Dissolved Organic Matter Enhances Transport of PAHs to Aquatic

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Environ. Sci. Technol. 2009, 43, 7212–7217

Dissolved Organic Matter Enhances Transport of PAHs to Aquatic Organisms THOMAS L. TER LAAK,* MARTIN A. TER BEKKE, AND JOOP L. M. HERMENS Institute for Risk Assessment Sciences (IRAS), Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands

Received December 28, 2008. Revised manuscript received March 11, 2009. Accepted March 13, 2009.

In this study, the uptake of pyrene and benzo[b]fluoranthene by an aquatic worm (Lumbriculus variegatus) and a poly(dimethylsiloxane) coated glass fiber was studied at different humic acid concentrations. The accumulation of pyrene was not affected by the presence of the humic matrix. However, the accumulation rate of benzo[b]fluoranthene increased a factor of 3 for the fiber and a factor of 4 when 55 mg L-1 dissolved organic carbon was added in the form of humic acid. The difference between the two chemicals can be explained by the higher affinity of benzo[b]fluoranthene for the dissolved humic material. A comparison of modeled transport enhancement of benzo[b]fluoranthene by humic acid and the experimental results suggested that the benzo[b]fluoranthene complexed with the humic phase was not completely labile.

Introduction Knowledge on transport kinetics of hydrophobic organic chemicals between organisms and the aqueous phase is essential for the correct interpretation of bioaccumulation studies, toxicity tests, and exposure of organisms in the field. Jonker and van der Heijden nicely illustrated how insufficient equilibration time of aquatic worms exposed to very hydrophobic organic chemicals can lead to underestimation of bioaccumulation factors (1). Understanding transport mechanisms of chemicals between aqueous organisms and their environment (2, 3) and elucidating routes of uptake (4) can help interpreting and designing bioaccumulation and toxicity experiments, interpret data, model bioaccumulation, use passive samplers as biomimetic tools, and estimate effects of dynamic exposure concentrations (5-7). The transport of chemicals between aqueous organisms and their aqueous environment can be limited by the aqueous phase. If the aqueous phase limits the transport of chemicals between organisms and their environment, a concentration gradient will occur in the aqueous layer near the organism. The layer in which this gradient occurs is the aqueous diffusion layer (8). Aqueous diffusion layers can contain dissolved and colloidal organic and mineral materials. These materials can enhance the transport of hydrophobic organic chemicals over these layers by associating with the chemical where its chemical activity () free concentration) is high, diffuse, and releasing the chemical where its activity is low (9, 10). Theoretically, a dissolved or suspended material will * Corresponding author current address: KWR Watercycle Research Institute P.O. Box 1072, 3430 BB Nieuwegein, The Netherlands; phone: +31 (0)30 60 69 657; fax: +31 (0)30 60 61 165; e-mail: [email protected]. 7212

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only contribute to the transport of chemicals between the aqueous environment and an organism (1) if the aqueous diffusion layer in the aqueous phase limits the transport process, (2) if a substantial part of the chemical is associated with this dissolved or suspended material, and (3) if the complexes are labile to a certain extent (11). The lability of a complex is defined by association and dissociation kinetics in relation to its residence time in the aqueous diffusion layer (12, 13), and can range from inert to completely labile (12). Inert complexes can not contribute to the transport of chemicals between an aqueous phase and a sorbent because the association and dissociation kinetics is too slow in relation to the residence time of chemicals and complexes in the diffusive boundary layer. Contrastingly, the association and dissociation kinetics of fully labile complexes is instantaneous in relation to the residence time in the diffusive boundary layer. Consequently, these complexes maximally contribute to the transport of chemicals between an aqueous phase and a sorbent. Various studies have shown that the presence of dissolved and suspended materials in an aqueous phase enhance the transport of hydrophobic organic compounds between water and passive samplers (9, 10, 13-17). Similar phenomena are expected for aqueous organisms. However, studies on these passive diffusion-driven transport phenomena with biota are scarce and generally focused on the accumulation of metals (18-20). This study investigates the effect of humic acid on the accumulation of two hydrophobic chemicals (pyrene and benzo[b]fluoranthene) by an aquatic worm and a passive sampler. The chemicals were applied to the test system by passive dosing (21) using a silicone sheet spiked with the test chemicals. The objective of this research is to (1) study how the uptake of the chemicals into worms and passive samplers is affected by the humic acids, (2) compare the accumulation of the chemicals by the fibers and organisms, (3) relate the effect of humic acid on transport to the affinity of two chemicals for humic acid, and (4) explain the effect of humic acid on the transport of the chemicals with a theoretical transport model.

