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Bioaccumulation potential of polycyclic aromatic hydrocarbons in Daphnia pulex
Water Research Vol. 12, pp. 973 to 977. Pergamon Press Ltd. 1978. Printed in Great Britain.
B I O A C C U M U L A T I O N P O T E N T I A L OF P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S IN DAPHNIA PULEX*t G. R. SOUTHWORTH,J. J. BEAUCHAMP~and P. K. SCHMIEDER§ Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, U.S.A.
(Received in revised form 13 April 1978) Abstract--The bioaccumulation potentials by aquatic biota from aqueous solution were determined for seven polycyclic aromatic hydrocarbons (PAH). The PAH were tested using Daphnia pulex and consisted of the following compounds: naphthalene, anthracene, phenanthrene, pyrene, 9-methylanthracene, benz(a)anthracene and perylene. Bioaccumulation kinetics were described as a first order approach to equilibrium in a two-compartment model (water and Daphnia), using a two-stage technique to estimate uptake and elimination rates, while accounting for decreasing aqueous concentrations. Estimates of equilibrium concentration factors were obtained by two methods: (1) evaluating the kinetic model as t tends to infinity and (2) direct measurement of concentration factor at t = 24 h. Estimations of equilibrium concentration factors obtained by the two methods were in good agreement, and increased dramatically with increasing molecular weight within the series of compounds. The calculated n-octanol-water partition coefficient was shown to be a good predictor of bioaccumulation potential of PAH in Daphnia. PAH were concentrated from a high of about 10,000-fold for benz(a)anthracene to a low of about 100-fold for naphthalene.
Large numbers of different PAH compounds are produced in synthetic fuels processes such as coal liquefaction (Guerin, 1975). This makes studying the environmental transport and transformation of all PAH components of an effluent a prodigious task. However, since PAH constitute homologous series of compounds varying by consistent changes in chemical structure (either number of aromatic rings or methyl side chains), their behavior might be reasonably well predicted by correlating the observed behavior of a series of model PAH compounds with a readily available physical property, such as n-octanol-water partition coefficient. This partition coefficient has been shown to be correlated with bioaccumulation of pesticides by fish (Neely et al., 1974), and is readily obtainable for PAH either through calculation or in previous determinations (Leo, 1975).
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAH) are commonly found in the environment as a result of combustion of fossil fuels and industrial processes such as coking (Herbes et al., 1976). The possible large scale commercialization of synthetic fuel production from coal and oil shale creates a potential for even greater release of PAH to air, water and land (Gehrs et al., 1976). Since many PAH have known carcinogenic properties towards humans and other mammals, (Christensen et al., 1975) the fate of PAH in the environment and identification of sites where they may accumulate to significant concentrations is important. The objective of this study was to evaluate the extent and rate at which PAH in aquatic environments are removed from solution and accumulated by the zooplankter, Daphnia pulex, a representative component of aquatic food webs. Daphnia have been shown to accumulate hydrophobic organics such as D D T (Crosby & Tucker, 1971), hexachlorocyclohexane (Canton et al., 1975), and the PAH, anthracene (Herbes & Risi, 1978). Methyl mercury, a common contaminant in fish, also exhibits bioaccumulation several thousand fold by Daphnia (Huckabee, 1975).
METHODS
Experimental
* Research sponsored by the Energy Research and Development Administration under contract with Union Carbide Corporation. t Publication No. 1232, Environmental Sciences Division, Oak Ridge, National Laboratory. :~ Computer Sciences Division, Union Carbide Corporation Nuclear Division. § College of Natural Resources, University of Wisconsin, Stevens Point, WI 54481, U.S.A.
PAH were obtained from Eastman Kodak, Matheson, Coleman, and Bell, Fisher Scientific, Aldrich and Amersham-Searle and used without further purification. Absorption and fluorescence emission-excitation spectra of each compound were compared with literature data to verif), that the correct compound was being monitored in these experiments (Sawicki et al., 1960; Schwarz & Wasik, 1976). Data for constructing bioaccumulation curves were developed by placing 200 Daphnia in 6 I. of filtered (Whatman No. 40) spring water containing a known concentration of dissolved PAH. PAH was introduced to solution by the addition of a small amount of stock solution of PAH in methanol, which resulted in aqueous methanol concentrations of < 100mg -1. The solutions were then maintained at 25°C in a water bath, and under a regimen
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G.R.
