by PSP Volume 15 No Fresenius Environmental Bulletin

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin THE EFFECT OF AGE ON THE CONCENTRATION OF POLY- CHLORINATED DIBENZO-P-DIOXINS, DIBENZOFURANS AND DIOXIN-LIKE POLYCHLORINATED BIPHENYLS IN BALTIC HERRING AND SPRAT Ott Roots and Vladimir Zitko Estonian Environmental Research Centre, Marja D, 67, Tallinn, Estonia Consultant, Reed Ave, St. Andrews, NB, E5B A, Canada SUMMARY This paper reports on the concentrations of polychlorinated dibenzo-p-dioxins (CDDs), polychlorinated dibenzofurans (CDFs), and dioxin-like (planar) polychlorinated biphenyls (PPCBs) in -8 years old herring (Clupea harengus membras L.) and sprat (Sprattus sprattus balticus Schn.), caught in. The concentrations are compared with those found in fish in and in the 99s. The concentrations in fish of the same age do not differ noticeably between and. The concentrations (pg/g lipid) of CDD and CDF in herring from and are lower than the concentrations in herring landed in the 99s, partly because of a higher lipid content in the former. Consequently, the loads (pg/fish) are closer to one another in all sets. In contrast, the concentrations as well as the loads of the chlorobiphenyls 5, 8, and 56 are much higher in the herring than in the 99s fish. In most cases, the concentrations in fish collected in a given year, increase with its age. This suggests that the age of the fish is more important than the year of capture for the concentrations of CDDs, CDFs, and PPCBs. The determination of long-term trends of persistent chemicals in Baltic fish requires monitoring of age-stratified samples, collected several years apart. The equation C or L = age/(a+b*age), where C and L are concentrations (pg/g lipid) or loads (pg/ fish) and a and b are constants, well describes the data. KEYWORDS: Persistent organic pollutants, Baltic herring, Baltic sprat, polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, dioxin-like polychlorinated biphenyls. INTRODUCTION The Baltic Sea is highly contaminated with polychlorinated dibenzo-p-dioxins (CDDs), polychlorinated dibenzo- furans (CDFs) and dioxin-like (planar) polychlorinated biphenyls (PPCBs), see for example []. The concentrations of these compounds are most often measured [-5] in the Baltic herring (Clupea harengus membras L.) and the Baltic sprat (Sprattus sprattus balticus Schn.) These fish are very good species for monitoring purposes, since they can be caught in all parts of the Baltic, their biology is fairly well-known [6], and they are of suitable size for pre-analytical sample treatment []. Baltic herring and sprat are also the most important fish species in the Baltic Sea, and are of considerable importance for the Estonian fish-processing industry. Consequently, the presence of toxicants in these fish is of concern from the point of view of human health [7]. A previous study [5] suggested that the age of the fish is a major factor affecting the levels, and, to some extent, also the congener profiles of CDDs and CDFs in herring and sprat. This paper reports the results of a similar study, performed a year later, and compares them with results, obtained by another laboratory, on herring collected in the 99s []. Samples MATERIALS AND METHODS Baltic herring and Baltic sprat were obtained by Dr. Mart Simm from Estonian Marine Institute, University of Tartu, between May and June from industrial trawlers operating along the Estonian coast []. The fish were immediately frozen. Before analysis, their length, weight, gender, and the maturity of gonads were determined. Samples of muscle were submitted for chemical analysis. Baltic herring (Table ) were collected in the Central Baltic (sample R9), in the western Gulf of Finland (sample R), and in the Gulf of Riga (samples R, R, R5 7

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin and R6). Baltic sprat (Table ) were collected in the Gulf of Riga. In comparison with the samples collected in and in the 99s, the codes of the former are as in the reference [5]. For the latter, the samples in Table of reference [] have the codes F-F7, F85 and B85, and, those in Table have the codes FS, FS9, FL9, BS9, and BL9. The letters F and B refer to the Gulf of Finland and Gulf of Bothnia, respectively, the numbers -7 are ages of the fish; the number 85 indicates that the age ranged from 8 to 5 years. S, S9, and L9 refer to small and large herring, caught in 99 and in 999, respectively. Chemical analyses The determination of dioxins and polychlorinated biphenyls was carried out by the National Research Centre for Environment and