Hydration State of the Moss Hylocomium splendens and the Lichen

Article pubs.acs.org/est

Hydration State of the Moss Hylocomium splendens and the Lichen Cladina stellaris Governs Uptake and Revolatilization of Airborne αand γ‑Hexachlorocyclohexane Henrik Kylin*,†,‡ and Henk Bouwman§ †

Department of Water and Environmental Studies, Linköping University, SE-58183 Linköping, Sweden Norwegian Institute for Air Research, Fram Centre, NO-9296 Tromsø, Norway § School of Biological Sciences, North-West University, PBag X 6001, Potchefstroom ZA-2520, South Africa ‡

S Supporting Information *

ABSTRACT: The partitioning of α- and γ-hexachlorocyclohexane between air and the moss Hylocomium splendens and the lichen Cladina stellaris were studied under laboratory conditions. After cultivation of the sample material to obtain a common starting point free from outside influence, the material was divided into four different treatment categories with different hydration/desiccation regimes. The concentrations of the analytes were 3−5 times higher in the hydrated moss or lichen than in the desiccated material. The results are in contrast to how these compounds are taken up by pine needles in which there is a continuous accumulation, more rapid during periods with high temperatures and dry weather. In general, the different adaptations to water economy is a more important explanatory factor for the concentration of airborne hydrophobic pollutants in mosses, lichens, and vascular plants than their designation as “plants” in a broad sense. It is, therefore, not advisible to mix data from different organism groups for monitoring or modeling purposes.

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via their hydrophilic surface.4−6 Adding to the complexity, lichens are symbiotic organisms, each consisting of a fungus, the mycobiont, and a photosynthetic alga or cyanobacterium, the photobiont.7 Because the mycobiont constitutes the bulk of the organism, it is reasonable to view lichens as fungi when studying the uptake of POPs from the air. Today, fungi are regarded as more closely related to animals than to vascular plants or mosses.3 In discussions and models of the partitioning of hydrophobic compounds, the octanol−air partitioning coefficient is used to characterize the partitioning between air and hydrophobic surfaces.8 Doing so for “plants” does not take into account that many have hydrophilic, not hydrophobic, surfaces. Even so, in some investigations, “plants” are considered as a stationary lipid phase in air into which POPs are passively partitioned,9 while other investigations mix data from vascular plants, mosses, and lichens without accommodating for the fundamental differences in physiology.10 A further complication is that typical POPs are semivolatile organic compounds (SOCs) that in the air are present both in the gas phase and bound to particles.11 We suggest that recognizing a more diversified “plant” phase, consisting of both hydrophilic and hydrophobic surfaces and

INTRODUCTION The Linnaean kingdoms “Vegetabilia”1 (later renamed “Plantae”2) and “Animalia”1 were defined in the early 18th century by characteristics based on morphology and anatomy. In contrast to Linnaeus, systematists today strive toward phylogenetically relevant taxa reflecting evolutionary relationships.3 The Linnaean “plants” have been split into separate phylogenetic branches reflecting many evolutionary lineages and adaptations.3 However, this separation is usually not reflected in how “plants” are treated when the environmental fate of persistent organic pollutants (POPs) is investigated. In such investigations, lichens, mosses, and vascular plants are usually considered as “plants”. In contrast, although Linnaeus’s animal kingdom is essentially intact, in investigations of the fate of POPs involving animals, these are separated into, for example, vertebrates or invertebrates, and usually even lower taxa, to account for different adaptations. In the discussions here, we will use citation marks to designate “plants” in a broad sense (i.e., organisms placed by Linnaeus in the plant kingdom) and reserve plants without citation marks for organisms that today are regarded as plants in a strict sense. Most vascular plants are adapted to a homoihydric lifestyle, taking up water via roots and having hydrophobic surfaces to limit water loss and maintain turgor. Other “plants”, e.g., most mosses and lichens, have adapted a poikilohydric lifestyle, withstanding desiccation and loss of turgor during drought, to rapidly rehydrate upon precipitation events by taking up water © 2012 American Chemical Society