SOUTHWORTH,J. J. BEAUCHAMP and P. K. SCHMIEDER
of 9 h light 15 h dark using gold fluorescent light (cutoff ~ 500 nm) to prevent photodegradation of PAH. Two or three replicates of five animals each were removed at varying intervals until equilibrium was reached, rinsed in distilled water, and placed in 5 ml of methanol. The samples were shaken for 30rain and the methanol extracts then analyzed for PAH using fluorescence spectrophotometry. Homogenization and reextraction of PAH from Daphnia extracted by this procedure indicated that virtually all of the P A H had been removed in the initial extraction of intact Daphnia. Concentration factors (concentration P A H in Daphnia/ concentration PAH in water) for the P A H studied were determined for each c o m p o u n d by placing 25 Daphnia in each of two beakers containing 500ml of filtered spring water containing PAH at concentrations and conditions used in kinetic experiments. The Daphnia were obtained by visually selecting individuals of similar size from a stock culture. A sample of 25 animals was then removed from this group, filtered onto W h a t m a n No. 40 paper, and then weighed individually on an Aiasworth Type 24n balance to estimate the m e a n live weight of organisms used in the study. After 24 h, three 5 animal replicates were taken from each beaker, and the PAH content of water and Daphnia determined using fluorescence spectrophotometry. Aqueous P A H concentrations used in this experiment were selected on the basis of water solubility, analytical sensitivity, and observed bioaccumulation in preliminary experiments. Thus, concentrations of approximately 6 ppb were used for benz(a)anthracene, anthracene, and 9-methyl anthracene, 0.3 ppb for perylene, 30 ppb for phenanthrene, 50 ppb for pyrene and 1.00 ppm for naphthalene. A control set of animals was maintained in spring water without added PAH. The possible variation of equilibrium concentration factor with aqueous PAH concentration was investigated by determining anthracene bioaccumulation factors at concentrations of 30, 3, and 0.3 ppb using t4C-labelled anthracene. Conditions were the same as those used for the 24-h concentration factor determinations on the other PAH. A longer bioaccumulation run was carried out for 7 days using 9-methyl anthracene to assess the longer term bioaccumulation of PAH. Animals were fed a trout chow suspension (Gehrs, 1973) every other day in this experiment. Water was not changed, and dissolved oxygen remained above 4 ppm throughout the experiment.
Analytical A suitable e x c i t a t i o ~ e m i s s i o n wavelength combination was determined for each PAH in water and methanol using a Perkin-Elmer M P F - 4 4 fluorescence spectrophotometer (Table 15. Dual calibration curves were constructed for methanol extracts--one in methanol only, the other in a sample extract from five Daphnia. If the slopes of the two
Table 1. Fluorimetric analysis of PAH Excitation wavelength, nm Naphthalene
H20 MeOH Phenanthrene' H20 MeOH Anthracene H20 MeOH 9-Methylanthracene H20 MeOH Benz(alanthracene H20 MeOH Perylene H20 MeOH Pyrene 80% HzO 20°. MeOH
Emission wavelenglh. nm
Absorption at 220 nm 278 317 251 364 251 364 251 399 251 399 366 388 255 388 287 386 287 386 252 438 252 438 272 382
calibration curves were not significantly different, P A H in the Daphnia-methanol extract would be determined directly from the calibration curve in methanol. The possibility of materials extracted from the Daphnia quenching P A H fluorescence would thus be detected. In all cases, addition of P A H to methanol and methanol-Daphnia extract produced calibration curves having slopes not significantly different. Since excitation-emission spectra for each P A H are characteristic, it was possible to verify that the P A H extracted from the Daphnia was in fact the same P A H present in the water. Bioaccumulation data were analyzed as a first-order approach to equilibrium in a two-compartment model (water and Daphnia). The uptake was assumed to be a first-order process with respect to aqueous P A H concentration, and the elimination rate first order with respect to P A H concentration in the Daphnia. These assumptions lead to the following differential equation as our model: dY(t)
dt
= CZ(t) - kY(t),
(1)
where:
Z(t) = aqueous P A H concentration at time t after the start of the experiment,
Y(t) = Daphnia P A H concentration at time t after the start of the experiment,
k = Daphnia elimination rate, constant, h - : , and C = uptake rate constant, h - 2 Since aqueous PAH concentration tended to decrease with time due to uncharacterized removal and degradation processes, Z(t) was approximated by the function:
Z(t) = c~ + fie-;",
(2)
to give a description of the behavior of the aqueous concentration over time. Substitution of equation (2) into equation (1) and solving this differential equation yields the expression:
Ck]exp(-kt) Cfl (2 E k ) e x p ( - 2 t )
Ca + k-"
(3)
For an unchanging aqueous concentration, i.e. Z(t) = ~ = Z(0), fi = 0 and 2 = pc, the expression takes the form:
Y(t) = CZ(O) [1 - e x p ( - kt)].
(4)
The estimates of the parameters C and k were obtained using a two-stage iterative least squares technique. The first stage used the observed (t, Z(t)) values to obtain estimates of c~, fl, and 2 or Z(0) for the unchanging aqueous concentration case. (The estimate of Z(0) was equal to the sample average of the observed Z(t) values.) By substituting the parameter estimates from the first stage into equation (3) or equation (45, a nonlinear iterative least squares procedure was used to determine the estimates of C and k (Draper & Smith, 19665. The bioaccumulation curve (concentration factor vs time) was derived from the ratio of equations (3) and (2). The n-octanol water partition coefficients (P.C.) were calculated using the method of Leo (1975).
RESULTS All P A H s t u d i e d were r a p i d l y t a k e n u p by Daphnia a n d c o n c e n t r a t e d by several o r d e r s o f m a g n i t u d e
Polycyclic aromatic hydrocarbons in Daphnia pulex above ambient aqueous PAH concentrations. The concentration factor (concentration of PAH in Daphnia/concentration PAH in water) increased with time until approaching an apparent equilibrium sometime within 24 h. Representative bioaccumulation curves for three PAH are depicted in Fig. 1. The combined effect of increasing PAH concentration in the Daphnia and decreasing PAH concentration in the water produce a bioaccumulation curve with a small maximum which then decreases to equilibrium. In all cases the 24-h concentration factor was within 10% of the equilibrium concentration factor. Resolution of log C vs log (P.C.) yields a rather small correlation, log C = 0.219 log(P.C.) + 1.642, R z = 0.45,
(5)
while log 1/k vs log P gives the expression: log 1/k = 0.418 log(P.C.) - 1.596, R 2 = 0.98.
