Environ. Sci. Technol. 2009, 43, 2295–2301
Accumulation and Cycling of Polycyclic Aromatic Hydrocarbons in Zooplankton NAIARA BERROJALBIZ,† SILVIA LACORTE,† ALBERT CALBET,‡ ENRIC SAIZ,‡ CARLOS BARATA,† AND J O R D I D A C H S * ,† Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, 08034, Barcelona, Catalunya, Spain, and Institut de Cie`ncies del Mar, ICM-CSIC, P. Marı´tim de la Barceloneta 37-49, 08003, Barcelona, Catalunya, Spain
Received July 1, 2008. Revised manuscript received January 8, 2009. Accepted January 9, 2009.
Planktonic food webs play an important role driving the environmental fate of persistent organic pollutants, and POP accumulation in phytoplankton has been previously studied for its importance as a first step in the aquatic food webs. However, little is known about the accumulation and cycling of organic pollutants between zooplankton and water. The present study shows the results of laboratory experiments on the bioconcentration (by passive uptake) of polycyclic aromatic hydrocarbons in phytoplankton (Rhodomonas salina) and accumulation in copepods (Paracartia(acartia) grani), by ingestion and diffusion. Both bioconcentration (BCF) and bioaccumulation (BAF) factors show significant correlation with the octanol-water partition coefficient (Kow) for phytoplankton and zooplankton. The BCF values for phytoplankton were 2 orders of magnitude higher than those for copepods. The analysis of fecal pellets shows that elimination by defecation is mainly significant for PAHs taken up from ingested phytoplankton but not due to passive uptake. However, the dominant elimination mechanisms are by far metabolism and diffusive depuration. Indeed, the mass balance suggests that metabolism of PAHs by copepods is a significant process that could play a role in the fate of PAHs in the water column. Uptake, depuration, eggestion, and ingestion rates increased with hydrophobicity of the chemical, while the metabolism rate was slightly higher for the less hydrophobic compounds. Passive partitioning dominated the accumulation of POPs in zooplankton. The derivation of all the uptake and loss rate constants for PAHs opens the door to future modeling studies of the role of zooplankton in PAH cycling in the marine environment.
Introduction Planktonic organisms play a key role driving the environmental fate and sinks of persistent organic pollutants (POPs) in the marine environment (1-3). During the past decade, several studies have dealt with the bioconcentration of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in phytoplankton (4-7). However, much less knowledge is known on the processes driving POP * Corresponding author e-mail:
[email protected]. † IDAEA-CSIC. ‡ ICM-CSIC. 10.1021/es8018226 CCC: $40.75
Published on Web 02/19/2009
2009 American Chemical Society
accumulation and elimination in zooplankton. Indeed, it is not clear whether zooplankton, and other heterotrophic organisms, accumulate POPs from feeding on phytoplankton or passively from surrounding waters. Recently, Sobek and co-workers (8) have suggested that diffusive uptake dominates accumulation of PCBs in zooplankton. However, their results contrast with the findings of Magnusson et al. (9) which using C-14 labeled PCBs found that both diffusive uptake and feeding contributed to PCB accumulation in zooplankton. So far, most information on PAH dynamics in marine zooplankton is restricted to naphthalene. Using radiolabeled C-14 naphthalene, uptake and depuration experiments were conducted in several copepod species (10). It was also reported that copepods were able to take up naphthalene from food and water and that half of the assimilated naphthalene during feeding was released to the water in some form other than the parental hydrocarbon, thus suggesting that copepods may also be able to metabolize PAHs (11). However, these processes have not been studied for PAHs with a wide range of physical-chemical properties, and previous studies (10-12) did not account for volatilization and other losses during the experiments. Given that fecal pellets efficiently transport PAHs, PCBs, and other POPs in the water column (13-15), it is important to study the transfer mechanisms of POPs to fecal pellets as well. In addition, the role of egg production has not been parametrized. M. C. Manus et al. (16) found faster elimination rates of PCBs in females of the planktonic copepode Acartia tonsa during egg production, as the compounds pass out to the lipids of the egg. Lotufo (17) exposed meiobenthic copepods to fluoranthene and also observed that 50% of the hydrocarbon body residue was in eggs, being released when these were extruded. Nevertheless, the copepod’s eggs hatch quite fast in the photic zone (18) and hence except in shallow waters its role as a dominant sinking mechanism of POPs is questionable. The objectives of this work are (i) to study the short-term uptake and accumulation of PAHs into zooplankton by diffusive uptake and feeding on phytoplankton, (ii) to quantify accumulation potential of PAHs in zooplankton, fecal pellets and eggs, and (iii) to parametrize the dynamics of PAHs in zooplankton.