Received: Revised: Accepted: Published: 10982

June 12, 2012 August 22, 2012 September 19, 2012 September 19, 2012 dx.doi.org/10.1021/es302363g | Environ. Sci. Technol. 2012, 46, 10982−10989

Environmental Science & Technology

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stellaris was included, as lichens have similar poikilohydric adaptations as mosses.5,29 Samples. H. splendens and C. stellaris were collected in September 1995 in a mixed forest with conifer dominance on Väddö, an island off the Swedish coast in the Baltic Sea approximately 80 km north of Stockholm. Preliminary investigations to set experimental conditions (analytical protocol, water regime, etc.) were conducted during September 1995 to January 1996, during which time the remaining material was stored outdoors under a fine-meshed net to avoid deposition of additional debris used in the experiment. The collected material was transplanted to polyethylene trays (30 × 50 × 5 cm) and debris removed before cultivation commenced in early January 1996. Exposure Conditions. The experiments were carried out in a laboratory previously used for quality control of pesticide formulations. The test substances were chosen because of the high concentrations present in the laboratory and as representatives of POPs that are present almost entirely in the gas phase at temperatures normally found in the environment.11 The γ-isomer of HCH is the insecticide lindane, while the α-isomer constitutes the bulk of the technical mixture in the production of lindane. 30 The relative composition of HCHs in the laboratory air (Table S1, Supporting Information) indicates the presence of some technical HCH but mostly pure lindane. This is consistent with the phase-out of technical HCH in the Nordic countries in 1979.30 Lab and outdoor air concentrations of HCHs were monitored with a low-volume air sampler consisting of an SKC Model 224-PCXR8 pump (SKC Inc., Eighty Four, PA) drawing air (approximately 0.5 L/min indoors and 5 L/min outdoor, 10 m3 total volume indoors and 100 m3 outdoors) through a sampling train of a glass-fiber filter (Whatman 0.7 μm cutoff, 70 mm diameter, from VWR-International, Spånga, Sweden) and two polyurethane foam plugs (75 × 85 mm, 75 × 85 mm, a gift from the Swedish environmental monitoring program for POPs in air).21 There was no active climate control in the laboratory, but temperature variations were monitored (20−22 °C). Air concentrations of HCHs were 2 orders of magnitude higher in indoor (approximately 4000 and 6000 pg/m3for αand γ-HCH, respectively) than in outdoor air (Table S1). While outdoors there was no significant difference (p = 0.1638, paired t test) between mean α- and γ-HCH concentrations, indoor mean α-HCH was significantly less (p < 0.0001, paired t test) than mean γ-HCH, with an α/γ ratio of 0.63. Experimental Procedure. To obtain a standardized starting point for the experiment, the samples were first hydrated by filling the trays with water and then decanting the water after 12 h. The samples were kept damp by sprinkling with water equivalent to 6 mm of rain daily. During the first two weeks, the water used for the moss contained a dilute nutrient mixture,31 but for the lichen and for the remaining experiment with the moss, deionized water filtered through activated carbon (Elgastat, High Wycombe, England) was used. No traces of HCHs were found in 5-L samples of the water. Both moss and lichen were illuminated with high-pressure sodium lamps (simulated daylight lamps, Philips, Stockholm, Sweden) 20/4 h light/dark regime. The artificial light was used to simulate a spring/summer daylight regime to stimulate growth but was turned off after the growth phase to stop further growth and avoid growth dilution. To avoid desiccation