Health, Institute of Ecological Chemistry, 8576 Neuherberg, Germany, under quality control according to EN 75 (accreditation license No. DAC-P- --, valid until November, 6). Chemicals All solvents were of trace analysis quality and purchased from LGC Promochem (Wesel, Germany), as were the silica, alumina and Florisil adsorbents. The C 8 -modified silica (Isolute C8) was purchased from Separtis GmbH (Grenzach-Wyhlen, Germany). All C-labeled standards were from Cambridge Isotope Laboratories (Andover, MA, USA) or Wellington Laboratories (Guelph, Ontario, Canada). Sample processing Measurement of lipids and CDDs, CDFs, and PPCBs Each pool of the fish samples was freeze-dried and homogenized. Of each pool, 7.5 g were spiked with C- labeled CDD and CDF standards and extracted by pressurized liquid extraction (ASE, Dionex GmbH, Idstein, Germany) with n-hexane/acetone 75/. The lipid concentration in the extract was determined gravimetrically [8]. Cleanup The fractions A and B from the alumina column (Fig. ) were used for the measurement of PCBs. The solvents were changed to. ml acetonitrile. This acetonitrile solution was placed on a SPE cartridge filled with g C 8 -modified silica (conditioned with 6 ml acetonitrile) and rinsed firstly with two portions of. ml, and then with ml of acetonitrile through the cartridge. All acetonitrile fractions were combined and reduced to µl by a gentle stream of nitrogen. Instrumentation The measurements were performed with a high-resolution mass spectrometer (MS) Finnigan MAT 95S (Thermo Electron GmbH, Bremen, Germany) coupled to an Agilent GC 689 (Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation was achieved by a splitless injection (cold injection system CIS, Gerstel GmbH, Mülheim, Germany) of µl on a Restek Rtx- column (length 6 m, ID.5 mm, film thickness (ft). µm, Restek GmbH, Sulzbach, Germany). The GC oven was programmed as follows: 9 C initially, held for.5 min, increase at a rate of C/min to C, followed by an increase of C/min to 8 C, then C/min to 6 C, and a final hold at 6 C for 5 min. The MS was operated in the SIM mode, at a resolution of, and the two most intense ions of the molecular ion cluster were monitored for the unlabeled and labeled isomers. Sample C-labelled internal PCDD/F standards ASE extraction n-hexane/acetone (75/5 v/v) A. 8 ml toluene B. ml n-hexane/ dichloromethane (98/ v/v) PCB analysis waste Recovery standard C -,,,-TCDD Sandwich-Column g SiO g SiO /% HSO g SiO, 5 g Na SO Elution with: Florisil-Column 5 g Florisil 8 g Na SO Elution with: A. 8 ml n-hexane concentration to µl HRGC-HRMS Elution with: 5 ml n-hexane C. ml n-hexane/ dichloromethane (5/5 v/v) B. ml dichloromethane FIGURE - The cleanup procedure. The PCB measurements were performed with a highresolution MS Finnigan MAT 95 (Thermo Electron GmbH, Bremen, Germany) coupled to an Agilent GC 589 Series II (Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation was achieved by a splitless injection (cold injection system CIS, Gerstel GmbH, Mülheim, Germany) of µl on a J&W DB-XLB column (length 6 m, ID.5 mm, ft.5 µm, Agilent Technologies, Palo Alto, CA, USA). The GC-oven was programmed as follows: 9 C initially, held for.5 min, increase at a rate of 5 C/min to C, followed by an increase of C/min to 5 C, held for min, then 8 C/min to C, and a final hold at C for 5 min. The MS was operated in the SIM mode at a resolution of 8, and the two most intense ions of the molecular ion cluster were monitored for the unlabeled and labeled isomers. 8

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin Data evaluation The relationship between the age (years) of the fish and the concentration (pg/g lipid) or load (pg/fish) of CDDs, CDFs, and PPCBs was evaluated by fitting the equation (A) to the data: C or L = age/(a + b*age) (A) The constants a and b were determined by linear least squares regression of the transformed equation /y = a/age + b, where y = C or L. In a few cases, the fit was improved when some visually determined outliers were not considered, or by a preliminary smoothing of the data with a quadratic polynomial. The relationship between the age of the fish and their contaminant profiles (concentrations scaled to a sum of ) was examined by Principal Component Analysis (PCA) of the samples as well as of theoretical samples of different ages, predicted by the equation (A). The PCA was performed by PLS_Toolbox [9], running in Matlab 5.. Graphs were prepared in Excel (Microsoft). Labeler [] labeled the points in the graphs. Not detectable concentrations of CDDs, CDFs, and PPCBs were replaced