( 0.01 0.58 0.99 0.55 ( 0.14 0.83 0.72 0.73 0.78 0.22 ( 0.05 0.39 ( 0.08 0.31 ( 0.02 1.7 ( 0.09 0.90 ( 0.04 1.6 ( 0.11 2.6 ( 0.31 1.3 ( 0.07 0.55 ( 0.08
1.7 ( 0.39 1.5 ( 0.10 1.5 ( 0.03 2.8 6.0 8.8 ( 0.67 24 18 0.93 ( 0.13 0.83 ( 0.04 9.7( 0.67 2.0 ( 0.15 0.84 ( 0.11 1.2 ( 0.20 3.4 ( 0.18 1.7 ( 0.22 10 ( 0.37 2.4 2.1 ( 0.23 2.9 ( 0.16 2.3 ( 0.53 1.8 ( 0.23 4.0 ( 0.51 5.3 ( 0.33 4.0 ( 0.21 5.8 ( 0.20 3.5 3.1 ( 0.02 2.5 ( 0.01 1.6 ( 0.06 1.1 ( 0.03 2.0 3.5 2.1 ( 0.27 3.3 2.7 3.8 3.5 0.96 ( 0.27 1.3 ( 0.17 0.96 ( 0.04 6.3 ( 0.26 3.4 ( 0.16 6.1 ( 0.52 3.3 ( 0.57 5.7 ( 0.69 2.7 ( 0.54
1.1 ( 0.25 1.1 ( 0.22 0.83 ( 0.08 0.69 0.91 3.9 ( 0.23 12 5.7 1.1 ( 0.10 0.71 ( 0.09 5.7 ( 0.37 1.3 ( 0.09 0.97 ( 0.06 0.87 ( 0.12 2.4 ( 0.13 0.64 ( 0.52 6.2 ( 0.15 1.1 1.2 ( 0.05 1.5 ( 0.05 1.1 ( 0.20 1.1 ( 0.04 1.5 ( 0.16 2.3 ( 0.09 2.1 ( 0.16 2.0 ( 0.09 2.3 1.9 ( 0.03 1.4 ( 0.10 1.1 ( 0.03 0.43 ( 0.35 1.5 2.0 0.85 ( 0.44 1.8 1.5 2.0 1.9 0.40 ( 0.29 0.60 ( 0.22 0.18 ( 0.14 2.8 ( 0.15 1.8 ( 0.04 1.8 ( 0.09 1.4 ( 0.40 2.4 ( 0.11 1.2 ( 0.13
1.4 ( 0.31 1.9 ( 0.17 1.2 ( 0.09 2.9 4.8 5.5 ( 0.37 16 10 1.2 ( 0.03 0.60 ( 0.04 7.2 ( 0.38 1.6 ( 0.13 0.69 ( 0.09 1.0 ( 0.15 3.4 ( 0.17 4.0 ( 0.82 6.3 ( 0.23 2.1 1.9 ( 0.13 2.2 ( 0.15 2.1 ( 0.46 1.8 ( 0.11 3.5 ( 0.55 4.1 ( 0.30 3.5 ( 0.20 3.6 ( 0.18 5.2 7.0 ( 0.12 3.2 ( 1.4 3.0 ( 0.07 0.98 ( 0.01 2.1 3.6 2.8 ( 0.40 3.2 2.7 2.9 3.2 1.2 ( 0.20 1.1 ( 0.12 1.3 ( 0.03 5.3 ( 0.52 3.3 ( 0.17 4.3 ( 0.25 1.8 ( 0.45 3.6 ( 0.12 1.9 ( 0.27
0.82 ( 0.18 0.61 ( 0.04 0.76 ( 0.06 1.7 3.2 4.4 ( 0.33 13 5.9 0.81 ( 0.04 0.60( 0.02 4.3 ( 0.29 1.0 ( 0.07 0.56 ( 0.06 0.75 ( 0.10 1.7 ( 0.09 0.71 ( 0.10 2.3 ( 0.10 0.96 1.1 ( 0.04 1.2 ( 0.02 1.3 ( 0.28 1.2 ( 0.05 1.9 ( 0.28 1.8 ( 0.15 1.6 ( 0.08 1.8 ( 0.10 3.2 1.9 ( 0.08 1.0 ( 0.04 0.82 ( 0.03 0.51 ( 0.01 0.89 1.3 0.80 ( 0.16 1.2 1.0 1.4 1.3 0.52 ( 0.08 0.60 ( 0.07 0.51 ( 0.04 2.46 ( 0.20 1.3 ( 0.03 1.8 ( 0.13 3.3 ( 0.15 2.0 ( 0.12 0.82 ( 0.12
2.0 ( 0.48 1.7 ( 0.24 1.9 ( 0.08 3.1 5.5 6.6 ( 0.45 20 11 0.99 ( 0.15 0.82 ( 0.06 11 ( 0.68 2.1 ( 0.21 1.6 ( 0.11 1.5 ( 0.44 3.8 ( 0.39 1.6 ( 0.29 13 ( 0.32 2.5 2.0 ( 0.11 2.6 ( 0.13 2.0 ( 0.38 2.5 ( 0.10 2.8 ( 0.28 4.3 ( 0.35 2.6 ( 0.24 5.9 ( 0.27 3.1 3.0 ( 0.00 3.0 ( 0.12 2.0 ( 0.01 1.6 ( 0.00 3.5 5.2 2.0 ( 0.42 4.5 4.0 2.9 3.9 1.6 ( 0.34 1.4 ( 0.19 1.2 ( 0.11 4.7 ( 0.25 4.4 ( 0.09 5.5 ( 0.43 1.4 ( 0.20 4.4 ( 0.14 4.0 ( 0.97
4.5 ( 1.00 4.2 ( 0.31 4.5 ( 0.30 4.5 8.9 11 ( 0.86 28 19 1.8 ( 0.22 1.8 ( 0.23 20 ( 0.99 4.2 ( 0.36 3.0 ( 0.27 3.2 ( 1.15 9.0 ( 1.05 3.8 ( 0.37 29 ( 0.51 5.7 4.2 ( 0.19 6.3 ( 0.39 3.9 ( 0.66 5.6 ( 0.20 5.8 ( 0.59 9.7 ( 65 6.0 ( 0.69 14 ( 0.63 4.2 6.9 ( 0.04 6.4 ( 0.09 4.9 ( 0.12 3.7 ( 0.00 7.1 10 5.1 ( 0.50 9.1 8.3 5.6 7.4 3.4 ( 0.51 3.9 ( 0.18 2.9 ( 0.24 11 ( 0.80 9.2 ( 0.33 13 ( 0.92 3.8 ( 0.95 8.9 ( 0.34 6.7 ( 0.78
< 0.80 < 0.80 < 0.80 < 0.80 < 0.80 0.95 ( 0.05 2.2 1.4 < 0.80 < 0.80 1.6 ( 0.09 < 0.80 < 0.80 < 0.80 < 0.80 < 0.80 1.9 ( 0.04 < 0.80 < 0.80 < 0.80