Experimental Procedures Chemicals, Samplers, Solvents, and Organisms. Pyrene (99%), benzo[b]fluoranthene (98%), and humic acid sodium salt were purchased at Sigma Aldrich Chemie BV (Zwijndrecht, Netherlands), and benzo[k]fluoranthene (99.5%) was purchased at Dr Ehrenstorfer GmbH (Augsburg, Germany). A fiber with a 99.75 µm poly(dimethylsiloxane) (PDMS) coating (66.67 µL PDMS m-1) and a glass core with a diameter of 113 µm was obtained from Poly Micro Industries (Phoenix, AZ). Solvents (n-hexane, acetone, acetonitrile; LaboratoryScan, Dublin, Ireland) were of Pestiscan grade, and 500 µm thick silicone sheets were purchased at Altec (http:// www.altecweb.com). Highly pure water (R g 18 MΩ) was prepared by a Millipore water purification system, equipped with organic free kit (Millipore Waters, Amsterdam, The Netherlands). The aquatic worm Lumbriculus variegatus was cultured at a temperature of 23 ( 1 °C in 45 L flow-through aquaria containing cellulose substrate and tap water. The organisms were fed with flake fish food (King British, UK) once a week. Before testing, organisms were left for 16 h under running tap water to empty their guts. Sheet-Water Partition Coefficients. Six silicone sheets (0.6 × 4.0 cm, 151 ( 4 mg) were washed three times with acetone, acetonitrile, and Millipore water before they were loaded with 29.3 ( 0.4 mg L-1 pyrene and 29.7 ( 0.3 mg L-1 10.1021/es803684f CCC: $40.75

 2009 American Chemical Society

Published on Web 04/13/2009

FIGURE 1. Experimental setup in a 40 mL vial with a 32 cm2 silicone sheet loaded with pyrene and benzo[b]fluoranthene, 5 worms (L. variegatus), and a 3.0 cm long PDMS coated fiber. benzo[b]fluoranthene by exposing them for 7 days to 5 mL spiked acetonitrile-Millipore water solution (1:1). Loaded sheets were shortly rinsed with Millipore water, placed in 40 mL vials (Supelco, Bellefonte, CA) containing 30 mL Millipore water with 50 mg L-1 sodium azide as bacterial inhibitor (Merck, Amsterdam, The Netherlands), and equilibrated (3) for 40 d on a roller mixer (Stuart SRT9D, Warrenville, IL) at 20 ( 1 C°. After equilibration, the sheets were extracted in 20 mL of acetonitrile for 5 days. Extraction recoveries using this volume ratio of acetonitrile and silicone rubber were considered 100% (3, 22). Aqueous samples of 20 mL were extracted according to ref (3) with the difference that the concentrated hexane extract is converted into acetonitrile. Extraction recoveries of PAHs from pure water with a bactericide were considered 100% (22). Kinetic Experiment. Humic acid sodium salt (250 mg L-1) was dissolved in filtered (0.45 µm) tap water, alkalinized to pH 10.1 (1.0 M KOH, Merck, Darmstadt, Germany), ultrasonicated for 45 min, stored overnight, and filtered at 0.45 µm to remove particulate humic material. The filtrate was acidified to pH 7.8 (1.0 M HCl, Merck, Darmstadt, Germany). The final dissolved organic carbon (DOC) content in the stock solution was 76.5 mg L-1 (Shimadzu TOC-5050A analyzer). Forty silicone sheets of 4 × 8 cm (1.98 ( 0.07 g) were washed, loaded, and rinsed as described for the 0.6 × 4.0 cm silicone slices above, with the difference that the volume of the spiking solution was 200 mL. Final concentrations in the sheets were 36.1 ( 0.4 mg L-1 pyrene and 21.7 ( 0.1 mg L-1 benzo[b]fluoranthene. After washing, the sheets were placed in 40 mL vials as a passive dosing phase. Subsequently, 30 mL of tap water with 0, 20.9, and 76.5 mg L-1 DOC was added to the vial (later referred to as 0, low, and high DOC), and the system was incubated for 24 h. After 24 h, 5 worms (L. variegatus) and one 3.0 cm long fiber were added to each test vial (Figure 1). The sheets were positioned at the top of the vial (75% of the sheet was submerged) to avoid direct contact with the worms and fibers exposed in the vials. The experiment was performed at 20 ( 1 C°. After exposing the worms in solution, actual DOC concentrations were 4.5 ( 0.1, 17.4 ( 2.4, or 54.9 ( 3.4 mg L-1 for the 0, low, and high DOC treatments, respectively. Passive dosing sheets were extracted in 20.0 mL acetonitrile. Extraction recoveries were 98.4 ( 0.1 and 99.1 ( 0.1% for pyrene and benzo[b]fluoranthene, respectively. The worms and fibers were sampled after 1, 2, 4, 8, 17, 31, 55, 96, 144, 240, 386, and 530 h exposure. One vial was sampled for every DOC treatment/exposure time combination, except for the 530 h exposure where two vials were sampled per treatment. Fibers were removed from the test vials, gently wiped with a piece of prewetted tissue, and extracted in 1000 µL of acetonitrile containing 10 µg L-1 benzo[k]fluoranthene as injection standard. The sampled worms were gently blotted dry with a tissue, weighed, and frozen at -20 °C until extraction. The average wet weight of