by.5 * LOD values. The concentrations were then converted to profiles, centered (mean = ), (std = ), and subjected to the PCA. Individual CCDs and CDFs were identified by their shorthand code (see Table ). The IUPAC numbers were used for the chlorobiphenyls. RESULTS AND DISCUSSION Age and weight Biological characteristics of the samples as well CDD, CDF and PPCB concentrations are in Tables and. With the exception of three samples, the relationship (B) weight [g] =.76*age[years] + 7.78 (B) describes the increase of weight with age of the herring in all three data sets (this paper, [], and [5], altogether samples). The exceptions are the sample R, in which the predicted median weight is 6 g, and the samples B85 and F85 [] with reported median weights of 6 g and 5 g, respectively, as opposed to the median weight of 6 g, predicted by (B). TABLE - Biological characteristics and CDD, CDF, and PPCB concentrations (pg/g lipid) in herring. Sample R R R5 R6 R9 R Dry matter (%).6..5..8. Lipids (%).8..7.6.6.9 Number in pool 7 5 7 8 Length, cm.5.5 5.7 6 6. 5.5 (.-7.) (.-.8) (.8-6.5) (.5-8.) (.6-8.) (.9-7.7) Weight, g 9.95 6.7.7.8..85 (78.8-6.7) (57.-7.8) (.-8,7) (.-.) (6.-9.8) (.7-.) Age, years 8 8.5 5 (8-9) (5-) (-6) (-5) (-6) (-8) Gender M=,F=.5 Maturity 5 5 5 (-5) (-5) (-5) (-6) (5-6) (-6) 78D 5.7 6.8 6. 7.6.5.9 78D 9.8 7.6 9. 9. 9.. 78D...6... 678D.7.9 6 7. 7.9 789D.88.7..8..77 678D.6.7.6..5 OCDD.6.7..5 7 7. 78F 86.9 88. 85.9 6. 7. 78F.5. 5.5 7.. 8.6 78F 9 95 89. 7. 8 78F 5.... 5.7 678F 7.9.8. 5.8. 9. 789F..55.96.98.68.5 678F 8.9.6. 5. 5.. 678F...7.8.5 5. 789F..99.65.5.7.9 OCDF.7... CDD/F TEQ 88.9 9.5 7. 69. 55.5 9.8 77 88 86 56 8 99 8 5.7 6.7 9 8.5 7..7 6 79 79 5 97 66 69 5 5 7 86 5 7 5 858 7 687 99 8 7 79 7 88 7 8 88 9769 866 9786 75 875 878 88 86 95 798 77 56 995 59 59 8 95 59 57 79 5895 5 997 6 55 67 556 75 66 678 57 75 89 98 8 85 966 77 7 PPCB TEQ. 5.9 59. 67. 5. 77.58 Total TEQ. 5. 9.8 6. 9.5 7. 9

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin TABLE - Biological characteristics and CDD, CDF, and PPCB concentrations (pg/g lipid) in sprat. Sample L L L L L5 Dry matter (%) 7. 7 9.9 9. 8. Lipids (%) 9.7 9.8.. 9. Number in pool 8 6 6 Length, cm.6..9.7 (.8-.9) (.-.7) (.-.7) (.-.7) (.-.6) Weight, g.5.5 8.5 8.8 8.5 (7.-.) (8.9-5.) (6.-.) (7.8-.9) (6.-.) Age, years.5 6 (-) (-) (-8) (-9) (-9) Gender M=,F= Maturity (.5-) (-) (-) (-) (-) 78D...6.5.8 78D 5. 9.7. 5.9 5 78D.8.5..79.5 678D 5.6.7. 5.7 5.9 789D.9.7.9.5.5 678D....8 OCDD.9. 5.. 78F.8 55. 6.8 7.9 78F 6.6 5.8 6 8.7 8.7 78F.9 9.5.8.8.7 78F..5.8. 678F.9.7.8.. 789F.5...5.7 678F. 6.6.8 678F 6...8..8 789F.6.5.5.6.5 OCDF.8.6..96. CDD/F TEQ.67 7.9 7..7.56 77 5 68 7 8 5.7 7.8.7 6.6 8. 6 9 8 5 69 86. 86.6 67 89.9 5 7 967 7 87 85 99 87 65 859 867 8 58 76 5 6 7779 69 58 66 557 5 56 66 5975 567 5859 57 8 985 8 67 8 97 9 5 89 97 76 5 5 9 PPCB TEQ 5.9.9 7.7 6.5 7.9 Total TEQ 66.75 7.8 5.9 69. 7.6 The concentrations of CDDs, CDFs, and PPCBs in this and previous studies The concentrations in pg/g lipid of all CDDs and CDFs in herring from the 99s [] are higher than those in the herring collected in [5] and this work (Table ). A lower concentration of lipids in the herring analyzed by Kiviranta et al. [] causes, at least in part, this difference. The difference is somewhat diminished by expressing the concentrations as loads (pg/fish). Surprisingly, the concentrations of the chlorobiphenyls 5, 8, and 56 are much higher in the fish studied in this work, than in those reported in []. Unfortunately, a more detailed evaluation of complete chlorobiphenyl profiles cannot be carried out, because concentrations of nonplanar chlorobiphenyls are not available for herring in this work. Effect of age on the concentration of CDDs, CDFs, and PPCBs The CDD, CDF, and PPCB concentrations increased with the age of the fish in this as well as previous studies [, 5]. Tables and 5 present the constants a and b of the equation (A). This equation is formally the same as the Langmuir adsorption isotherm. It is worth-noting that, according to (A), the concentration of the contaminant will approach /b in infinitely old fish when b>. The initial rise in the concentration is proportional to /a. When the constant b is negative, the concentration increase with age shows no signs of slowing down. When b is very small, the concentration increase approximates a straight line through the origin. The curve (A) has a discontinuity at x = -a/b, which may present fitting problems when b<. The discontinuity may be avoided by removing outliers, or by smoothing the concentration vs age relationship by a quadratic polynomial.

