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.
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.
Figure 3. Lethality to Folsomia candida after 7 d of exposure to naphthalene (closed circle), phenanthrene (open circle), and pyrene (cross) as a function of (A) freely dissolved polycyclic aromatic hydrocarbon (PAH) concentration in water, (B) PAH concentration in air, (C) PAH chemical activity, and (D) equilibrium partitioning PAH concentration in lipid. Error bars represent standard error of the mean (n = 5). The shaded areas are (C) chemical activity range 0.01 to 0.1 and (D) membrane burden range 40 to 160 mmol PAH kg−1 lipid. The broken line is a tentative fitting for pyrene toxicity. Notice the different scales for the x axes.
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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)
| ||LCfree50a (µg L−1)||LCair50b (µg L−1)||La50c||LClipid50d (mmol kg−1)||LBB50e (mmol kg−1 fresh wt)|
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. candida 11.
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.