Materials and Methods Experimental Design. Three types of incubation experiments were conducted to assess the accumulation routes and cycling of PAH in zooplankton: (A) one containing a culture of a phytoplankton species, Rhodomonas salina (2 L; 300 000 cells/mL); (B) a second one containing adults of the copepod Paracartia (acartia) grani without phytoplankton (10 L, 4000 adults), and (C) a third one containing adults of P. grani (10 L, 4000 adults) fed with R. salina (300 000 cels/mL). The incubations took place in Pyrex glass (with Teflon caps) bottles filled with 0.5 µm filtered seawater and incubated for 48 h at 20 °C in an 12 h:12 h day/night cycle. In experiments C phytoplankton levels were selected to ensure that the bulk of algae were fully grazed at the end of 48 h exposure treatments (19, 20). Each one of these three experimental treatments consisted of two replicates spiked with a mixture of the selected PAH (spiking level between 15.3 and 0.5 µg/L, Supporting Information, Annex I) and two replicated controls with and without PAH. Individual PAHs within the mixture were dosed proportionally to their log Kow and below toxic levels to ensure the lack of any effect of the mixture on copepod swimming and grazing behavior and guarantee VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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copepod survival (21, 22) to maximize egg and fecal pellet production. Water samples were taken at the beginning and at the end (48 h) of the experiment. At t ) 48 h copepods were separated from water with fecal pellets, eggs, and debris using a 150 µm mesh sieve and placed in preweighed and precombusted (350 °C, 12 h) 47 mm Whatman GF/F glass fiber filters. Phytoplankton or the mixture of fecal pellets and eggs were then separated from water by filtration (Whatman GF/F) in incubations A and B. In incubation experiment C, the complete absence of algae was corroborated microscopically at the end of the incubation, and then fecal pellets, eggs, and debris were filtered onto Whatman GF/F. Filter samples were frozen (-18 °C) until analysis, whereas filtered water samples were kept at 4 °C and extracted later within 5 days. Chemical Analysis. Dissolved and particulate phase and copepods were analyzed for nine low molecular weight PAHs (methylnaphthalene, dimethylnaphthalene, fluorene, dibenzonthiophene, phenanthrene, methylphenanthrene, dimethylphenanthrene, fluoranthrene, pyrene). A mix containing three deuterated PAHs (acenaphthene-d10, phenanthrened10, and crysene-d12; Sigma-Aldrich) was used as surrogate. Water samples were analyzed using a slight modification of the method described by Martinez et al. (23). Briefly, 500 mL of each sample was spiked with 100 ng of the surrogate standard, preconcentrated in solid-phase C18 extraction cartridges (500 mg, Merck) using a Baker vacuum system, dried under vacuum, and then eluted with 50 mL of hexane: dichloromethane (1:1, v/v). Before the preconcentration step, SPE cartridges were conditioned with 5 mL of hexane followed by 5 mL of propanol and 5 mL of HPLC grade water containing 2% of propanol. The extract was concentrated to 0.5 mL by vacuum rotary evaporation, transferred to a 1.7 amber vial with hexane, and evaporated to 400 µL under a nitrogen stream. At this step, 100 ng of the internal standard anthracene-d10 was added to the extract. Filters were freeze-dried during 24 h and Soxhlet-extracted with hexane/dichloromethane (1:1, v/v) for 24 h. Extracts were rotary evaporated, purified on a column filled with 1-2 g of anhydrous sodium sulfate over 5 g of silica gel (silica 60, 200 mesh, activated at 250 C° for 24 h) and 3 g of 3% deactivated neutral alumina (aluminum oxide 90, activated at 250 °C for 12 h), and eluted with 60 mL of hexane/ dichloromethane (2:1, v/v) (24). The