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Hydrophobic organic contaminants (HOCs) are ubiquitous and constitute both an environmental and a human risk. It is well established that nominal or total measured concentrations of HOCs in soil are not sufficient to define exposure due to the numerous interactions between the HOCs and the heterogeneous soil matrix 1, 2. The freely dissolved concentration (Cfree) takes sorption into account and is therefore frequently perceived as the effective concentration for bioconcentration and toxicity 3–5, and the concept is clear and straightforward for aqueous media and sediments. However, it seems more challenging to define the exposure of HOCs in soils, particularly in unsaturated soils, where only limited free water is present or when the target organism mainly inhabits the interstitial pore space. In these cases, it might make better sense to define exposure in terms of chemical activity (denoted a) 2.
Chemical activity is a medium-independent exposure parameter that quantifies the energetic level, and not the concentration, of an HOC relative to its energetic level in a chosen reference state. The pure subcooled liquid of the HOC was chosen as the reference state (a = 1), and the chemical activity is then defined between 0 and 1 2. In this way, chemical activity takes into account losses and matrix interactions that can either increase or decrease the exposure. At equilibrium, chemical activity is equal in all compartments (e.g., asoil = aair = aorganism) in contrast to the concentration (e.g., Csoil ≠ Cair ≠ Corganism). Therefore, a difference in chemical activity between compartments determines the potential, direction, and extent of spontaneous physicochemical processes such as diffusion, partitioning, and chemical reactions 2. This implies that the diffusive bioconcentration of HOCs in small soil organisms is driven by a difference in chemical activity between the organism and the surrounding environment. The maximum chemical activity (amax) of a solid HOC can be estimated from its melting temperature (Tm, K) and the ambient temperature (T, K) according to Yalkowsky et al. 6, assuming the entropy of melting to be 56 J mol−1 K−1 (i.e., Walden's rule)
As seen from Equation 1, HOCs that are solid at a given ambient temperature have an amax below 1, and amax decreases with increasing melting temperature. Chemical activity (aHOC, dimensionless) can be related to the concentration (CHOC, mol L−1) via a compound- and medium-specific activity coefficient (γHOC, L mol−1) 7
Chemical activity is an established concept in physical chemistry and the basis for the equilibrium partitioning theory that is often used to link total soil and sediment concentrations to actual concentrations in organisms 8. More recently, various passive sampling techniques have been developed to measure chemical activity 2, 9, 10, and passive dosing techniques have been developed to control chemical activity in laboratory tests 11–13.
The exposure of soil organisms to HOCs often decreases with time due to sorption, aging, (bio)transformation, and other depletive processes 1, 14. Though many of these processes are environmentally relevant, they also lead to great difficulties in measuring and defining exposure when working with test systems containing soil. Passive dosing vials with silicone were therefore applied in the present study as a simple and practical method for providing well-defined and constant exposure. In passive dosing, a polymer is loaded with the test compound(s) and then applied to control HOC exposure by equilibrium partitioning without coexposing the test organisms to solvents. In this way, the exposure is kept constant since various losses are efficiently buffered by repartitioning from the polymer. Here, the test compounds were six polycyclic aromatic hydrocarbons (PAHs), and the test organism was the terrestrial springtail Folsomia candida, which inhabits the interstitial pore space of topsoil and is a standard test species 15. The applied passive dosing system has previously been used in F. candida toxicity studies of 10 PAHs, tested at their respective maximum exposure levels (i.e., their amax) 11.