(6)
The equilibrium concentration factor was determined by evaluating Z(t) and Y(t) as t = zc in equations (2) and (3), and is equal to C/k. The observed relationship between equilibrium concentratiofi factor and partition coefficient is given by: log C/k = 0.656 log(P.C.) + 0.00051, R z = 0.85. (7) Thus, elimination rate and equilibrium concentration factor are closely tied to chemical structure, and variations in equilibrium concentration factor with changes in PAH structure are associated in most part with the variation in elimination rate. Elimination rates were rapid for all PAH, with half lives ranging from 0.4 to 5 h. Analysis of variance of anthracene 24h concentration factors obtained at 30, 3 and 0.3 ppb anthracene indicated no significant difference (P > 0.05) in observed concentration factors, with mean (n = 6) concentration factor ranging from a high of 1085 at 3 ppb to a low of 988 at 0.3 ppb. In the longer term bioaccumulation experiment, the slope of the concentration factor vs time from t = 6 h to t = 167 h does not significantly differ from unity (P > 0.05). This indicates that the 24-h concentration 7000
t
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6000 r- 5000 z 0
4000
3000 ~z 2000 7/J 8 1000 ~00~ 0 ~__ I 0
ANTHRACENE
t }
NAPHTHALENE
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20
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30 40 50 TIME (hr) Fig. l. Concentration factor I-concentration PAH in animal (wet weight)/concentration PAH in water] vs time for naphthalene, anthracene, and benz(a)anthracene in Daphnia pulex at 25~C. Error bars are 4-1 S.E.
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factor adequately represents concentration factors resultant from long-term exposure. Results of the direct measurement of concentration factor are depicted in Table 2, and agree reasonably well with values obtained in kinetic experiments. Regression of concentration factor (C.F.) (24 h) vs log partition coefficient (Fig. 2) yields the expression: log C.F. = 0.7520 log P - 0.4362, R 2 = 0.85. (8) The bioaccumulation potential of PAH increases by nearly a factor of 10 with each additional ring structure; thus two-ring naphthalene is accumulated about 100-fold while four ring benz(a)anthracene is concentrated about 10,000-fold. The 95% prediction interval about an estimated value of concentration factor can be seen to encompass a band slightly greater than one order of magnitude centered about the predicted value. Statistical comparison of equations (7) and (8) indicate that their slopes and intercepts do not vary significantly (P > 0.05). Predicted concentration factors obtained from each equation for any PAH within the range of partition coefficients studied vary by less than 32%. Since possible temporal variations in lipid content of the Daphnia population in the stock culture would undoubtedly affect equilibrium concentration factors, the data derived using animals from a single population on the same day, equation (8), is a more reliable estimate of the variation in bioaccumulation potential with chemical structure. DISCUSSION
Bioaccumulation potential of other PAH in Daphnia pulex can be predicted using equation (8). Thus, carcinogenic materials such as benz(a)pyrene and benzo(c)phenanthrene would be expected to accumulate about 13,000 and 6000-fold, respectively, in Daphnia. This degree of bioaccumulation is of similar magnitude to that exhibited by other toxicants which are objectionable in aquatic ecosystems in part because of their propensity to bioaccumulate. Thus, DDT was concentrated 16,000-fold by Daphnia magna (Crosby, 1971), while another pesticide commonly found in the environmeht, 6-hexachlorocyclohexane, was accumulated up to 350-fold by the same species (Canton et al., 1975). Methyl mercury has been shown to be accumulated about 4000-fold by Daphnia pulex at equilibrium (Huckabee, 1975). While kinetic parameters such as uptake and elimination rates are of value in describing the change in animal body burdens of a foreign substance over time, equilibrium concentrations within the organisms probably result from the equilibrium partitioning of the material between animal lipids and external water (Clayton et al., 1977). If n-octanol/water partition coefficient accurately approximates lipid/water partitioning, then Daphnia lipid content should be estimated by the ratio of equilibrium concentration factor to partition coefficient. When applied to these
976
G. R. SOUTHWORTH,J. J. BEAUCHAMPand P. K. SCHMIEDER Table 2. Bioaccumulation of PAH by Daphnia pulex
('ompourLd
Molccular v, eighl
Number of rings
128 178 178 192 202 228 252
2 3 3 3 4 4 5
Napl'dhalene Phcrtanlhrcr, c Anlhraccne 9-Melhyhnllhraccnc Pyrcne Bcnz(a)anthracenc Perylenc
Partitic, n* coelliciem
Concentrationt factor. 4-S.E. 24 h
2.1/0 2.82 2.82 1.32 7.94 3,98 1.15
131 325 917 4583 2702 10,109 7191
x 111s x 10~ x 10"~ x 10 s x l04 x 105 x I(Y'
4- 10 4- 56 4- 48 4- 1004 _+ 245 4- 507 4- 804
Uptake rate, C++ h - l, + S,E, 197 203 702 561 1126 669 752
+ 51 + 47 4- 77 4- 65 + 123 _ 90 4- 42
Elimination rate. k++ h - ~. +S.E. 1.667 0.543 0.589 0.144 0.343 0.144 0.139
+ 0.467 4- 0.176 _q_ 0.078 + 0.064 _+ 0.049 4- 0.022 4- 0.011
C,' ~; 118 374 1192 3896 3283 4646 5410
* Octanol/water partition, coefficient, calculated from Leo, 1975. I" Ratio, concentration PAH in Daphnia (wet weight)/concentration PAH in water. Kinetic parameters from bioaccumulation model, dY(t)/dt = C Z ( t ) - k Y(t), where Z(t)= aqueous PAH concentration at time t after the start of the experiment, Y(t) = Daphnia PAH concentration (wet weight) at time t after the start of the experiment. § Kinetic estimation of concentration factor as t--~ :<.