extract was concentrated to 0.5 mL by vacuum rotary evaporation, transferred to an amber vial, and processed as indicated for water samples. PAH analysis was conducted by gas chromatography coupled to a mass spectrometer and quantified with the internal standard procedure. The system was operated in electron impact mode (EI, 70 eV), and the injection was performed in the splitless mode. The separation was achieved with a 30 m × 0.25 mm i.d. × 0.25 µm TRB-5MS capillary column (Teknokroma, Spain). The oven temperature was programmed from 90 °C (holding time 1 min) to 175 at 6 °C/min (holding time 4 min) to 235 at 3 °C/min and finally to 300 at 9 °C/min, keeping the final temperature for 5 min. The analytical procedure for both water samples and filters was validated by determining the recovery rates. With this objective, filters with algae and copepods and ultrapure water were spiked with a solution containing 100 ng of PAH and were processed in the same way as samples. The observed good recoveries for all the cases (above 80%) and good reproducibility (between 4% and 6%) for both the dissolved phase and filter samples prove the suitability of the analytical method. Moreover, procedural blanks were prepared with each series of water and filter samples. Concentrations of PAH in algae and copepods exposed to the PAH mixture were 10-100 fold higher than those grown in clean seawater. 2296
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Control conditions (water, phytoplankton, and zooplankton) contained negligible concentrations of PAH.
Results and Discussion PAH Bioconcentration in Phytoplankton. Because of the large surface/volume ratio of phytoplankton and their high organic carbon content, freely dissolved PAH can be bioconcentrated by diffusive sorption (4, 5). The bioconcentration factor (BCF, L kg-1) describes the tendency of a chemical to accumulate passively in aquatic organisms and is given by: BCF ) CP/CW
(1)
where CP (ng kg-1) is the PAH concentration in the organism, and CW (ng L-1) is the PAH concentration in the dissolved phase determined as the initial PAH exposure levels. Figure 1A shows the significant (p < 0.01) log BCF-log Kow correlations for bioconcentration of PAHs in phytoplankton (see Annex II in Supporting Information for potential influence of the colloidal phase on CW). The BCF for phenanthrene measured here for Rhodomonas salina is not significantly different than those reported for Thalassiosira weissflogii, Dunaliella tertiolecta, and T. pseudonana (6). PAH Accumulation in Zooplankton. The same passive uptake partitioning is expected in treatments B, which contain copepods without food (phytoplankton) (25-27). Indeed there was a significant correlation between BCF and Kow values of PAHs when only diffusive uptake occurs (Figure 1, p < 0.01). There are no previous reports of PAH accumulation in copepods, but previous laboratory experiments with the freshwater cladoceran Daphnia pulex exposed to PAHs with Kow ranging from 3 to 6 gave similar BCF values (28). Figure 2 shows that BCFs of PAHs in Paracartia grani are from 2 to 3 orders of magnitude lower than BCFs in phytoplankton (Rhodomonas salina). These results agree with previous findings (9), where a study of the bioaccumulation of radiolabeled PCB and PBDE reported significantly higher values in two species of phytoplankton than in the copepod Calanus finmarchichus. Bioconcentration differences between phytoplankton and zooplankton can be assessed by evaluating the mass balance of PAHs in zooplankton, which is given by: dCzoo/dt ) kuCW+kfoodCW - kdCzoo - kegCzoo - kmetCzoo (2) where Czoo is the PAH concentration in zooplankton (ng kg-1), ku is the uptake constant due to diffusive uptake (m3 kg-1 d-1), kfood is the uptake constant due to feeding on phytoplankton (m3 kg-1 d-1), kd is the diffusive depuration constant (d-1), keg is the depuration due to egestion of fecal pellets and laying of eggs (d-1), and kmet is the depuration due to metabolism of PAHs (d-1). The positive terms in eq 2 are the inputs of PAHs to the organism, while the negative terms are the output fluxes from the organism to the dissolved phase. Therefore, bioaccumulation factors (BAF, L kg-1), under steady-state conditions (see Annex III in Supporting Information), are given by: BAF ) (ku+kfood)/(kd+keg+kmet)