the worms was 4.6 mg, the dry fraction was 23.0 ( 2.1%, and the lipid content of the worms was 2.63 ( 0.07% on a wet weight basis. The worms were extracted by adding 0.5 mL of 1 M KOH solution and 1.2 mL of N-hexane. After ultrasonication (45 min) and shaking by a roller mixer, the vials were centrifuged for 10 min at 1000 rpm. Subsequently, 0.5 mL of the supernatant (n-hexane) was added to 1.0 mL of acetonitrile and the n-hexane was evaporated in the fume hood. Furthermore, 0.2 mL of acetonitrile containing 10 µg L-1 benzo[k]fluoranthene was added as injection standard. The recoveries of this extraction procedure were 93.9 ( 5.0% for pyrene and 88.4 ( 4.5% benzo[b]fluoranthene. Concentrations were corrected for these extraction efficiencies. The oxygen content was measured twice a week (thereby allowing fresh air into the headspace). The average oxygen saturation was 79 ( 8% for all treatments. Furthermore, mucus was removed from the vials and the fibers were wiped with a piece of tissue after 7 and 15 days, because this mucus (probably gut material from the worms) can be eaten by the worms and foul the fiber-surface, thereby affecting uptake rates of the chemicals (23, 24). The pH was determined after all exposure times. A pH drop from 7.80 ( 0.02 to 7.27 ( 0.05 was observed during the first 2 days for all treatments, because the solutions were not buffered. Aqueous samples for the determination of pyrene and benzo[b]fluoranthene were taken at the end of 1, 4, 8, 386, and 530 h exposure. Volumes of 5.0, 1.0, and 0.5 mL were sampled for the 0, low, and high DOC solutions, respectively. The aqueous samples were extracted with 1.0 mL of n-hexane. The rest of the extraction is identical to the procedure described in the section “Sheet-Water Partition Coefficients”. A separate recovery experiment showed that 90.7 ( 1.4% and 97.3 ( 2.3% was recovered at 0 DOC, 78.6 ( 7.7% and 87.8 ( 7.7% at low DOC, and 77.4 ( 4.6% and 83.2 ( 2.4% at high DOC solutions for pyrene and benzo[b]fluoranthene, respectively. Concentrations were corrected for their extraction efficiencies. Analysis. Concentrations in fiber extracts, worm extracts, aqueous extracts, and sheet extracts were determined by a HPLC system (22). Separation was performed at 35 °C using a Supelcosil LC-PAH column (Supelco, Bellefonte, CA, length 100 mm, internal diameter 3.0 mm, particles 5 µm) The chemicals were separated using a 80/20 acetonitrile/water mixture at a flow rate of 450 µL min-1. The excitation and emission wavelengths of pyrene were 270/390 nm, while benzo[b]fluoranthene and benzo[k]fluoranthene were detected at 270/435 nm.