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin TABLE - A comparison of the median CDD, CDF, and PCB concentrations (pg/g lipid and pg/ fish) in herring, reported by Kiviranta et al. [], Roots et al. [5], and in this work. Reference [] Reference [5] This work Reference [] Reference [5] This work Lipids (%).9 9.5. Length, cm 7..75 6.5 Weight, g.5.5 Age, years 5.5.5 Code pg/g lipid pg/fish 66d 78D 5.65 5.9 8.55.5.8 76d 78D 6.65.6 6.5 5.6. F6d 78D...5..8.7 77d 678D 66. 9. 7.6.88 7.6 7Ed 789D 5.8.975.7...56 F7d 678D..5 6.7.55.66 FFd OCDD 5.6.5 9..89. 66f 78F 76. 86.. 7. 65.6 76f 78F 5.5 7.9 9.6 5..6 E6f 78F 9. 7 7. 8. F6f 78F.85.75..77.6 77f 678F.885 6.85..88 5. 7Ef 789F.75.8.6.6 E7f 678F.5 7..68.8 5.9 F7f 678F..5 6.7.55.86 FEf 789F.75.68.5.5 FFf OCDF.9..95.65 5.. CDD/F TEQ 7 8. 79.6 55 8.5 6. 77 57 68 5 8 8.. 6.5 59 8 69 8 78.5 7 87 5 88 768 5.6 7 96 55 8 7 7. 78 9 769 56 8 895.6 55 57 5 69 67 7 59 89 97 8 PPCB TEQ 9 7. 6. 5.9 Total TEQ 8 5 7 6 TABLE - The effect of age on CDD, CDF, and PPCB concentrations (pg/g lipid) in herring, C at age = age/(a + b*age), where a and b are constants. Mean values are shown where trends were not apparent. Reference [] Reference [5] This work a b a b a b 78D.E+ -.E-.5E+.65E- 5.9E+ mean 78D.79E- -5.7E-.7E+ -6.8E-.7E-.5E- 78D.E+ -.E+.5E+ -.9E+ 7.96E-.E- 678D.5E- -.7E-.7E+.78E-.5E-.E- 789D.9E+ -.E-.E+.58E+.E+.E+ 678D 9.E- -5.E-.8E+ mean.7e-.5e- OCDD.5E+ mean 5.6E+ mean.e-.7e- 78F 6.9E- 9.E- 5.98E-.6E-.8E-.6E- 78F 6.E- -7.5E-.E+ -7.E-.E-.7E- 78F.E- -.E-.E- -.E-.E- 7.E- 78F.E+ -.58E-.E+ 9.56E-.E-.8E- 678F.5E+ -.97E-.5E+.87E-.E- 6.8E- 789F.8E- mean 7.97E- mean 678F 9.E- -.7E-.7E+.5E- 6.9E-.7E- 678F.7E-.E-.E+ mean 9.78E-.5E- 789F 9.E- mean.8e+ 6.66E- OCDF.8E+ mean.e+ mean.77e-.e- CDD/F TEQ.E- -.6E-.5E-.9E-.66E- 9.E- 77 5.E-.6E-.E- 5.9E- 8.7E-.7E- 6.75E- -.56E- 6.7E- 7.7E- 69.6E- -8.6E-.6E-.67E- 5.65E- -.67E- 6.E-5 8.E-6.68E-.8E- 8.7E- -8.78E-.9E-5.7E-6.E-.8E-5 56.E+ -.57E-.6E-.6E-5 57.E- 7.E-5 67.E-.7E-5 89.68E-.9E- PPCB TEQ.7E- -.56E-.96E-.E- Total TEQ 7.E- -8.6E-.E-.E-

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin TABLE 5 - The effect of age on the concentrations (pg/g lipid) of CDDs, CDFs, and PPCBs in sprat, C at age = age/(a + b*age), where a and b are constants. Mean values are shown where trends were not apparent. For CDDs and CDFs, the constants were derived from the combined data from [5] and this work. Concentrations Loads a b a b 78D.8E+ -.5E- 9.E-.57E- 78D.E+ -.7E- 5.E- 5.6E- 78D 5.89E+.58E+ 5.8E- mean 678D.5E+ -8.E-.7E- 9.7E- 789D.E- mean.7e+ -.7E+ 678D.6E+ mean.5e+ mean OCDD.6E+ mean 8.5E+ mean 78F.7E- -7.E-.E-.E- 78F.6E+ -.E-.5E- 5.58E- 78F.85E- -.58E- 6.5E-.E- 78F.68E+ -.96E-.E+.9E- 678F.9E+ -.7E- 6.5E-.6E- 789F.E- mean.7e- mean 678F.67E+ -.68E- 5.58E- 9.6E- 678F.5E+ -.E-.E-.E- 789F.9E- mean 9.E+.55E+ OCDF.7E+ 9.9E-.8E+ mean CDD/F TEQ.E- -.6E- 6.88E-.66E- 77.E-.E-.9E-.77E- 8 6.E-.55E-.E-.6E- 6 5.9E-.E-.9E-.E- 69.99E- 6.96E-.E-.5E- 5.8E-.8E-5.8E- 7.65E-6.5E- 6.E-.98E-.E- 8 5.9E-5.E-5 7.86E-5.8E-6.59E-.7E- 7.57E- 6.95E-5 56.E- 7.7E-5 6.56E-.5E-5 57.88E-.6E-.8E-.7E- 67 5.97E-.9E-.E-.E- 89.7E-.E- 7.E- 5.85E- PPCB TEQ.67E-.8E- 8.E-.E- TABLE 6 - The effect of age on the loads (pg/fish) of CDDs, CDFs, and PPCBs in herring, C at age = age/ (a + b*age), where a and b are constants. Mean values are shown where trends were not apparent. Reference [] Reference [5] This work a b a