PAH data, most estimates of lipid content fall in the 1-3.5~o range, which is consistent with observed Daphnia lipid content (S. E. Herbes, unpublished data). This suggests that PAH content of Daphnia lipids is in equilibrium with aqueous PAH concentration; i.e. that Daphnia-PAH bioaccumulation behavior is explainable primarily in terms of lipid/H20 PAH partitioning. Bioaccumulation in higher aquatic organisms such as fish, is complicated by the more complex transport pathway from external water to internal lipids, as well as the presence of metabolic elimination/detoxification mechanisms. Overall uptake and elimination kinetics in such a complex system with a much smaller
10 5
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surface area/volume ratio would be slower, making attainment of an apparent equilibrium concentration slower. In addition, the presence of a second elimination mechanism would likely prevent lipid PAH concentrations from attaining levels representative of simple equilibrium lipid/water phase partitioning. In such a system, the equilibrium concentration factor is likely to be a true dynamic equilibrium resulting from offsetting uptake and elimination rates. Before the bioaccumulation behavior of a class of compounds in a simple system such as Daphnia can be used to make inferences about the behavior of the compounds in an organism such as a fish, further research must be carried out comparing the behavior of given substances in each system.
REFERENCES
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-
1163-1169.
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Z
_o FI1:
t03
Z t/3 O o
10 z
=
//
/
/
_=
:/4 _--
/
/ io ~
I 1o 3
=
/
I
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n~OCTANOL-H20
Canton J. H., Greve P. A., Scoof W. & Van Esch G. J. (1975) Toxicity, accumulation, and elimination studies of 3-hexachlorocyclohexane (6-HCH) with freshwater orgamsms of different trophic levels. Water Res. 9,
I I I IIll
I
105
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lo s
PARTITION COEFFICIENT
Fig. 2. Log concentration factor (24h) vs log n-octanolwater partition coefficient (calculated from Leo, 1975) for several PAH in Daphnia pulex at 25°C. Dashed lines delineate 95% prediction interval.
Christensen H. E., Lugin Byhl T. T. & Carroll B. S. (1975) Registry of toxic effects of chemical substances. U.S. Dept. H.E.W., Rockville, Maryland. Clayton J. R., Jr., Pavlou S. P. & Breitner N. F. (19771 Polychlorinated biphenyls in coastal marine zooplankton: bioaccumulation by equilibrium partitioning. Environ. Sci. Teehnol. 11, 676-682. Crosby D. G. & Tucker R. K. (1971) Accumulation of DDT by Daphnia Magna. Environ. Sci. Technol. 5, 714-716. Draper N. R. & Smith H. (19661 Applied Regression Analysis. pp. 263-304. Wiley, New York. Gehrs C, W. (1972) Aspects of the Population Dynamics of the Calanoid Copepod, Diaptomus Clavipes (Schacht). Ph.D. Thesis, University of Oklahoma. Gehrs C. W., Coordinator. (19761 Coal conversion, description of technologies and necessary biomedical and environmental research. ORNL-5192. Guerin M. 119751 Analytical R and D for coal-conversion technology. In Coal Technology Program Progress Report. January 1975. ORNL/TM-4850, Oak Ridge National Laboratory. Herbes S. E., Southworth G. R. & Gehrs C. W. (1976) Organic contaminants in aqueous coal conversion efffluents--environmental consequences and research
Polycyclic aromatic hydrocarbons in Daphnia pulex priorities. In Trace Substances in Environmental Health--X. (symp.) (Edited by Hemphill D. D.) University of Missouri, Columbia. Herbes S. E, & Risi G. F. (1978) Metabolic alteration and excretion of anthracene by Daphnia pulex. Bull. Envir. Cont. Tox. 19, 147-155. Huckabee J. W., Goldstein R. A., Janzen S. A. & Woock S. E. (1975) Methylmercury in a fresh food chain. In Proceedinys of the International Conference on Heavy Metals in the Environment. pp. 199-216. Oct. 23-27. Toronto. Leo A. J. (1975) Calculation of partition coefficients useful in the evaluation of the relative hazards of various chemicals in the environment. In Symposium on Structure-Activity Correlations in Studies of Toxicity and
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Bioconcentration with Aquatic Organisms (Edited by Veith G. D. & Konasewich D. E.) International Joint Commission, Ontario. Neeley W. R., Branson D. R. & Blau G. E. (1974) Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. Technol. 8, 1113-1115. Sawicki E., Hauser T. R. & Stanley T. W. (1960) Ultraviolet, visible, and fluorescence spectral analysis of polynuclear hydrocarbons. Int. J. Air. Pollut. 2, 253272. Schwarz F. P. & Wasik S. P. (1976) Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene and benzo(e)pyrene in water. Analyt. Chem. 48, 524-528.