Results and Discussion Passive Dosing. Maintaining constant concentrations of hydrophobic chemicals in aqueous solutions is cumbersome. Concentrations spiked to aqueous solutions by a carrier solvent often decrease during exposure, while generator column spiking is expensive and labor-intensive (25). The dosing sheets used in this study can maintain constant free aqueous concentrations in solution, provided that the potential transport rate from the sheets is far greater than the uptake rate by worms and fibers. A pilot study illustrated that 24 h incubation of the loaded sheets with the DOC solutions is sufficient to reach a constant total concentration in the medium (Figure 2). Furthermore 97.8 ( 1.4% of pyrene and 97.8 ( 1.2% of benzo[b]fluoranthene was recovered from the sheets after exposure. Additionally, the transport of chemicals from the sheets through the aqueous phase to the worms and fibers is most likely limited by the transport over the worm-water and fiberwater interface because the surface of the sheet is far greater (∼24 cm2, if only the submerged side that is facing the solution is considered) than the surface of the worms and fiber together (∼2.4 cm2). Consequently, the passive dosing VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Concentrations of pyrene (A) and benzo[b]fluoranthene (B) in medium as a function of time elapsed since the passive dosing sheet was inserted in DOC solutions at nominal DOC concentrations of 0 (circle), 5 (triangle), 20 (square), and 100 (diamond) mg L-1 DOC.

FIGURE 3. Uptake of pyrene by the fibers and worms. The square represents 0 DOC, the triangle represents low DOC, and the circle represents high DOC. The lines represent the fit of eq 2. method generates constant free aqueous concentrations in all DOC treatments during the exposure of the worms and fibers. This dosing method is far less labor-intensive than a generator column setup (26). Sheet-water partition coefficients were determined in a separate experiment. The partition coefficients between the silicone rubber and Millipore water (Ksw) and their standard deviations were 4.69 ( 0.01 for pyrene and 5.70 ( 0.05 (n ) 6) for benzo[b]fluoranthene. With these coefficients, concentrations in the silicone rubber sheets (Cs, mg L-1) can be used to determine freely dissolved aqueous concentrations (Caq, mg L-1) (26): Caq )

Cs Ksw

(1)

Accumulation of Pyrene. Figure 3 shows the uptake of pyrene by the PDMS coated fibers and the aquatic worms. Since the passive dosing resulted in constant freely dissolved concentration of 0.72 ( 0.02 µg L-1 (eq 1), a one-compartment model can be used to describe the accumulation of chemicals: C(t) ) Caq ·

ku ·(1 - e-ket) ke

(2)

Here the concentration in the PDMS or worm tissue in time (Ct, mg L-1) is a function of the free aqueous concentration, the uptake rate constant (ku, h-1), the elimination rate constant (ke, h-1) and time (t, h). The full curves in Figure 3 are the fits of eq 2 to the experimental data of pyrene. Rate constants of these fits are listed in Table 1. The kinetics and the equilibrium concentration of pyrene in the fibers did not statistically differ between DOC treatments. Apparently, the presence of DOC did not have a significant effect on uptake kinetics or free concentrations. The free concentration (0.72 ( 0.02 µg L-1) and average equilibrium concentration in the PDMS fiber coating (14.32 ( 0.43 mg L-1) revealed a PDMS-water partition coefficient of 4.30 ( 0.02, which is comparable to values in the literature (1, 27, 28). 7214