The first aim of the present study was to determine the bioconcentration of naphthalene, anthracene, pyrene, benz[a]anthracene, and benzo[a]pyrene in F. candida. It was investigated whether stable internal PAH concentrations could be reached within the duration of a 7-day toxicity test. Equilibrium partitioning calculations were then applied to distinguish between thermodynamic equilibrium and steady state. The second aim of the study was to determine the springtail toxicity of naphthalene, phenanthrene, and pyrene and then relate this toxicity to various exposure parameters, including chemical activity and equilibrium partitioning PAH concentrations in lipids. These three PAHs were selected because their individual amax was found to be sufficient to exert acute toxicity to F. candida11 and since stable (near) equilibrium concentrations of naphthalene, anthracene (the not acutely toxic isomer of phenanthrene), and pyrene were reached in the springtail during a 7-d period. Furthermore, the three PAHs are found in relatively high concentrations in the environment 16. Chemical activity and equilibrium partitioning PAH concentrations in lipid were selected as exposure parameters since chemical activity drives diffusion and partitioning of HOCs into small soil organisms and membrane lipid is the site of toxic action for these compounds. The working hypothesis was that the toxicity of PAHs is initiated in the chemical activity range for baseline toxicity of 0.01 to 0.1 2, 11 and, when expressed on a lipid basis, in the range of lethal membrane burden for baseline toxicity of 40 to 160 mmol kg−1 lipid 17.
MATERIALS AND METHODS
Passive dosing vials
Passive dosing vials were prepared and loaded as described earlier 11. Medical-grade poly(dimethylsiloxane) silicone was made with the MDX4-4210 kit (Factor II), which contained a prepolymer and catalyst. The silicone was made by mixing the two components according to the supplier's instructions. Passive dosing vials were prepared by adding 500 mg (±1%) silicone to the bottom of each 10-ml glass vial (Mikrolab Aarhus). During silicone curing, the vials were kept at 5°C for 72 h (reduces air bubbles in the silicone), then at room temperature for 72 h, and finally at 110°C overnight. Before loading, the silicone was cleaned three times with excess ethanol (96%; Kemetyl) to remove impurities and subsequently three times with excess Milli-Q water (Super Q–treated; Millipore) to remove the ethanol. Total contact times were at least 48 and 24 h for ethanol and Milli-Q water, respectively.
Naphthalene (99%; Aldrich), phenanthrene (99.5%; Aldrich), anthracene (99%; Fluka), pyrene (99%; Sigma), benz[a]anthracene (99%; Aldrich), and benzo[a]pyrene (99%; Cerilliant) were used as test compounds. Methanol (99.9%; Merck) loading solutions were prepared as saturated solutions (amax) and dilutions thereof (a < amax), as specified in the following sections. An amount of 1.000 ml loading solution was added to each passive dosing vial, and the vials were closed with airtight screw caps (with Teflon-coated septa) to prevent methanol evaporation. Control vials were loaded with 1,000 µl pure methanol. The PAHs were transferred to silicone by equilibrium partitioning from the loading solution for at least 48 h at room temperature (∼21°C). The silicone was loaded either to saturation (amax) or to defined chemical activities below saturation (a < amax). Loading to saturation was ensured by the presence of PAH crystals in the loading solution during the entire loading period, whereas no PAH crystals were present in passive dosing vials loaded below saturation. The loading solution was renewed after 48 h when loading below saturation to compensate for potential PAH depletion in the first volume of loading solution. The spent loading solutions were then removed from the passive dosing vials, and the loaded silicone was cleaned with lint-free tissues (Assistent) and small volumes of Milli-Q water to remove any PAH crystals, if present. Approximately 2 ml of Milli-Q water was then applied at least three times with a total contact time of 48 h or more to remove any methanol traces. Finally, the PAHs in the loaded silicone were equilibrated with the air in closed passive dosing vials for at least 12 h before the test start.
We cultured F. candida in closed Petri dishes containing a mixture of charcoal and plaster of Paris (1:8). The cast was moistened, and the springtails were fed a diet of dried yeast. The Petri dishes were stored at 20°C with a 12:12 h light:dark photoperiod. The springtails were age-synchronized and had an average fresh weight of 141 µg and an average dry weight of 58 µg at an age of 39 to 42 d (average weight of 500 individuals).