B I O A C C U M U L A T I O N P O T E N T I A L OF P O L Y C Y C L I C A R O M A T I C H Y D R O C A R B O N S IN DAPHNIA PULEX*t G. R. SOUTHWORTH,J. J. BEAUCHAMP~and P. K. SCHMIEDER§ Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, U.S.A.
(Received in revised form 13 April 1978) Abstract--The bioaccumulation potentials by aquatic biota from aqueous solution were determined for seven polycyclic aromatic hydrocarbons (PAH). The PAH were tested using Daphnia pulex and consisted of the following compounds: naphthalene, anthracene, phenanthrene, pyrene, 9-methylanthracene, benz(a)anthracene and perylene. Bioaccumulation kinetics were described as a first order approach to equilibrium in a two-compartment model (water and Daphnia), using a two-stage technique to estimate uptake and elimination rates, while accounting for decreasing aqueous concentrations. Estimates of equilibrium concentration factors were obtained by two methods: (1) evaluating the kinetic model as t tends to infinity and (2) direct measurement of concentration factor at t = 24 h. Estimations of equilibrium concentration factors obtained by the two methods were in good agreement, and increased dramatically with increasing molecular weight within the series of compounds. The calculated n-octanol-water partition coefficient was shown to be a good predictor of bioaccumulation potential of PAH in Daphnia. PAH were concentrated from a high of about 10,000-fold for benz(a)anthracene to a low of about 100-fold for naphthalene.
Large numbers of different PAH compounds are produced in synthetic fuels processes such as coal liquefaction (Guerin, 1975). This makes studying the environmental transport and transformation of all PAH components of an effluent a prodigious task. However, since PAH constitute homologous series of compounds varying by consistent changes in chemical structure (either number of aromatic rings or methyl side chains), their behavior might be reasonably well predicted by correlating the observed behavior of a series of model PAH compounds with a readily available physical property, such as n-octanol-water partition coefficient. This partition coefficient has been shown to be correlated with bioaccumulation of pesticides by fish (Neely et al., 1974), and is readily obtainable for PAH either through calculation or in previous determinations (Leo, 1975).
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAH) are commonly found in the environment as a result of combustion of fossil fuels and industrial processes such as coking (Herbes et al., 1976). The possible large scale commercialization of synthetic fuel production from coal and oil shale creates a potential for even greater release of PAH to air, water and land (Gehrs et al., 1976). Since many PAH have known carcinogenic properties towards humans and other mammals, (Christensen et al., 1975) the fate of PAH in the environment and identification of sites where they may accumulate to significant concentrations is important. The objective of this study was to evaluate the extent and rate at which PAH in aquatic environments are removed from solution and accumulated by the zooplankter, Daphnia pulex, a representative component of aquatic food webs. Daphnia have been shown to accumulate hydrophobic organics such as D D T (Crosby & Tucker, 1971), hexachlorocyclohexane (Canton et al., 1975), and the PAH, anthracene (Herbes & Risi, 1978). Methyl mercury, a common contaminant in fish, also exhibits bioaccumulation several thousand fold by Daphnia (Huckabee, 1975).
METHODS
Experimental
* Research sponsored by the Energy Research and Development Administration under contract with Union Carbide Corporation. t Publication No. 1232, Environmental Sciences Division, Oak Ridge, National Laboratory. :~ Computer Sciences Division, Union Carbide Corporation Nuclear Division. § College of Natural Resources, University of Wisconsin, Stevens Point, WI 54481, U.S.A.
PAH were obtained from Eastman Kodak, Matheson, Coleman, and Bell, Fisher Scientific, Aldrich and Amersham-Searle and used without further purification. Absorption and fluorescence emission-excitation spectra of each compound were compared with literature data to verif), that the correct compound was being monitored in these experiments (Sawicki et al., 1960; Schwarz & Wasik, 1976). Data for constructing bioaccumulation curves were developed by placing 200 Daphnia in 6 I. of filtered (Whatman No. 40) spring water containing a known concentration of dissolved PAH. PAH was introduced to solution by the addition of a small amount of stock solution of PAH in methanol, which resulted in aqueous methanol concentrations of < 100mg -1. The solutions were then maintained at 25°C in a water bath, and under a regimen
973
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G.R.