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The uptake kinetics of pyrene into the worms was also not affected by DOC and the equilibrium concentrations were similar as well. The weight normalized lipid content (flip, 2.63%) and the uptake and elimination rate constants obtained from fitting eq 2 to the data can be used to calculate the bioconcentration factor (BCF): BCF )

ku(worm) ke(worm) ·flip

(3)

The average log BCF for pyrene was 5.54 ( 0.08 (values obtained at individual DOC treatments are listed in Table 1). This value is similar to the BCF obtained by Jonker and Van Der Heijden (1) when using worms from the same culture and PDMS coated fibers to determine freely dissolved aqueous concentrations in the test system. Accumulation of Benzo[b]fluoranthene. Figure 4 shows the uptake of benzo[b]fluoranthene by the PDMS coated fibers and the aquatic worms. Both the concentrations in the fiber coating and worm did not come close to a steady state within 22 d exposure, so elimination rate constants and steady state levels could not be fitted with eq 2. However, since all dosing sheets contained the same concentration and generated an identical freely dissolved concentration of 0.043 µg L-1 (eq 1), equilibrium concentrations in the fiber coating and worm tissue are expected to be similar in the different DOC treatments (29). Subsequently, a reduced form of eq 2 can be used to model the accumulation of benzo[b]fluoranthene: C(t) ≈ Caq ·ku ·t

(4)

The freely dissolved aqueous concentration can be inserted in eq 4 to estimate the apparent uptake rate constant for the worm and the fiber. An estimation of the uptake rate constant by eq 4 is acceptable if the uptake is still in the linear phase, which means that the elimination from the worm and fiber is negligible compared to the uptake. This is the case if the concentration in the sampler or organism does not exceed ∼25% of the final equilibrium

TABLE 1. Kinetic Parameters and Distribution Coefficients of Fibers, Worms, and Humic Acida water and DOC -1

Cm (µg L-1)

Cm/Caq

Log KDOC (L kg-1)

0 DOC low DOC high DOC

0.72 (0.02) 0.72 (0.02) 0.72 (0.02)

0.73 (0.06) 1.89 (0.04) 4.59 (0.04)

1.02 (0.12) 2.64 (0.11) 6.40 (0.26)

4.97 (0.07) 5.00 (0.04)

0 DOC low DOC high DOC

0.043 (0.005) 0.043 (0.005) 0.043 (0.005)

0.064 (0.09) 1.49 (0.08) 5.22 (0.04)

1.50 (0.18) 34.2 (4.5) 12.0 (14.4)

6.27 (0.03) 6.34 (0.04)

Ceq (mg L-1)

ke (h-1)

ku (h-1)

13.96 (0.24) 14.80 (0.22) 14.21 (0.25)

0.0066 (0.0003) 0.0065 (0.0003) 0.0077 (0.0004)

Caq (µg L ) pyrene

benzo[b]fluoranthene

fiber

pyrene

0 DOC low DOC high DOC

benzo[b]fluoranthene

0 DOC low DOC high DOC

129 (5) 133 (4) 152 (6) 785 (1.9) 151 (5) 220 (16)

worm

pyrene

0 DOC low DOC high DOC

benzo[b]fluoranthene

0 DOC low DOC high DOC

Ceq (mg kg-1 ww)

ke (h-1)

ku (L kg-1 ww h-1)

Log BCF (L kg-1)

8.04 (0.29) 5.63 (0.29) 7.32 (0.51)

0.0058 (0.0006) 0.0076 (0.0011) 0.0094 (0.0020)

65.4 (4.5) 74.2 (11.2) 96.2 (17.4)

5.60 (0.02) 5.45 (0.02) 5.56 (0.03)

47.8 (1.4) 10.4 (3) 18.9 (5)

a Caq (the freely dissolved aqueous concentration) is calculated from the concentration in the silicone dosing sheet (eq 1). Cm (the medium concentration) is determined by extracting the medium. Cm/Caq ) 1 + KDOC DOC. KDOC values (sorption coefficients to dissolved organic carbon) are not shown for the 0 DOC treatments since the nature of the low DOC present in this system is unknown. Ceq is the equilibrium concentration, ke is the elimination rate constant, and ku is the uptake rate constant of the worm and fiber. The BCF (bioconcentration factor) was calculated with lipid-corrected equilibrium concentrations in the worms and Caq. Standard deviations (KDOC and Cm/Caq) or standard errors (Cm, Caq, Ceq, ke, ku, log BCF) are given between brackets.