(3)
The BCF is a particular case of BAF when there are no dietary sources (kfood ) 0). Therefore, BAF for zooplankton are lower than for phytoplankton because of the importance of egestion and metabolism processes or higher depuration rates. A different lipid composition between phytoplankton and zooplankton could explain only a small fraction of the differences in accumulation factors. The accumulation of PAH in zooplankton due to dietary uptake can be assessed using experiments C, where both zooplankton and phytoplankton were exposed to PAHs. Figure 1C shows that there is a significant correlation between
For example, Fisk et al. (27) measured HCH and PCB concentrations in samples of Calanus hyperboreus (Arctic marine zooplankton) and found a linear correlation between BCF and Kow for those compounds (slope ) 0.72). Hoekstra et al. (29) described a significant linear correlation between BAF and Kow (slope ) 1.04) for organochlorine contaminants with a log Kow 3 to 6 in Calanus hyperboreus from the Arctic. Similar relationships were reported for Arctic and North Pacific marine zooplankton (30, 31). All these studies suggested partitioning as the dominating process. Conversely Berglund et al. (32) found similar slopes (0.75) for PCB BAFs of freshwater zooplankton (dominated by Daphnia ssp. and copepods) predominantly fed by phytoplankton. This work suggests that POP concentration in zooplankton could be predicted by direct partitioning and equilibrium with the dissolved phase as has been shown for PCBs (8). Still, the lack of significantly higher BAF than BCF does not mean that PAH could not be transfered from food to the organism, but that depuration and other elimination processes are fast enough so the final PAH levels in the organism are dominated by water-zooplankton partitioning and/or metabolism. In fact, as estimated below, these re-equilibration factors are very fast. PAH Accumulation in Eggs and Fecal Pellets. POPs can be eliminated by egg production or/and the egestion of fecal pellets. PAH partitioning between eggs-fecal pellets and dissolved phase (KP, L kg-1) is given by: KP)CFP/CW
FIGURE 1. Bioconcentration factors (BCF) for treatments with Rhodomonas salina (BCFA1 and BCFA2) and Paracartia (Acartia grani) (BCFB1 and BCFB2) on its own and bioaccumulation factor (BAF) for treatment made with P. grani in presence of Rhodomonas salina (BAFC1 and BAFC2) for each compound related to the octanol-water partitioning coefficient (Kow). Note that in Figures 1-3, units for BCF and BAF are in L kg-1, in order to be compared to KOW, and not in m3 kg-1 as used in the manuscript equations. BCFs and Kow values (p < 0.01). In experiments C, in addition to accumulation due to feeding on phytoplankton, there will also be diffusive uptake. In fact, PAH BAF and BCF values for copepods are not statistically different (p < 0.01, based on t tests comparing BAF and BCF of each studied PAH; Figure 2). Therefore, even if copepods were exposed to contaminated phytoplankton in addition to dissolved PAHs, this did not result in higher concentrations of PAHs in them (see Annex IV in Supporting Information for an assessment of biomagnification potential). This is consistent with other studies that have reported a near 1:1 relationship for BCFKow for POPs in small herbivorous zooplankton (9, 26, 27).