SOUTHWORTH,J. J. BEAUCHAMP and P. K. SCHMIEDER
of 9 h light 15 h dark using gold fluorescent light (cutoff ~ 500 nm) to prevent photodegradation of PAH. Two or three replicates of five animals each were removed at varying intervals until equilibrium was reached, rinsed in distilled water, and placed in 5 ml of methanol. The samples were shaken for 30rain and the methanol extracts then analyzed for PAH using fluorescence spectrophotometry. Homogenization and reextraction of PAH from Daphnia extracted by this procedure indicated that virtually all of the P A H had been removed in the initial extraction of intact Daphnia. Concentration factors (concentration P A H in Daphnia/ concentration PAH in water) for the P A H studied were determined for each c o m p o u n d by placing 25 Daphnia in each of two beakers containing 500ml of filtered spring water containing PAH at concentrations and conditions used in kinetic experiments. The Daphnia were obtained by visually selecting individuals of similar size from a stock culture. A sample of 25 animals was then removed from this group, filtered onto W h a t m a n No. 40 paper, and then weighed individually on an Aiasworth Type 24n balance to estimate the m e a n live weight of organisms used in the study. After 24 h, three 5 animal replicates were taken from each beaker, and the PAH content of water and Daphnia determined using fluorescence spectrophotometry. Aqueous P A H concentrations used in this experiment were selected on the basis of water solubility, analytical sensitivity, and observed bioaccumulation in preliminary experiments. Thus, concentrations of approximately 6 ppb were used for benz(a)anthracene, anthracene, and 9-methyl anthracene, 0.3 ppb for perylene, 30 ppb for phenanthrene, 50 ppb for pyrene and 1.00 ppm for naphthalene. A control set of animals was maintained in spring water without added PAH. The possible variation of equilibrium concentration factor with aqueous PAH concentration was investigated by determining anthracene bioaccumulation factors at concentrations of 30, 3, and 0.3 ppb using t4C-labelled anthracene. Conditions were the same as those used for the 24-h concentration factor determinations on the other PAH. A longer bioaccumulation run was carried out for 7 days using 9-methyl anthracene to assess the longer term bioaccumulation of PAH. Animals were fed a trout chow suspension (Gehrs, 1973) every other day in this experiment. Water was not changed, and dissolved oxygen remained above 4 ppm throughout the experiment.
Analytical A suitable e x c i t a t i o ~ e m i s s i o n wavelength combination was determined for each PAH in water and methanol using a Perkin-Elmer M P F - 4 4 fluorescence spectrophotometer (Table 15. Dual calibration curves were constructed for methanol extracts--one in methanol only, the other in a sample extract from five Daphnia. If the slopes of the two
Table 1. Fluorimetric analysis of PAH Excitation wavelength, nm Naphthalene
H20 MeOH Phenanthrene' H20 MeOH Anthracene H20 MeOH 9-Methylanthracene H20 MeOH Benz(alanthracene H20 MeOH Perylene H20 MeOH Pyrene 80% HzO 20°. MeOH
Emission wavelenglh. nm
Absorption at 220 nm 278 317 251 364 251 364 251 399 251 399 366 388 255 388 287 386 287 386 252 438 252 438 272 382
calibration curves were not significantly different, P A H in the Daphnia-methanol extract would be determined directly from the calibration curve in methanol. The possibility of materials extracted from the Daphnia quenching P A H fluorescence would thus be detected. In all cases, addition of P A H to methanol and methanol-Daphnia extract produced calibration curves having slopes not significantly different. Since excitation-emission spectra for each P A H are characteristic, it was possible to verify that the P A H extracted from the Daphnia was in fact the same P A H present in the water. Bioaccumulation data were analyzed as a first-order approach to equilibrium in a two-compartment model (water and Daphnia). The uptake was assumed to be a first-order process with respect to aqueous P A H concentration, and the elimination rate first order with respect to P A H concentration in the Daphnia. These assumptions lead to the following differential equation as our model: dY(t)
dt
= CZ(t) - kY(t),
(1)
where:
Z(t) = aqueous P A H concentration at time t after the start of the experiment,
Y(t) = Daphnia P A H concentration at time t after the start of the experiment,
k = Daphnia elimination rate, constant, h - : , and C = uptake rate constant, h - 2 Since aqueous PAH concentration tended to decrease with time due to uncharacterized removal and degradation processes, Z(t) was approximated by the function:
Z(t) = c~ + fie-;",
(2)
to give a description of the behavior of the aqueous concentration over time. Substitution of equation (2) into equation (1) and solving this differential equation yields the expression:
Ck]exp(-kt) Cfl (2 E k ) e x p ( - 2 t )
Ca + k-"
(3)
For an unchanging aqueous concentration, i.e. Z(t) = ~ = Z(0), fi = 0 and 2 = pc, the expression takes the form:
Y(t) = CZ(O) [1 - e x p ( - kt)].
(4)
The estimates of the parameters C and k were obtained using a two-stage iterative least squares technique. The first stage used the observed (t, Z(t)) values to obtain estimates of c~, fl, and 2 or Z(0) for the unchanging aqueous concentration case. (The estimate of Z(0) was equal to the sample average of the observed Z(t) values.) By substituting the parameter estimates from the first stage into equation (3) or equation (45, a nonlinear iterative least squares procedure was used to determine the estimates of C and k (Draper & Smith, 19665. The bioaccumulation curve (concentration factor vs time) was derived from the ratio of equations (3) and (2). The n-octanol water partition coefficients (P.C.) were calculated using the method of Leo (1975).
RESULTS All P A H s t u d i e d were r a p i d l y t a k e n u p by Daphnia a n d c o n c e n t r a t e d by several o r d e r s o f m a g n i t u d e
Polycyclic aromatic hydrocarbons in Daphnia pulex above ambient aqueous PAH concentrations. The concentration factor (concentration of PAH in Daphnia/concentration PAH in water) increased with time until approaching an apparent equilibrium sometime within 24 h. Representative bioaccumulation curves for three PAH are depicted in Fig. 1. The combined effect of increasing PAH concentration in the Daphnia and decreasing PAH concentration in the water produce a bioaccumulation curve with a small maximum which then decreases to equilibrium. In all cases the 24-h concentration factor was within 10% of the equilibrium concentration factor. Resolution of log C vs log (P.C.) yields a rather small correlation, log C = 0.219 log(P.C.) + 1.642, R z = 0.45,
(5)
while log 1/k vs log P gives the expression: log 1/k = 0.418 log(P.C.) - 1.596, R 2 = 0.98.