level. However, in this study the equilibrium level could not be determined. Alternatively, uptake rate constants were determined for several specific time intervals (0-96, 0-144, 0-240, 0-384, and 0-530 h) and it was tested whether confidence limits (95%) of the fitted (eq 4) uptake rate constants of a particular time interval covered the fitted uptake rate constants of all shorter time intervals. The largest time interval where the uptake rate constant did not significantly differ from shorter time intervals was selected to determine the uptake rate constant. This procedure selected an exposure time interval of 0-144 h for the fibers exposed to the high DOC treatment, an interval of 0-240 h for the worms at the high DOC treatment and the fibers at the 0 and low DOC treatment, and an interval of 0-384 h for the worms in the 0 and low DOC treatment. The solid symbols are the selected data and the lines represent the fits (eq 4) through these data (Figure 4). The obtained uptake rate constants of these specific time intervals are listed in Table 1. Aqueous Diffusion Layers. The uptake rate constants of hydrophobic chemicals such as pyrene and benzo[b]fluoranthene are limited by diffusion through an aqueous diffusion layer (δw, m) (2, 3, 13). The thickness of this layer can be estimated from the uptake rate constant (ku, h-1), the area (A, m2), and volume (V, m3) of the worm or fiber and the diffusivity of the chemical in water (Dw, m2 h-1): δw ≈

ADw Vku

(5)

This equation considers a planar diffusion layer. If the diffusion layer is tubular shaped (e.g., around the cylindrically shaped fiber or worm), the calculated δw (eq 5) should be corrected (δw(corr), m) for the radius of the worm or fiber (R, m):

( ( ) )

δw(corr) ) R exp

δw -1 R

(6)

The aqueous diffusion layers of pyrene and benzo[b]fluoranthene can be calculated from their uptake rate constants (Table 1), dimensions of the fibers and worms, and aqueous diffusivities of the chemicals (eqs 5 and 6). The aqueous diffusivities of pyrene (4.1 × 10-10 m2 s-1) and benzo[b]fluoranthene (3.7 × 10-10 m2 s-1) are estimated from the diffusivity of phenanthrene (30) with a correction for molecular weight differences (eq 18.55 in ref (31)). If the worms are considered 3.0 cm long cylinders with a density of 1.0 L kg-1 and a volume of 4.6 µL, the obtained diffusion layer thicknesses of the fibers and worms are 334 and 362 µm for pyrene and 625 and 470 µm for benzo[b]fluoranthene, respectively. Diffusion layers of several hundreds of micrometers are expected for stagnant aqueous systems (32). The similar dimensions of the fiber and worm and their similar affinities for pyrene result in similar equilibration kinetics (Figure 3). Sorption to DOC. The organic carbon normalized sorption coefficient to the dissolved humic material (KDOC, L kg-1) can be calculated with the following equation: VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Uptake of benzo[b]fluoranthene by the fibers and worms. The square represents 0 DOC, the triangle represents low DOC, and the circle represents high DOC. The lines represent the fits of eq 4, only the solid data points are used for this fit.

FIGURE 5. Facilitation of transport of pyrene (A) and benzo[b]fluoranthene (B) in relation to the ratio of the bound and free concentration. Error bars represent standard deviations. The solid line illustrates the situation when DOC does not facilitate transport and the gray area illustrates the expected transport facilitation under the assumption of complete lability of the complexes. KDOC )