(4)
where CFP is the PAH concentration in the fecal pellets and eggs (ng kg-1). It is important to consider that only females produce eggs, that under comparable conditions, Paracartia grani produce approximately the same amount of eggs and fecal pellets (22), and that eggs and fecal pellets have similar sizes (33, 34). Specifically, in our experiment, only 60% of the copepods were females. This means that it is reasonable to assume a 40% greater contribution in weight of fecal pellets than eggs. Nevertheless, differences in lipid content are likely to occur between fecal pellets composed mainly of undigested algae (35) and eggs that are known to be rich in lipids (36). Unfortunately, our experimental set up was unable to separate between these two fractions. Figure 3 shows the correlation of KP versus Kow for experiments B and C. There is a highly significant positive relationship between KP and Kow for PAHs in eggs-fecal pellets (R2 ) 0.89; p < 0.01) of copepods fed with the algae (treatment C, Figure 3). The KP-KOW relationship is also statistically significant for the treatments with copepods exposed to PAH by only diffusive uptake (treatment B, R2 ) 0.7; p < 0.05), even though the concentrations are very low. Unlike copepods fed with Rhodomonas salina, P. grani without food do not produce a significant amount of eggs nor fecal pellets (22). Nevertheless, because of the experimental setup, during the first 1-2 h of exposure without food, the copepod may have produced low quantities of eggs and fecal pellets due to its previous feeding history since they came from bulk cultures with algae. Contrarily, PAHs concentrations are 10 to 100 fold higher in fecal pellets when P. grani was grazing on R. salina than passively from the surrounding waters (Figure 3 and Annex V in Supporting Information, Figure V.1). A potential artifact in these results would be particle-water repartitioning after egg or fecal pellet production. If this was significant, then KP values would be similar for the two sets of experiments, which it is not. This suggests that fecal pellets contain organic material not easily available from the dissolved phase, thus leading to slow particle-water partitioning. This would also explain the high relevance of fecal pellets as a transport vector of organic pollutants in the marine environment (14). PAH Processing in Zooplankton. Equation 2 provides the framework for evaluating the cycling and processing of VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Comparison between BCF and BAF values for different treatments: (A) a culture of Rhodomonas salina and two treatments made in presence of zooplankton (B) with or (C) without phytoplankton. Phe: phenanthrene; MNaph: methylnaphthalene; DMNaph: dimethylnaphthalene; Fluo: fluorine; DBT: dibenzothiophene; Phe: phenanthrene; MPhe: methylphenanthrene; DMPhe: dimethylphenanthrene; Flth: fluoranthrene; Pyr: pyrene.
FIGURE 3. Fecal pellets-eggs water partition coeficient (KP) values for treatments containing (i) P. grani. alone (KpB) (ii) P. grani fed with Rhodomonas salina (KpC1 and KpC2), for each compound related to the octanol-water partition coefficient (Kow). It also includes BCF values for treatments with Rhodomonas salina (BCFA1 and BCFA2) for each compound versus the octanol-water partitioning coefficient (Kow) for comparison purposes. POPs in zooplankton and estimation of the different parameters appearing in this equations is performed here. The losses by metabolism can be estimated by a comparison of the initial and final mass balance of PAHs under the presence or absence of copepods, for phytoplankton incubation experiments and controls (see Annex I in Supporting Information). For the control, there was a loss of 11% on average of PAHs in water at the final time, compared to the total initial inventory (Annex I). These losses can be related to a number of factors such as volatilization, adsorption to glass walls, photodegradation, and other unknown processes. It is assumed that these losses also occur in the other treatments. Therefore, the 30% losses in the PAH inventory observed for the phytoplankton incubations suggest that there is some extra PAH degradation, accounting for a 19% of the PAH mass, which may be due to microbial degradation (37) and/or oxidation triggered by radicals from phytoplankton (38). When copepods were present (with and without phytoplankton), an extra 45% loss of PAHs was found, attributed to possible metabolism by zooplankton or the microbial communities associated to zooplankton (39). Indeed, Harris et al. (12) reported that 55-77% of naphthalene 2298