(6)
The equilibrium concentration factor was determined by evaluating Z(t) and Y(t) as t = zc in equations (2) and (3), and is equal to C/k. The observed relationship between equilibrium concentratiofi factor and partition coefficient is given by: log C/k = 0.656 log(P.C.) + 0.00051, R z = 0.85. (7) Thus, elimination rate and equilibrium concentration factor are closely tied to chemical structure, and variations in equilibrium concentration factor with changes in PAH structure are associated in most part with the variation in elimination rate. Elimination rates were rapid for all PAH, with half lives ranging from 0.4 to 5 h. Analysis of variance of anthracene 24h concentration factors obtained at 30, 3 and 0.3 ppb anthracene indicated no significant difference (P > 0.05) in observed concentration factors, with mean (n = 6) concentration factor ranging from a high of 1085 at 3 ppb to a low of 988 at 0.3 ppb. In the longer term bioaccumulation experiment, the slope of the concentration factor vs time from t = 6 h to t = 167 h does not significantly differ from unity (P > 0.05). This indicates that the 24-h concentration 7000
t
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ANTHRACENE
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NAPHTHALENE
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30 40 50 TIME (hr) Fig. l. Concentration factor I-concentration PAH in animal (wet weight)/concentration PAH in water] vs time for naphthalene, anthracene, and benz(a)anthracene in Daphnia pulex at 25~C. Error bars are 4-1 S.E.
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factor adequately represents concentration factors resultant from long-term exposure. Results of the direct measurement of concentration factor are depicted in Table 2, and agree reasonably well with values obtained in kinetic experiments. Regression of concentration factor (C.F.) (24 h) vs log partition coefficient (Fig. 2) yields the expression: log C.F. = 0.7520 log P - 0.4362, R 2 = 0.85. (8) The bioaccumulation potential of PAH increases by nearly a factor of 10 with each additional ring structure; thus two-ring naphthalene is accumulated about 100-fold while four ring benz(a)anthracene is concentrated about 10,000-fold. The 95% prediction interval about an estimated value of concentration factor can be seen to encompass a band slightly greater than one order of magnitude centered about the predicted value. Statistical comparison of equations (7) and (8) indicate that their slopes and intercepts do not vary significantly (P > 0.05). Predicted concentration factors obtained from each equation for any PAH within the range of partition coefficients studied vary by less than 32%. Since possible temporal variations in lipid content of the Daphnia population in the stock culture would undoubtedly affect equilibrium concentration factors, the data derived using animals from a single population on the same day, equation (8), is a more reliable estimate of the variation in bioaccumulation potential with chemical structure. DISCUSSION
Bioaccumulation potential of other PAH in Daphnia pulex can be predicted using equation (8). Thus, carcinogenic materials such as benz(a)pyrene and benzo(c)phenanthrene would be expected to accumulate about 13,000 and 6000-fold, respectively, in Daphnia. This degree of bioaccumulation is of similar magnitude to that exhibited by other toxicants which are objectionable in aquatic ecosystems in part because of their propensity to bioaccumulate. Thus, DDT was concentrated 16,000-fold by Daphnia magna (Crosby, 1971), while another pesticide commonly found in the environmeht, 6-hexachlorocyclohexane, was accumulated up to 350-fold by the same species (Canton et al., 1975). Methyl mercury has been shown to be accumulated about 4000-fold by Daphnia pulex at equilibrium (Huckabee, 1975). While kinetic parameters such as uptake and elimination rates are of value in describing the change in animal body burdens of a foreign substance over time, equilibrium concentrations within the organisms probably result from the equilibrium partitioning of the material between animal lipids and external water (Clayton et al., 1977). If n-octanol/water partition coefficient accurately approximates lipid/water partitioning, then Daphnia lipid content should be estimated by the ratio of equilibrium concentration factor to partition coefficient. When applied to these
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G. R. SOUTHWORTH,J. J. BEAUCHAMPand P. K. SCHMIEDER Table 2. Bioaccumulation of PAH by Daphnia pulex
('ompourLd
Molccular v, eighl
Number of rings
128 178 178 192 202 228 252
2 3 3 3 4 4 5
Napl'dhalene Phcrtanlhrcr, c Anlhraccne 9-Melhyhnllhraccnc Pyrcne Bcnz(a)anthracenc Perylenc
Partitic, n* coelliciem
Concentrationt factor. 4-S.E. 24 h
2.1/0 2.82 2.82 1.32 7.94 3,98 1.15
131 325 917 4583 2702 10,109 7191
x 111s x 10~ x 10"~ x 10 s x l04 x 105 x I(Y'
4- 10 4- 56 4- 48 4- 1004 _+ 245 4- 507 4- 804
Uptake rate, C++ h - l, + S,E, 197 203 702 561 1126 669 752
+ 51 + 47 4- 77 4- 65 + 123 _ 90 4- 42
Elimination rate. k++ h - ~. +S.E. 1.667 0.543 0.589 0.144 0.343 0.144 0.139
+ 0.467 4- 0.176 _q_ 0.078 + 0.064 _+ 0.049 4- 0.022 4- 0.011
C,' ~; 118 374 1192 3896 3283 4646 5410
* Octanol/water partition, coefficient, calculated from Leo, 1975. I" Ratio, concentration PAH in Daphnia (wet weight)/concentration PAH in water. Kinetic parameters from bioaccumulation model, dY(t)/dt = C Z ( t ) - k Y(t), where Z(t)= aqueous PAH concentration at time t after the start of the experiment, Y(t) = Daphnia PAH concentration (wet weight) at time t after the start of the experiment. § Kinetic estimation of concentration factor as t--~ :<.