Before starting the experiments, a 2-µl droplet of Milli-Q water was placed at the center of each loaded silicone to ensure sufficient humidity during the exposure period. Ten springtails were transferred to each passive dosing vial. The springtails could move freely within the vial, and most springtails were observed on the silicone surface. The uptake of PAHs via water was negligible due to the tiny water volume, limited water uptake, and low aqueous concentrations. This leaves exposure through air and via direct contact with silicone as the two possible routes of PAH uptake in the passive dosing system. The springtail experiments were conducted at 20°C with a 12:12 h light:dark photoperiod. Control passive dosing vials were included in the experiments, as mentioned above. Springtail survival in the 20 control vials (10 in each experiment) was 99.0%, ensuring a sound basis for investigating the springtail bioconcentration and toxicity of PAHs.
The bioconcentration experiment was conducted to investigate whether stable PAH concentrations were reached in the springtails within a 7-d toxicity test and whether stable concentrations characterized steady state or thermodynamic equilibrium. Naphthalene, anthracene, pyrene, benz[a]anthracene, and benzo[a]pyrene were selected as test compounds to cover a wide range of physicochemical properties (Table 1). Anthracene was used instead of phenanthrene due to its low amax (Table 1), since results from several studies have shown that only PAHs exceeding a certain amax can exert acute toxicity 11, 18–20. Phenanthrene and anthracene are isomers with very similar partitioning and diffusion properties, and the bioconcentration is therefore expected to be rather similar for these two PAHs. In the experiment, naphthalene exposure was 5% of its amax (Eqn. 1, a5% = 0.013 at 21°C) to avoid lethality, while F. candida was exposed to amax of the remaining four PAHs (Table 1). The springtails were 42 to 45 d old at the beginning of the bioconcentration experiment. Springtails from triplicate passive dosing vials were harvested at five to six exposure times between 0 and 14 d, and springtail survival was examined in each vial immediately after harvesting. The average springtail survival within the entire bioconcentration experiment was 99.7%. Live springtails were transferred to 2-ml amber glass vials, and the PAHs were extracted in 500 µl methanol by sonicating for 90 min. Extracts were kept at room temperature for 24 h before storage at −18°C. After storage, methanol extracts were kept at room temperature for 24 h, before an additional 90-min sonication. Fluoranthene (99%; Aldrich) was used as an internal volumetric standard, and 20 µl of a 51.0 mg L−1 solution of fluoranthene in methanol was added to each of the extracts before high performance liquid chromatography (HPLC) analysis. Springtails from the 10 control passive dosing vials were also analyzed, and no PAHs were detected.
Table 1. Physicochemical properties of the six polycyclic aromatic hydrocarbons used in the two experiments
Springtail lethality has previously been linked to the respective amax of 10 PAHs 11. The main focus in the present toxicity study was to investigate the acute (7 d) springtail lethality of naphthalene, phenanthrene, and pyrene separately to establish actual activity–response relationships and, furthermore, to determine and compare effective lethal activities (La50s) of single PAHs. The toxicity experiment was conducted as an activity–response test, and the loading solutions were consequently prepared as dilution series with five to six dilutions per PAH. Loading solutions with naphthalene and phenanthrene, respectively, were prepared with chemical activities covering the 0.01 to 0.1 range. The amax of pyrene is 0.049 at 21°C (Table 1), and based on previous results, full toxicity of this compound was not expected within 7 d 11. Each treatment was done in five replicates, and the springtails were 43 to 46 d at the beginning of the toxicity experiment. Springtail lethality was examined after 7 d of exposure, and the springtails were allowed to recover for 2 h in Petri dishes with a clean and moistened charcoal and plaster of Paris mixture before examination. Springtails were characterized as living when able to walk in a coordinated manner, after gentle stimulation with a fine brush, if necessary.