b a b 78D.E+ -.E-.8E+ -.5E- 9.9E- -.5E- 78D.79E- -5.7E- 9.E- -.5E-.5E- -.6E- 78D.E+ -.E+.E+ -.E+.87E+ -.8E- 678D.5E- -.7E- 8.7E- -.7E-.E+ -9.98E- 789D.9E+ -.E-.59E+ -6.E-.7E+ -8.6E- 678D 9.E- -5.E-.6E+ mean.e+ -7.6E- OCDD.5E+ mean.78e-.8e-.5e+ -.9E- 78F 6.9E- 9.E- 6.5E- -6.6E- 8.6E- -.7E- 78F 6.E- -7.5E-.E+ -.E-.5E- -.9E- 78F.E- -.E-.9E- -.6E- 6.7E- -.9E- 78F.E+ -.58E-.8E+ -.98E-.56E+ -.6E- 678F.5E+ -.97E-.95E+ -.E-.5E+ -.E- 789F.66E+ 9.99E+ 6.7E+.9E- 678F 9.E- -.7E-.E+ -.7E-.7E+ -.9E- 678F.7E-.E-.87E+ mean.58e+ -.6E- 789F.88E- mean 9.58E+ -7.5E- OCDF.8E+ mean 8.6E+ mean.e+ -.5E- CDD/F TEQ.E- -.6E-.8E- -.9E- 8.95E- -5.88E- 77 5.E-.6E-.E- -.77E- 8 5.9E- -6.66E- 6.75E- -.56E-.86E- -.6E- 69 6.6E- -6.97E-.88E- -.E- 5.65E- -.67E-.8E- -.8E-5.8E- -.87E- 8.7E- -8.78E- 7.66E-5-6.5E-6 7.98E- -7.9E-5 56.E+ -.57E- 6.9E- -5.57E-5 57.57E- -.9E- 67.E- -9.5E-5 89 7.6E- -6.E- PPCB TEQ.7E- -.56E-.E- -9.9E- Total TEQ 7.E- -8.6E- 5.8E- -.96E-

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin As can be seen from Tables and 5, the values of the constants are quite close for almost all CDDs and CDFs in herring and sprat from this and the study [5]. This confirms the suggestion [5] that the age of the fish is more important than the year of collection in two successive years. On the other hand, the values of a in herring from the 99s [] are generally higher, and values of b are negative, since the concentrations increase, without a tendency to level off, with age of the fish. In contrast to herring, the concentrations of almost all CDDs and CDFs increase with the age of sprat (Table 5), without a tendency to level off. This may mean that sprat are more efficient than herring in the accumulation of these compounds, but less efficient than herring in the accumulation of PPCBs. The loads (pg/fish) of all the compounds in sprat show signs of leveling off with increasing age (b>, Table 5). This is generally not the case with herring (Table 6). A few examples of the concentration vs age of herring relationships from this work and from [] are in Fig, those of loads vs age in Fig., and those of TEQ concentrations and loads in Fig.. As can be seen from Figs. and, the loads have changed slightly from the 99s to. The concentration vs age of sprat relationships for combined [5] and data are shown in Fig. 5 There is a good agreement between the and data. 9 8 7 6 5 C A 6 8 5 9 7 5 - B 6 8 8 6 C CC 6 8 6 8 6 D 6 8 FIGURE Concentration vs. age of herring. A - 78F, B - 678D, C - 78F, D - 6. Diamonds data from [], triangles present work. Lines fitted relationships C = age/(a+b*age). 9 8 7 6 5 A BL9 958 6 8 8 6 B BL9 9 6 8 5 C 5 5 D 5 5 5 5 6 8 6 8 FIGURE Loads (pg/fish) vs. age of herring. A - 78F, B - 678D, C - 78F, D - 6. Diamonds data from [], triangles present work. Lines fitted curves L = age/(a+b*age).

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin 6 A 5 6 8 8 B 6 8 6 5 6 7 8 9 8 7 6 C BL9 7 5 D 5 5 5 5 6 8 5 6 7 8 9 FIGURE - Concentrations (pg/g lipid), A, B, and loads (pg/fish), C, D, of CDD/F TEQ (A, C) and PPCB TEQ (B, D) vs age of herring. Diamonds - data from [], triangles - this work. 9. 8. A 7. 6. 5........... 5. 6. 7.. B.. 8. 6......... 5. 6. 7. 9. 8. C 7. 6. 5........... 5. 6. 7. 8. 7. D 6. 5........... 5. 6. 