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retained in the copepod Calanus helgolandicus exposed over a period of 4-6 days was metabolized, and Lee (40) found that copepods were able to convert parental PAHs to hydroxylated derivatives as fish do. In addition, Walters et al. (41) detected the activity of AHH (aryl hydrocarbon hydroxylase) involved in the metabolism of petroleum hydrocarbons in cell-free extracts of mixed zooplankton (two species of calanoid copepod and mixed microplankton). The metabolism rate can thus be estimated from the losses in the incubations (see Annex VI in Supporting Information) and are depicted in Table 1. The uncertainties associated with these rates were estimated from the different replicates. The cycling of PAHs in copepods depends as well on the other processes depicted in eq 2. Keg can be estimated from the concentrations measured in eggs and fecal pellets (Table 1 and see Annex VI in Supporting Information for estimation method). This confirms the capability of copepods to eliminate effectively ingested PAHs through eggs and fecal pellets. Harris et al. (11) found that 39-42% of ingested C-14 naphthalene was released into fecal pellets. Furthermore, the existence of intact or even live phytoplankton cells in fecal pellets of zooplankton of different species suggest that
TABLE 1. Uptake and Elimination Rate Constants of PAHs in copepodsa MNaph
DMNaph
Fluo
DBT
Phe
MPhe
DMPhe
Flth
Pyr
kfood (m3 kg-1 d-1) 0.24 ((0.03) 1.23 ((0.09) 1.21 ((0.01) 2.63 ((0.71) 5.39 ((0.89) 31.20 ((9.57) 142.57 ((39.04) 115.5 ((21.87) 127.32 ((43.86) (n ) 2) keg (d-1) (n ) 2)
1.67 ((0.15) 6.58 ((0.05) 10.76 ((2.26) 5.45
K*met (d-1) (n ) 3)
0.02 ((0.06) 0.23 ((0.04) 0.55 ((0.10)
0.33 ((0.02) 0.42 ((0.02)
Kmet (d-1)
89
10.34 ((1.69) 21.72 ((1.65) 25.85 ((2.05)
19.89 ((1.89)
18.17 ((1.46)
0.62 ((0.06) 1.06 ((0.11)
1.02 ((0.04)
1.05 ((0.04)
616
2800
238
227
119
200
299
211
ku (m3kg-1 d-1) 0.79
7
15
10
33
59
91
142
138
kd (d-1)
73
68
60
167
366
284
518
369
11
a
Phe: phenanthrene; MNaph: methylnaphthalene; DMNaph: dimethylnaphthalene; Fluo: fluorine; DBT: dibenzothiophene; Phe: phenanthrene; MPhe: methylphenanthrene; DMPhe: dimethylphenanthrene; Flth: fluoranthrene; Pyr: pyrene. K*met corresponds to the overal metabolism constant for the incubation. Uncertainty is given as standard deviation and were estimated from variability between the various experiment replicates. Kmet, ku, and kd have been calculated with the average value of the rest of the constants, and they have an uncertainty of a factor of 2.
under conditions of abundant food, zooplankton has a low efficiency of cycling the organic matter ingested, thus not accumulating PAHs (42). Under feeding conditions, metabolism is increased and so is the production of fecal pellets, which will both induce a higher elimination of PAHs. In the present experiments, the KP values of Figure 3 are in fact for a mixture of fecal pellets and eggs. It is also likely that under abundance of food, there is a higher production of eggs, and this would also induce an enhancement of the depuration efficiency of PAHs. Because we know the amount of phytoplankton cells that were ingested we can calculate Kfood: kfood ) BCF × I
(5)
where I (kg phytoplankton kg-1zooplankton d-1) is the ingestion rate for zooplankton, that is the phytoplankton biomass consumed per day by the zooplankton biomass present in the experiments, which in our experiment is 4.7 kg phytoplankton kg-1 zooplankton d-1assuming that half of the algae present was consumed daily and an algae and copepod individual weight of 0.055 ng C/cell and 4.4 µg C/cop, respectively (19, 20, 43). In other conditions of abundance of food, kfood will have different values. These consumption rates are higher than the typical daily rations reported for Paracartia grani in nature, but in the upper range of values found in the laboratory (44). The term kfoodCW in eq 2 gives the amount of PAHs that are introduced in the copepods. The efficiency of this uptake will be given by the difference of this term and the depuration terms. The other two parameters that appear in eq 2 are ku and kd, that can be obtained by applying eq 2 to the two incubation experiments (B and C) with copepods (with and without phytoplankton) as explained in Annex VII of Supporting Information. This leads to two equations with two variables the solutions of which are (see Annex VII): kd ) (kfood - BAFkeg)/(BAF - BCF)
(6)
ku)BCFkd+k′met/Bzoo
(7)
The obtained results of ku and kd for all individual PAHs are shown in Table 1. The uncertainty in ku and kd are of the order of two due the uncertainty of the other rate constants, of the zooplankton biomass, and of the other parameters used when estimating these rate constants. In any case, with kd values on the order of hundreds for most PAHs, the response time of zooplankton to varying dissolved phase