The PAH exposure was analytically confirmed at the end of both experiments by measuring PAH concentrations in equilibrated water (Cfinal) and then comparing these to target concentrations, which were calculated from aqueous solubilities and dilution factors. Passive dosing vials from the experiments were cleaned with a small volume of Milli-Q water and lint-free tissue to remove springtail waste. Milli-Q water (1.000 ml) was added to each vial, and the vials were closed and stored for at least 24 h for the PAHs to equilibrate. After equilibration, 500 µl water was transferred to a 1.5-ml amber HPLC vial, and 500 µl methanol was added to stabilize the sample. The water+methanol samples were stored at −18°C until chemical analysis.
One analytical method was used to quantify PAHs in the two types of samples: the methanol extracts with PAHs extracted from the springtails from the bioconcentration experiment and the water+methanol samples (50:50 v/v) used to determine PAH exposure at the end of both experiments. Extracts were analyzed by HPLC (Agilent 1100 Series) with fluorescence detection (G1321A FLD with an excitation wavelength of 260 nm and emission wavelengths of 350, 420, 440, and 500 nm). The HPLC was equipped with a CP-EcoSpher PAH column (Varian) and operated with a flow rate of 0.500 ml min−1 (30 µl injection, 28°C). The binary solvent system consisted of Milli-Q water and methanol and had the following steps: 50% methanol was held for 2 min, linear gradient to 75% methanol at t = 2 to 7 min, linear gradient to 100% methanol at t = 7 to 35 min, 100% methanol was held for 15 min, linear gradient to 50% methanol at t = 50 to 52 min, and finally reequilibration of the column with 50% methanol for 7 min. As mentioned, fluoranthene was used as an internal volumetric standard in the samples from the bioconcentration experiment, and fluoranthene was thus added to both samples and external calibration standards. In this way, analyte to fluoranthene ratios were used to quantify the PAHs in extracts from F. candida by external calibration with an internal volumetric standard. The PAHs in the water+methanol extracts from both experiments were quantified by external calibration without the use of an internal volumetric standard.
The PAH bioconcentration was investigated by plotting springtail PAH concentration (Cspringtail, mmol PAH kg−1 dry wt) as a function of time (d) and fitting the data with a two-compartment model with first-order kinetics using least-squares regression by the GraphPad Prism 5.0 software (GraphPad Software)
The stable concentrations (Cstable, mmol PAH kg−1 dry wt) and rate constants (k, d−1) are fitting parameters of the model, and the k values for the five PAHs were determined and used in the calculation of the time to reach 95% of the stable concentration (t95%)
The PAH toxicity to F. candida was plotted as a function of four different exposure parameters: freely dissolved PAH concentration in water (Cfree), PAH concentration in air (Cair), PAH chemical activity (a), and equilibrium partitioning PAH concentration in lipid (Clipid). PAH concentrations in water were measured in the passive dosing vials immediately after ending the experiment, as described above. PAH concentrations in air at saturation were calculated from the ideal gas law
where n is amount (mol), V is volume (L), p* is vapor pressure (Pa, Table 1), R is gas constant (L [kPa]/K [mol]), and T is temperature (K). The PAH concentrations in the air of the different dilution levels were then calculated using the respective dilution factors. Maximal chemical activities were calculated from Equation 1, and submaximal chemical activities were subsequently calculated according to dilution levels. Equilibrium partitioning concentrations of the PAHs in lipid (Clipid, mol L−1) were calculated from chemical activities (a, dimensionless, Eqn. 1) and compound-specific activity coefficients in lipid (γlipid, L mol−1, Table 1)
Springtail lethality was plotted as a function of either concentration or chemical activity, and the exposure–response relationship was fitted with a sigmoidal dose–response function with variable slope using least-squares regression by the GraphPad Prism 5.0 software
The effective lethal concentrations and activities causing 50% lethality (LCfree50, LCair50, La50, and LClipid50) were determined by this software as well. Finally, effective lethal body burdens (LBB50s) were estimated for naphthalene and pyrene as products of aqueous bioconcentration factors and LCfree50 values.