subject to physiological processes, should lead to an improved and more accurate understanding of the partitioning of POPs and other SOCs in this important environmental compartment. The Stockholm Convention12 restricts the use of POPs and in Article 16 calls for the Conference of Parties to evaluate its effectiveness by monitoring trends in environmental POP concentrations. It is, therefore, of interest to develop cheap and efficient methods to map the deposition of airborne POPs, e.g., by measuring the concentrations in “plants”. Vascular plants, especially pine needles, have often been used13 but sometimes also mosses or lichens.14−18 Although it may be hard to calculate absolute air concentrations based on the levels in the vegetation,19 vascular plants can be used to map relative differences in air concentration and deposition between different areas, although a better understanding of the uptake mechanisms is necessary to interpret the data.19−21 Since the 1960s, the deposition of heavy metals in Scandinavia has been mapped with mosses.22 Therefore, the Swedish Environmental Protection Agency in 1985 commissioned a feasibility study on the use of mosses to monitor the deposition of airborne SOCs, and lichens were included for comparison.23,24 The results were compared with the results from a parallel ongoing study on Scots pine (Pinus sylvestris) needles.21,25,26 While there was a continuous uptake of SOCs in the pine needles, the concentrations in mosses and lichens, especially of the more volatile analytes such as the hexachlorocyclohexanes (HCHs), varied erratically. It was concluded that mosses or lichens, although they might be of some use for particle-bound compounds, are not well suited for general monitoring of SOCs, and especially not of SOCs predominantly present in the gas phase.24 The opposite was concluded for pine needles that more efficiently retained gaseous than particle bound SOCs.21,25,26 Further scrutiny of the data from the feasibility study24 indicated that the seemingly erratic variations of the SOC concentrations in mosses and lichens were linked to variations in the weather. The concentrations, particularly of the HCHs, were higher after rain events than during hot and dry weather, while HCH concentrations remained almost constant in pine needles. This indicated that adaptation to poikilohydry or homoihydry influences how SOCs in the gas phase are taken up by “plants”. This study investigated the effect of the hydration state of two poikilohydric “plants” with different evolutionary histories, the moss Hylocomium splendens and the lichen Cladina stellaris, on the uptake of HCHs.

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EXPERIMENTAL SECTION Choice of Test Species. The primary test species, H. splendens, was chosen because its growth pattern makes identification of the most recent growth particularly easy27 (see Figure S1, Supporting Information). Mosses have no roots, and ectohydric mosses such as H. splendens generally have limited internal transport.28 By activating a new growth at the start of the experiment (see below), the test material was given a common starting point free from outside exposure or forest debris, so that the HCHs measured could only have come from the laboratory air. Consequently, analyzing the latest growth cultivated in the laboratory separately will give information on the partitioning of HCHs between laboratory air and moss without interference from previous uptake. To obtain additional information on how poikilohydry influences the uptake of airborne hydrophobic pollutants, C. 10983

dx.doi.org/10.1021/es302363g | Environ. Sci. Technol. 2012, 46, 10982−10989


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Figure 1. Concentrations (ng/g dry mass) and standard deviation of α-HCH and γ-HCH in the moss Hylocomium splendens and lichen Cladina stellaris depending on hydration. Black: Treatment 1, kept hydrated throughout. Red: Treatment 2, hydrated at the start and desiccated after day 22. Green: Treatment 3, dry at the beginning and hydrated on day 22. Blue: Treatment 4, kept desiccated throughout. Individual data are provided in Tables S2−S4.

variability within each treatment, the total number of subsamples analyzed for each treatment varied between one and four (see Tables S2−S4) to keep both within the capacity of the lab and provide sufficient data for statistics. The new-growth of H. splendens was separated from older growth with a pair of stainless steel scissors cleaned with dichloromethane (DCM, pestiscan grade, Labscan, Stillorgan, County Dublin, Ireland) between samples. Of the lichen, one or two subsamples were taken at each sampling occasion. Because new growth could not be easily identified in C. stellaris, the smaller branches were cut from the main stem and any remnants of debris removed. The sampled material was cut finely with the scissors and then transferred to DCM-filled brown glass vials with Teflon lined screw caps and stored in a freezer (−20 °C) until extraction. Extraction was performed by refluxing for 24 h with DCM in a specially designed extractor32 with addition of 1,1,1-trichloro2,2-bis(4-fluorophenyl)ethane, synthesized in-house, as surrogate standard. Water was removed with a Dean−Stark type 2 trap. For moss samples, the solvent was evaporated in a rotary evaporator and the dry mass determined by weighing the residues in the extractor and in the round flask after evaporating the DCM. The residue in the round flask was also used as a measure of the “soluble lipid content” of the individual samples. The extract was redissolved in pentane (pestiscan grade, Labscan) and passed through a column of silica gel with 30% (mass/mass) concentrated sulfuric acid.32 Undecane (25 μL, Kebo, Spånga, Sweden, redistilled twice) was added as keeper solvent, after which the pentane was evaporated under a gentle stream of nitrogen (99.99%, Aga, Lidingö, Sweden). Because raw DCM extracts of the lichen were difficult to redissolve in pentane, the DCM volume was first reduced to approximately 5