Cm - Caq Caq ·DOC

(7)

where DOC is the concentration of dissolved organic carbon in solution (kg L-1) and Cm is the total concentration of the chemical in the medium (both freely dissolved and complexed with humic acid). Since all parameters are known, the KDOC can be determined. The KDOC values of the low and high DOC treatment do not differ significantly, so the data of both treatments were pooled. The average KDOC values were 4.98 ( 0.05 for pyrene and 6.31 ( 0.04 benzo[b]fluoranthene, which is comparable to literature values (33). A concentration of 4.5 ( 0.1 mg L-1 organic carbon was found in the treatment without humic acid. However, since the source of this organic carbon is not Aldrich humic acid, but probably mucus and gut material from the worms, KDOC values were not determined for this phase. As discussed in the Introduction, the transport of a chemical can only be enhanced by humic material if aqueous diffusion layer limit the transport, a substantial part is associated with the humic material, and the complexes are labile. The accumulation of pyrene and benzo[b]fluoranthene in the PDMS and worm tissue are both limited by aqueous diffusion layers (34). The fraction that is complexed with the humic material can be calculated from sorption coefficients to DOC and DOC concentrations in solution (eq 7). The ratio of the complexed and free fraction of pyrene does not exceed a factor 6 at the tested DOC levels, while the ratio of the complexed and free concentration of benzo[b]fluoranthene is 33 and 120 at the low and high DOC concentration, respectively. If we consider that diffusivities of the freely dissolved organic chemicals are a factor 10-50 higher than the diffusivities of humic aggregates (31), complexed fractions of pyrene can only marginally contribute to the mass transport. Contrastingly, the larger complexed fractions of benzo[b]fluoranthene should be able to significantly contribute to the mass transport and affect the uptake rate constant. The maximum ratio of uptake rate constants with and without humic material (assuming fully labile complexes) 7216

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can be calculated from the concentration ratio of the freely dissolved and complexed chemical (i.e., DOC × KDOC) and the ratio of the diffusivities of the complex (Dcomplex) and the free chemical (Dfree) (11):

(

)

Dcomplex ku(+DOC) ) 1+ DOC·KDOC · ku(-DOC) Dfree

(

Dfree

Dfree + KDOC ·DOC·Dcomplex 1 + KDOC ·DOC

)

1/3

(8)

The gray area in Figure 5 shows the expected increase of the uptake with DOC of fully labile complexes (eq 8) under the assumption that diffusivities of the freely dissolved chemicals are a factor 10-50 higher than the diffusivities of the complexes (31). It can be observed that the uptake of pyrene will only be marginally affected by the addition of DOC, even if complexes are fully labile. Contrastingly, fully labile benzo[b]fluoranthene-DOC complexes will substantially affect uptake rates. Experimental uptake rates of benzo[b]fluoranthene fall below this gray area, suggesting that complexes are not fully labile. Additionally, uptake at high DOC deviates more from this zone of full lability than uptake at low DOC. Apparently, the lability decreases with increasing DOC concentration. This can be explained by the nature of the DOC at different DOC concentrations. Increasing DOC concentrations might result in larger humic aggregates (24). Larger aggregates have lower diffusivities (4, 23) which reduces their potential to facilitate transport of chemicals. Furthermore, increasing aggregate size might also affect the absorption and desorption kinetics of benzo[b]fluoranthene and DOC, thereby reducing the lability of benzo[b]fluoranthene even further. The enhancement of the uptake of benzo[b]fluoranthene is similar for the worm and fiber (Figure 5). This suggests that the same facilitated transport mechanisms determine the uptake of the passive sampler and organism. Nevertheless, a small but significant difference can be observed between

the worm and fiber at high DOC treatment. This difference might be related to the fact that especially at high DOC worm mucus and organic material from solution precipitated on the fiber surface. The fouling of this material might have increased the thickness of the diffusion layer and reduced the mass transfer of chemicals to the fiber. Environmental Relevance. In this study the aquatic worm Lumbriculus variegatus was exposed in the aqueous phase. We are aware that the normal habitat of these worms is in/ on the sediment surface. Additional exposure to sediment particles on the skin and gut wall of the worm will likely facilitate the transport of chemicals to an even larger extent (4, 35). Nevertheless, this study is focused on the facilitated transport phenomena generated by dissolved matrices in the aqueous phase. Therefore the observations are particularly relevant for pelagic species and passive samplers exposed in the water column.

Acknowledgments This work was funded by the European Union, ECODIS, Contract 518043. Barry Muijs is thanked for providing worm lipid contents.

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