RESULTS AND DISCUSSION
Folsomia candida inhabits the interstitial pore space of topsoil. This implies that the springtails are exposed not only via water and food but also via air and direct contact to soil particles. These two exposure routes are rarely considered or studied in terrestrial ecotoxicology. However, recent studies have shown that diffusive mass transfer of the two-ringed naphthalene is much more efficient through air than through water and that even three- and four-ringed PAHs are efficiently transferred through air 21, 22. The passive dosing system provided exposure via these two routes with partitioning of the HOCs from the loaded silicone to the air in the vial and the free movement of F. candida on the loaded silicone surface. The exposure system did not contain soil, food, a substantial water phase, other species, and so on, which, on the one hand, rendered the passive dosing system somewhat artificial but, on the other hand, made it possible to secure constant and defined PAH exposure by eliminating numerous physical, chemical, and biological interactions. This in turn allowed for the links between exposure, bioconcentration, and acute toxicity to be rigorously investigated. Furthermore, the passive dosing system ensured reproducible results and was easy to apply in the bioconcentration and toxicity experiments.
Regular bioconcentration curves were obtained for naphthalene, anthracene, pyrene, and benz[a] anthracene (Fig. 1 and Supplemental Data, Fig. S1), and the time to reach a stable internal concentration (t95%) ranged from 1 h for naphthalene to 5.3 d for pyrene (Table 2). The bioconcentration kinetics for benzo[a]pyrene was characterized by more scatter, and the obtained t95% value of 2.6 d is only tentative (Supplemental Data, Fig. S1, and Table 2). Therefore, the results demonstrate that stable internal PAH concentrations were reached in F. candida within the 7-d toxicity tests when applying passive dosing.
Table 2. Results from the bioconcentration experiment with Folsomia candida
The two main routes of PAH uptake into F. candida in the applied passive dosing system were expected to be through air and direct contact, and the bioconcentration data were therefore related to a previous study investigating the diffusive mass transfer of PAHs through air from a loaded silicone disk to an initially clean silicone disk 22. The time to reach near equilibrium (t95%) when diffusing through air was closely related to the octanol to air partition ratio (Koa) of the PAHs, as can be seen in the log–log plot of Figure 2A. Log t95% values for the bioconcentration kinetics of naphthalene, anthracene, and pyrene in F. candida were fitted to a linear regression that is parallel and thus follows the same relationship with Koa (Fig. 2A). In fact, the slopes of the linear regressions from the diffusive mass transfer through air 22 and the present data were statistically not significantly different (analysis of covariance, p = 0.285). The bioconcentration data were also related to the diffusive mass transfer of PAHs through water and via direct contact 22. Log t95% values for bioconcentration and diffusive mass transfer were plotted against the octanol to water partition ratio (KOW, Supplemental Data, Fig. S2), and the slope of the linear regression for bioconcentration was statistically significantly different from the slopes for diffusive mass transfer through water (analysis of covariance, p = 0.004) and direct contact (analysis of covariance, p < 0.0001). This analysis of the kinetic data suggests that diffusive mass transfer through air was rate-limiting for the uptake of the PAHs with lower molecular weight into the springtails, which in turn supports diffusive uptake through air as an important route. The obtained t95% values for benz[a]anthracene and benzo[a]pyrene were in all cases well below the regression lines (Fig. 2A and Supplemental Data, Fig. S2), which is in good agreement with a steady state that is below the thermodynamic equilibrium concentration.