7. FIGURE 5 - Concentration (pg/g lipid) of CDDFs vs the age of sprat. Combined data from [] and this work. A - 78F, B - 678F, C- 78F, D- TEQ The extrapolation from edible parts to the whole fish is justifiable for herring and sprat since both species have lipids quite evenly distributed throughout the body. The increase of the load with the age (and size) of the fish may also play a role in the food chain: a larger prey delivers a larger amount of the contaminant to the predator, than an equal weight of smaller prey. Chemical structure of CDDs, CDFs, and PPCBs and the constants a and b (equation A) 78-Tetrachloro- and 78-pentachloro-dibenzofuran (66f and E6f) have the lowest values of the constants in all examined data sets (Table 7). Compounds exhibiting the highest values of the constants are less consistent. It would be interesting to examine additional data to see what patterns will emerge.

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin The constants a and b showed no discernible relation to the structure of the PPCBs in the set []. On the other hand, there is a linear log-log relationship between a and b for PPCBs in both, herring and sprat (Fig. 6). Three (8, 6, and 69) of the four di-ortho unsubstituted chlorobiphenyls have the highest a and b values in herring as well as in sprat. The fourth one (77) is preceded by the two mono-ortho unsubstituted heptachlorobiphenyls (57 and 59), and by the only mono-ortho unsubstituted pentachlorobiphenyl with a single chlorine in one of the benzene rings (). More data have to be examined before making any conclusions. TABLE 7 - Minimal and maximal values of the constants a and b (equation (A)) for CDDs and CDFs. R and L are herring (Table ) and sprat (Table ), respectively, other data are from references [] and [5]. For CDD and CDF codes see Table. References 66f E6f 7Ed FEf a b a b a b a b.8.6..7...8.666 [5] 66f E6f 7Ed F6d a b a b a b a b.598.6. -...58 5. -.9 [] 66f E6f 7Ed F6d a b a b a b a b.6.9. -..9 -.. -. L 66f E6f F6d F6f a b a b a b a b.7 -.7.85 -.58 5.89.58.68 -.96 - - 8-69 77 89 6 log(b) - 57 67-5 56 5 8-6 -7-5 -.5 - -.5 - -.5 - -.5 - -.5 log (a) see equation (A) FIGURE 6 - The relationship of the constants a and b (equation (A)) of PPCBs in herring (diamonds and IUPAC codes) and sprat ( ). The order of PPCBs is the same in both series. Age and the profile of CDD, CDF and PPCB Projections of the CDD and CDF (combined as CDDF) profiles of herring ([] and present work) on the plane of the principal components and (pc- & pc-), and of the profiles calculated from (A) and marked by age in years (numbers -), are on the left-hand side of Fig. 7. It can be seen that the age is a major factor and accounts for 8% and 58% of the original variance. There is a good agreement between actual [] and predicted profiles. The F7 profile is closer to that predicted for 6-years-old herring, and the FS9 and BS9 profiles are similar to that of 8-years-old herring. For the samples from this work, the profile of the 8-years-old R herring is more similar to that of younger fish, and the profile of R (age years) to that of much older fish. The right-hand side of Fig. 7 shows the effect of the original variables (individual CDDs and CDFs, for the codes see Table ) on the principal components. Those with positive loadings in the pc- (ev->) are more prevalent in the younger fish. 5