concentrations is on the order of minutes, which is a very fast process. For modeling purposes, an uncertainty of a factor of 2 is not relevant, because it will be in any case a very fast process, with virtually instantaneous equilibration times. The comparison of all the elimination rate constants (kd, keg, kmet) shows that diffusive depuration and metabolism dominates as a loss term. However, depuration just returns the chemical to the dissolved phase, and thus the high values found for kd show that there is a very active cycling of PAHs between copepods and the surrounding waters. Conversely, egestion of fecal pellets will contribute to the marine sink of PAHs to the extent that they are capable of efficiently settling in the water column (14, 15) and have not been recycled in the photic zone. Finally, metabolism eliminates the PAHs from the environment and can thus be considered as an important sink, as evidenced for the first time in this study. This finding is corroborated by the fact that copepods do have PAH metabolizing capabilities as fish have (10, 11). Although biotransformation has been less studied in aquatic invertebrates than in other superior animals, some studies have been conducted in this direction, investigating the activity of cytochrome P450 (CYP) monooxygenases, which are the main enzymes responsible for the oxidative metabolism of PAH and other aromatic compounds (45). The depuration and eggestion rates are higher for the more hydrophobic compounds, while metabolism is faster for the lower molecular weight PAHs (see Annex VIII in Supporting Information). Implications of Zooplankton on PAH Cycle and Fate in the Marine Environment. This study shows that zooplankton may have important implications for the PAH environmental cycling and fate. Zooplankton, fecal pellets, and eggs are important vectors for the transport of PAHs and other POPs in the marine environment (13-15). The difference in KP values for concentrations of PAHs in fecal pellets depending on whether PAHs are accumulated diffusively or from phytoplankton suggests that the marine vertical fluxes associated with fecal pellets will depend strongly on zooplankton trophic status, an issue that will require further research in the future. Cycling in the photic zone plays an important role in PAH sink and fate. Tsapakis et al. (46) showed that sinking fluxes out of the photic zone account for only 10% of atmospheric inputs, and therefore 90% of PAHs disappear in the upper water column due to unknown processes. Metabolization of PAHs by zooplankton found here suggests that it can be an VOL. 43, NO. 7, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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important sink of PAHs in the ocean, the extent of which will require further studies. In addition, it will also be important to elucidate more quantitatively the role of zooplankton contribution to the vertical fluxes of PAHs and other POPs out of surface waters. This transport can be due to the settling of fecal pellets (14), but a non-negligible fraction of fecalpellet-bound PAHs will be recycled in the photic zone because fecal pellets degrade rapidly within the mixed layer (47) because of microbial decomposition and cropophagy (42). Active transport of POPs by zooplankton has not been studied so far, even though it has been suggested as a significant process in POP cycling (48). Especially important would be to elucidate the coupling of zooplankton diurnal vertical migration with PAH transport. During night time, PAHs would accumulate in zooplankton at surface waters, and will be released at deeper waters during the day due to partitioning to low concentration deep seawater and excretion processes. This active transport is feasible due to the fast water-zooplankton partitioning as shown here. It is now accepted that POP dynamics is closely related to the organic carbon cycle (49), where zooplankton plays an important role. The work described here opens the door to further field, laboratory, and modeling studies which should allow delimitating the apparent important role that zooplankton play in POP cycling and sinks.
Acknowledgments N.B. acknowledges a predoctoral fellowship from the Basque Autonomous Government. This research was funded by the Spanish Ministry of Education and Science project Petrozoo (VEM2003-20037) to A.C., and by the European commission through the THRESHOLDS (003933-2) to J.D. Dr. Roser Chaler and Dori Fanjul are acknowledged for GC-MS assistance.
Supporting Information Available PAH concentrations for the different experiments (Tables S1 and S2). Details of the estimations methods of BCF, BAF, keg, kmet, ku, and kd and discussion of uncertainty. This information is available free of charge via the Internet at http:// pubs.acs.org.
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