Steady state versus thermodynamic equilibrium
Two lipid-based PAH concentrations (mmol PAH kg−1 lipid) were determined in order to resolve whether the observed stable internal concentrations indicated steady state or thermodynamic equilibrium 23, 24. First, lipid-normalized PAH concentrations were calculated as ratios of the stable concentration (Cstable, Eqn. 3) and a lipid content of 9.9% (w/w, dry wt) in F. candida25. Second, equilibrium partitioning concentrations of the PAHs in lipids were calculated from Equation 6 as ratios of chemical activity and compound-specific activity coefficients in lipids (Table 1), assuming a lipid density of 0.9 kg L−1 lipid 26. Finally, the ratios between these two lipid-based concentrations were determined and plotted against Koa (Fig. 2B). For naphthalene, this ratio exceeded 1 by a factor of 3, and this can be explained by the lipid-normalized concentration exceeding the actual concentration in the lipids. This occurs when a significant fraction of the compound is distributed to compartments of the organism other than the lipids 27, 28. In F. candida, this other compartment is likely the cuticle, which is covered by a hydrophobic waxy layer to reduce evaporative water loss 29. The concentration ratio then decreased with increasing Koa of the PAHs (Fig. 2B). When combining these observations with the bioconcentration kinetics (Fig. 2A), it appears that naphthalene reached its thermodynamic equilibrium within the first hours of the experiment and that a considerable fraction of naphthalene was present in the waxy layer of the cuticle. The concentration ratios for anthracene and pyrene were near unity, which might be interpreted as near or at the equilibrium level. More likely, the ratios for anthracene and pyrene are the result of a combination of distribution to the waxy layer and a slight or moderate underequilibration. The concentration ratios for the heavier PAHs were well below 1, which demonstrated clear underequilibration of benz[a]anthracene and benzo[a]pyrene and that the internal concentrations of these two compounds were in fact characterized by steady state.
The (near) equilibration of naphthalene, anthracene, and pyrene within 7 d clearly demonstrates an efficient uptake via air even for compounds with relatively low vapor pressures and high Koa values (Table 1). The uptake through air is, based on these results, expected to be even more efficient and important for compounds with lower Koa values, such as alkanes, many substituted benzenes, and the lower chlorinated PCB congeners.
Bioconcentration factors (BCFs) were derived for the five PAHs, bearing in mind that the internal springtail concentrations of benz[a]anthracene and benzo[a]pyrene were characterized by steady state. The BCFs rather than the bioaccumulation factors were calculated due to the absence of food in the passive dosing system. The BCFs were calculated as the ratio of the PAH concentration in the springtails at d 7 (µg kg−1 fresh wt) and either water (Cfree, µg L−1) or air (Cair, µg L−1) as exposure mediumi
The BCFs from water were calculated due to the tradition of using water as an exposure medium and to allow comparisons to BCF values for small aquatic organisms. As expected, the obtained BCFs (log BCFwater) increased with increasing hydrophobicity and lipophilicity, ranging from 2.99 for naphthalene to 4.92 for benzo[a]pyrene (L kg−1 fresh wt, Table 2). The obtained BCF values were generally within 0.5 log units from BCF values for the freshwater isopod Asellus aquaticus30 and the marine amphipod Rhepoxynius abronius31. The BCFs from air were calculated since naphthalene, anthracene, and pyrene were efficiently taken up through air in the present study. The PAH concentrations in air were calculated from Equation 5, and as expected, the obtained BCFs (log BCFair) increased with decreasing vapor pressure and increasing lipophilicity, ranging from 4.63 for naphthalene to 9.13 for benzo[a]pyrene (L kg−1 fresh wt, Table 2). To our knowledge, these are the first BCFs expressed on an air concentration basis for invertebrates living in soil.
In summary, stable (near) equilibrium concentrations were reached in F. candida for naphthalene, anthracene, and pyrene within 7 d, and these were efficiently taken up through air. The toxicity experiment was conducted with naphthalene, phenanthrene (the acute toxic isomer of anthracene), and pyrene due to the results from the bioconcentration experiment and since their individual amax was found to be sufficient to exert acute toxicity to F. candida11.