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin 7. 6 5.. F6d 77f F7F FFf 66f FFf F7f FFd E6f pc- (%) 9 BL9 BS9 FL9 FS9 8 F F ev- -. -. -. 76f 7Ed F7d - - 7 F7 F6 6 FS 5 F5 F -. 77d 76d F6f E7f 66d - -6 - - 6 8 pc- (8%) -.5 -.5 -. -. -. -..... ev- 5.5 R. F7f 7Ed FFf F7d pc- (8%) - 9 8 7 6 R 5 R6 ev-... -. -. 77f 76dE7f 77d 76f F6f E6f FFd 66d 7Ef 66f R -. F6d - R9 R5 -. FEf - -6 - - 6 8 pc- (58%) rcn -.5 -. -. -. -..... ev- rcn FIGURE 7 - Changes of CDDF concentration profiles with age of herring. On the left are projections on the plane of pc-&pc-, on the right are loadings of CDDFs on the principal components. In the upper and lower row are data from [], and this work, respectively. For sample codes see Table and 'Samples' in text, for CDDF codes see Table. Numbers alone mark profiles predicted from fitted curves. pc- (%).5.5 -.5 - -.5-8 BL9 FL9 FS9 7 6 F7 F6 FS F5 5 BS9 F F F -.5 - - - - 5 pc- (75%) pcbhc ev-.6.. -. -. -.6 -.8 6 69 5 56 8 - -.6 -. -....6 ev- pcbhc 77 pc- (%) R R5 R 7 6 5 8 R6 - R9 - - - -5 R -6-6 - - 6 pc- (56%) pcbrcn ev-. 67. 5. 8 69 -. 77 57 -. -. 56 6 -. 89 -.5 8 -.6 -.5 -. -. -. -......5 ev- pcbrcn FIGURE 8 - Changes of PPCB profiles with the age of herring. Arrangements and sample codes are the same as in Fig. 7, and IUPAC codes are used for PPCBs. 6

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin pc- (6%) 7 b5 k5 6. L 5 L t5 L L5 L - t t7 t6 b k - - - - - - - pc- (5%) ev-.6.5 F7f.. F6f FFf. E7f 76d FEf. 7Ef 76f -. 77d FFd E6f 77f 66d -. F6d7Ed -. 66f F7d -. -.5 -. -. -. -..... ev-.8 L.6 8. pc- (%) - 7 6 5 L5 L L ev-. -. -. 5 57 89 69 77 6 67 8 - L -.6 56 - -6 - - 6 8 pc- (77%) scn -.8 -. -. -. -..... ev- scn FIGURE 9 - Changes of combined (this work and [5]) profiles of CDDFs (upper plots) and profiles of PPCB (this work) with the age of sprat. 5.. 5.. 5.. R 5.. 5.. 5 6 7 8 9 76d 66f E6f F7f 66f* E6f* 76d* F7f* FIGURE - Actual concentrations and that calculated (*) from (A) of 78-pentachlorodibenzo-p-dioxin (76d), 78-tetrachlorodibenzofuran (66f), 78-pentachlorodibenzofuran (E6f), and 678-heptachlorodibenzofuran (F7f), plotted against the age of herring. The changes of the PPCB profile with age of herring are shown in Fig. 8, and those of CDDFs and PPCBs of sprat in Fig. 9. The deviations from the age trend (pc-) are relatively minor, and illustrated in Fig. for the major components of the herring profile from this work. 7

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin TABLE 8 - A summary of positive and negative effects of individual CDDFs and PPCBs on the principal component pc-. For codes of CDDs &CDFs see Table, IUPAC number are used for PPCBs. Compounds with positive effect decrease, those with a negative effect increase with age in the profile (concentrations scaled to a sum of ). CDDF Positive effect on pc- herring this work 7Ef 66f 66d herring [] FFd FFf F7f 66f herring [5] FFd 7Ef FEf F7f F7d sprat this work FFd 7Ef FFf FEf Negative effect on pc- herring this work 76d E7f 77f 77d herring [] E6f 76f 77f F6d herring [5] E6f 76f 76d sprat this work E6f 76f 76d E7f PPCB Positive effect on pc- herring this work 77 69 5 herring [] 77 sprat this work 77 69 8 6 67 89 Negative effect on pc- herring this work 56 57 herring [] 56 5 6 69 sprat this work 56 5 57 The effect of the original variables (CDDFs and PPCBs) on the principal components is shown in the ev- vs ev- plots in Figs. 7-9. Some patterns appear to be present (Table 8), and additional work in this direction is desirable. ACKNOWLEDGEMENT The project