PAH data, most estimates of lipid content fall in the 1-3.5~o range, which is consistent with observed Daphnia lipid content (S. E. Herbes, unpublished data). This suggests that PAH content of Daphnia lipids is in equilibrium with aqueous PAH concentration; i.e. that Daphnia-PAH bioaccumulation behavior is explainable primarily in terms of lipid/H20 PAH partitioning. Bioaccumulation in higher aquatic organisms such as fish, is complicated by the more complex transport pathway from external water to internal lipids, as well as the presence of metabolic elimination/detoxification mechanisms. Overall uptake and elimination kinetics in such a complex system with a much smaller
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surface area/volume ratio would be slower, making attainment of an apparent equilibrium concentration slower. In addition, the presence of a second elimination mechanism would likely prevent lipid PAH concentrations from attaining levels representative of simple equilibrium lipid/water phase partitioning. In such a system, the equilibrium concentration factor is likely to be a true dynamic equilibrium resulting from offsetting uptake and elimination rates. Before the bioaccumulation behavior of a class of compounds in a simple system such as Daphnia can be used to make inferences about the behavior of the compounds in an organism such as a fish, further research must be carried out comparing the behavior of given substances in each system.
REFERENCES
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Canton J. H., Greve P. A., Scoof W. & Van Esch G. J. (1975) Toxicity, accumulation, and elimination studies of 3-hexachlorocyclohexane (6-HCH) with freshwater orgamsms of different trophic levels. Water Res. 9,
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lo s
PARTITION COEFFICIENT
Fig. 2. Log concentration factor (24h) vs log n-octanolwater partition coefficient (calculated from Leo, 1975) for several PAH in Daphnia pulex at 25°C. Dashed lines delineate 95% prediction interval.
Christensen H. E., Lugin Byhl T. T. & Carroll B. S. (1975) Registry of toxic effects of chemical substances. U.S. Dept. H.E.W., Rockville, Maryland. Clayton J. R., Jr., Pavlou S. P. & Breitner N. F. (19771 Polychlorinated biphenyls in coastal marine zooplankton: bioaccumulation by equilibrium partitioning. Environ. Sci. Teehnol. 11, 676-682. Crosby D. G. & Tucker R. K. (1971) Accumulation of DDT by Daphnia Magna. Environ. Sci. Technol. 5, 714-716. Draper N. R. & Smith H. (19661 Applied Regression Analysis. pp. 263-304. Wiley, New York. Gehrs C, W. (1972) Aspects of the Population Dynamics of the Calanoid Copepod, Diaptomus Clavipes (Schacht). Ph.D. Thesis, University of Oklahoma. Gehrs C. W., Coordinator. (19761 Coal conversion, description of technologies and necessary biomedical and environmental research. ORNL-5192. Guerin M. 119751 Analytical R and D for coal-conversion technology. In Coal Technology Program Progress Report. January 1975. ORNL/TM-4850, Oak Ridge National Laboratory. Herbes S. E., Southworth G. R. & Gehrs C. W. (1976) Organic contaminants in aqueous coal conversion efffluents--environmental consequences and research
Polycyclic aromatic hydrocarbons in Daphnia pulex priorities. In Trace Substances in Environmental Health--X. (symp.) (Edited by Hemphill D. D.) University of Missouri, Columbia. Herbes S. E, & Risi G. F. (1978) Metabolic alteration and excretion of anthracene by Daphnia pulex. Bull. Envir. Cont. Tox. 19, 147-155. Huckabee J. W., Goldstein R. A., Janzen S. A. & Woock S. E. (1975) Methylmercury in a fresh food chain. In Proceedinys of the International Conference on Heavy Metals in the Environment. pp. 199-216. Oct. 23-27. Toronto. Leo A. J. (1975) Calculation of partition coefficients useful in the evaluation of the relative hazards of various chemicals in the environment. In Symposium on Structure-Activity Correlations in Studies of Toxicity and
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Bioconcentration with Aquatic Organisms (Edited by Veith G. D. & Konasewich D. E.) International Joint Commission, Ontario. Neeley W. R., Branson D. R. & Blau G. E. (1974) Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. Technol. 8, 1113-1115. Sawicki E., Hauser T. R. & Stanley T. W. (1960) Ultraviolet, visible, and fluorescence spectral analysis of polynuclear hydrocarbons. Int. J. Air. Pollut. 2, 253272. Schwarz F. P. & Wasik S. P. (1976) Fluorescence measurements of benzene, naphthalene, anthracene, pyrene, fluoranthene and benzo(e)pyrene in water. Analyt. Chem. 48, 524-528.