Lethality to F. candida was examined after 7 d of exposure to naphthalene, phenanthrene, and pyrene and plotted as a function of four different exposure parameters: freely dissolved PAH concentration in water (Cfree), PAH concentration in air (Cair), PAH chemical activity (a), and equilibrium partitioning PAH concentration in lipid (Clipid). In this way, it was possible not only to establish actual exposure–response relationships for the individual PAHs but also to determine and compare median lethal concentrations (LC50s) and effective lethal activities (La50s) across PAHs. For all exposure parameters, regular sigmoidal exposure–response curves were obtained for naphthalene and phenanthrene, while maximal exposure to pyrene resulted in 44% springtail lethality and, thus, a partial sigmoidal exposure–response curve (Fig. 3A–D). The coefficients of determination (r2) were consistent across exposure parameters and were 0.91, 0.93, and 0.78 for naphthalene, phenanthrene, and pyrene respectively.
First, springtail lethality was fitted as a function of the measured PAH concentrations in water (Cfree, Fig. 3A). Acute effective lethal concentrations (LCfree50s) were 2,387, 176, and 95 µg L−1 for naphthalene, phenanthrene, and pyrene, respectively (Table 3), spanning more than one order of magnitude. No literature acute LC50 values based on water concentrations were available for comparison. However, Styrishave and coworkers 32 determined an effective pyrene concentration that caused 50% reduction in F. candida reproduction to be 23 µg L−1 based on a freely dissolved concentration basis. This chronic median effective concentration (EC50) value is, as expected, lower than the acute LC50 value from the present study, and the LC50 to EC50 ratio of 4.1 is well within the reported range of acute to chronic ratios (e.g., 1.1–11 with a geometric mean of 3.8 for nonhalogenated hydrocarbons 33). Therefore, the toxicity results from the passive dosing approach were in good correspondence with those from soil 32.
Table 3. Results from the toxicity experiment with Folsomia candida (d 7)
Folsomia candida inhabits the interstitial pore space of topsoil, and bioconcentration results indicated efficient uptake of low–molecular weight PAHs through air. As a consequence, springtail lethality was also fitted as a function of the calculated PAH concentrations in air (Cair, Fig. 3B). Acute effective lethal concentrations (LCair50s) were 44, 0.25, and 0.072 µg L−1 for naphthalene, phenanthrene, and pyrene, respectively (Table 3), spanning three orders of magnitude. To our knowledge, these are the first effective lethal concentrations expressed on an air concentration basis for invertebrates living in soil.
As mentioned, the diffusion and partitioning of HOCs into small soil organisms is driven by a difference in the chemical activity between the organism and the surrounding environment 2. Therefore, springtail lethality was linked to the chemical activities of naphthalene, phenanthrene, and pyrene as the third exposure parameter (Fig. 3C). Acute effective lethal activities (La50s) were 0.019, 0.026, and 0.052 for naphthalene, phenanthrene, and pyrene, respectively (Table 3), and were all within a factor of 2.8. This might be explained by chemical activity not only controlling the uptake in the organism but also the internal distribution of the contaminant to the site of toxic action. Naphthalene, phenanthrene, and pyrene are believed to be baseline toxicants and, thus, to have the same mode of action, that is, perturbation of the cell membrane integrity 17. Baseline toxicity is expected to initiate in the chemical activity range 0.01 to 0.1 2, 11, 34, and springtail lethality values of all three PAHs were within this range (Fig. 3C), which is also in good agreement with the results of a pilot experiment (Supplemental Data, Fig. S3) and the previously published study using the same passive dosing system, PAHs, and F. candida11.
Effective lethal body burdens (LBB50s) were calculated for naphthalene and pyrene as products of aqueous BCFs (Table 2) and LCfree50 values (Table 3). The LBB50 values were 18 and 11 mmol kg−1 fresh wt for naphthalene and pyrene, respectively (Table 3), and thereby slightly higher than the range of 2 to 8 mmol kg−1 fresh wt proposed earlier 35. These results are in agreement with other results of the present study indicating partitioning of the PAHs to the hydrophobic waxy layer covering the cuticle of F. candida.