was financially supported by the Ministry of Agriculture of Estonia. CONCLUSION The concentrations of CDDs and CDFs in the Baltic herring have not changed very much between the 99s and, and those in the Baltic sprat between and. The concentrations of most of the PPCBs in herring, reported in this paper, are much higher than those found by Kiviranta et al. [] in the 99s. Unfortunately, that data on other chlorobiphenyls are not available. It should be a standard practice to report the concentrations of all the detectable chlorobiphenyls. An important factor for the concentration of CDDs, CDFs, and PPCBs is the age of the fish. Monitoring programs should be using fish samples of well-defined age. The equation (A), formally the same as the Langmuir adsorption isotherm, was very useful in the evaluation of the effect of age on the concentration of the contaminants. Its constants may provide some insights into the relationships between chemical structure and the accumulation of organochlorine compounds. Both equation (A) and the Principal Component Analysis are good tools for the evaluation of the data, and it would be desirable to use them also as a quality control before the data leave the laboratory. The analyses are expensive, but funds should be available for duplicate analyses of extracts as well as samples, to have an idea about the uncertainties in the data. Small numbers of samples in this work did not warrant an examination of regional differences in the concentration of CDDs, CDFs, and PPCBs in the fish. REFERENCES [] The Baltic Marine Environment 999- (). Baltic Sea Environment Proceedings, Helsinki Commission, No. 87: -. [] Kiviranta, H., Vartiainen, T., Parmanne, R., Hallikainen, A., and Koistinen, J. (). PCDD/Fs and PCBs in Baltic herring during the 99s. Chemosphere: 5:-6. [] Otsa, E., Roots, O. and Simm, M. (). Level of dioxins and polychlorinated biphenyls, similar to dioxins, in the fish in the coastal sea of Estonia in the year. Estonian Environmental Research Centre. Contract No. [] http://www.agri.ee/eng. [5] Roots, O., Zitko, V. and Holoubek, I. (). Chlorinated dibenzo-p-dioxins and dibenzofurans patterns in the baltic herring and sprat. Organohalogen Compounds, 6:-. [6] Roots, O. and Zitko, V. (). Chlorinated dibenzo-p-dioxins and dibenzofurans in the baltic herring and sprat of Estonian coastal waters. Environmental Science & Pollution Research, :86-9. [7] Lankov, A. and Kukk, H. (). Feeding of herring in the Gulf of Finland in the 97s-9s. Proc. Estonian Acad. Sci. Biol. Ecol., 5: 77-9. [8] Kiviranta, H., Hallikainen, A., Ovaskainen, M.-L., Kumpulainen, J., Vartiainen, T. () Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans and polychlorinated biphenyls in Finland. Food Additives and Contaminants: 8:95-95. 8

by PSP Volume 5 No. 6 Fresenius Environmental Bulletin [9] Wu, W., Schramm, K.-W. and Kettrup, A. (). Bioaccumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans in the foodweb of Ya-Er lake area, China. Water Research 5:-8. [] Wise, B.M. and Gallagher, N.B. (998). PLS-Tool., Eigenvector Inc., Manson WA 988, USA. [] Billo, E.J. (). Excel for Chemists. A Comprehensive Guide. Wiley-VCH, New-York, 6p. Received: October, 5 Accepted: December 8, 5 CORRESPONDING AUTHOR Ott Roots Estonian Environmental Research Centre Marja D 67 Tallinn Estonia e-mail: Ott.Roots@klab.ee FEB/ Vol 5/ No / 6 pages 7-9 9