Since the lipid membranes are considered the site of toxic action for baseline toxicity 17, the chemical activities of the PAHs were converted into equilibrium partitioning concentrations in lipid using compound-specific activity coefficients in lipid (Eqn. 6). Activity coefficients were previously measured in fish, olive, rapeseed, and sunflower oils 7, and the values were almost identical in these four oils. The activity coefficients from the animal fish oil were used to calculate the equilibrium partitioning concentrations in springtail lipid. It has to be emphasized that, in case of underequilibration, these equilibrium partitioning concentrations will exceed the actual PAH concentrations in the lipids of the organism. Springtail lethality was then fitted as a function of this lipid-based exposure parameter (Clipid, Fig. 3D), and the acute effective lethal concentrations (LClipid50s) were 101 and 104 mmol kg−1 lipid for naphthalene and phenanthrene, respectively (Table 3), well within the expected range of lethal membrane burden for baseline toxicity (40–160 mmol kg−1 lipid 17). Pyrene toxicity was initiated at a higher equilibrium lipid concentration and had an LClipid50 value of 343 mmol kg−1 lipid, likely because the equilibrium estimate exceeded the actual concentrations in the lipid membranes due to underequilibration. This is in good agreement with results from the bioconcentration experiment.
Controlled exposure is essential in robust laboratory tests, and PAH exposure was therefore confirmed in the passive dosing vials after ending the two experiments. The final PAH concentrations (Cfinal) were measured and plotted against target concentrations (Ctarget; Supplemental Data, Figs. S4 and S5). Target concentrations were estimated from temperature-dependent solubility equations at 21°C 36 and the appropriate dilution factor. The Cfinal values were between 90 and 108% of the Ctarget values for the five PAHs in the bioconcentration experiment, while the naphthalene concentrations were 104 to 111%, phenanthrene concentrations were 137 to 144%, and pyrene concentrations were 85 to 107% of the Ctarget values in the toxicity experiment. The exceedance of the measured phenanthrene concentrations relative to calculated target values is most likely due to uncertainties in both the measured and the estimated concentrations of phenanthrene 36. Nevertheless, the passive dosing system was considered to accurately control the targeted exposure and keep it constant during the entire test duration.
In conclusion, passive dosing was successfully applied to expose the springtail F. candida in bioconcentration and toxicity experiments. Stable internal PAH concentrations were reached within 7 d, which for naphthalene and anthracene was governed by (near) equilibrium partitioning. Furthermore, naphthalene, anthracene, and pyrene were shown to be efficiently taken up via air. The acute toxicity of naphthalene, phenanthrene, and pyrene was closely linked to chemical activity, and the effective chemical activities (La50s) were within the expected chemical activity range for baseline toxicity of 0.01 to 0.1. The results of both the bioconcentration (Clipid normalized/Clipid equilibrium >1 for naphthalene) and toxicity (LBB50s >2–8 mmol kg−1) experiments suggest the presence of an additional significant sorbing phase for the PAHs in the springtails, which is likely the hydrophobic waxy layer covering the cuticle.
Both the applicability of passive dosing to terrestrial ecotoxicological tests and the use of chemical activity as an exposure parameter need further investigation. Ongoing experiments are directed at linking mixture toxicity to the sum of chemical activities and at exploring the potential of the passive dosing approach in multiple stressor studies.
Figure S1. Bioconcentration kinetics of benz[a]anthracene and benzo[a]pyrene in F. candida.
Figure S2. Uptake of PAHs through water and via direct contact.
Figure S3. A pilot experiment where springtail lethality caused by naphthalene and phenanthrene was determined.
Figure S4. Exposure confirmation in the bioconcentration experiment.
Figure S5. Exposure confirmation in the toxicity experiment. (49 KB PDF).
We greatly thank M.M. Fernqvist, E. Jørgensen, and Z. Gavor for their guidance and assistance with the passive dosing system and F. candida. The present research project was financially supported by the European Commission (MODELPROBE, 213161; OSIRIS, COGE-037017) and the PhD research program STAiR. Additionally, M. Holmstrup was supported by the Danish Council for Independent Research (contract 10-084579).