caused by the heat from the lamps, four standard household air humidifiers (Boneco, Stockholm, Sweden) were placed among the sample trays. After eight weeks, when it was judged visually that a new growth of moss had been completed, the lamps and humidifiers were removed. The samples were air-dried at 20− 22 °C for three weeks in darkness, after which subsamples were taken to determine α- and γ-HCH concentrations and soluble lipid content at the start of the experiment. The moss was subjected to four hydration treatments: 1. Kept hydrated during the whole experiment. 2. Hydrated at the beginning of the experiment as in treatment 1 and then allowed to desiccate after day 22. 3. Desiccated at the beginning of the experiment as in treatment 4 and then hydrated on day 22. 4. Kept desiccated during the whole experiment. Because it was not possible to easily identify and sample the recent growth of lichen, only two hydration treatments were tested on the lichen, corresponding to treatments 2 and 3. Trays assigned to the different treatments were placed randomly on the laboratory benches. Treatments 1 and 2 were first hydrated by filling the trays with water (no nutrients added) for 12 h after which the water was poured off and the state of hydration maintained by daily sprinkling with deionized water as above. Sprinkling of treatment 2 stopped on day 22 after which the material was allowed to dry up at its own rate. Treatment 3 samples were hydrated similarly as above after subsamples had been removed on day 22 and then kept damp by sprinkling. Treatment 4 received no water throughout. Subsamples were taken three times per week prior to the daily sprinkling with water. The total number of samples analyzed at each sampling occasion was 12−16 (Tables S2−S4, Supporting Information). To obtain a measurement of the 10984



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mL. This solution was then passed through the silica gel/ sulfuric acid column, undecane was added, and the remaining DCM was evaporated under a gentle stream of nitrogen. The dry mass of the lichen was determined on two separate samples (one hydrated and one desiccated) at each sampling occasion. The soluble lipid content of the lichen was determined according to the method of Bligh and Dyer,33 which was also used to cross-check (n = 5) the soluble lipid determinations of the moss. Instrumental Analysis. Quantifications were conducted with a Varian 3400 gas chromatograph (Varian, Walnut Creek, CA); temperature program: 80 °C for 2 min, 10 °C/min to 280 °C, isothermal 5 min); equipped with a split−splitless injector operated in the splitless mode (injector temp 250 °C), a DB-5 column (30 m, 0.25 mm ID, 0.25 μm phase thickness, J&W Scientific, Folsom, CA), and an electron capture detector at 360 °C. Carrier gas was hydrogen (99.999%, Aga, Lidingö, Sweden) and makeup gas nitrogen (99.999%, Aga, Lidingö, Sweden). Data were collected and processed with a PC-based ELDS900 system (Chromatography Data Systems, Svartsjö, Sweden). Quality Assurance and Control. To test for recovery and reproducibility, samples spiked with known amounts of the analytes were extracted, and the linearity was checked with standard additions. Statistical Analysis. Except where mentioned, data were normally distributed (using the D’Agostino-Pearson omnibus K2 normality test) and not transformed. Prism 4.03 (GraphPad Software, Inc.) was used for statistical analysis. All t tests were two-tailed. Nonlinear regression was performed using a fourparameter logistic equation with variable slopes (similar to sigmoidal dose−response). F-tests comparing Hill slopes (steepness) were performed using logical pairs of data sets for comparisons. Elimination/uptake time was calculated as the time in days from day 22 (the day when treatment 2 was allowed to desiccate and treatment 3 was hydrated), to reach a concentration bottom or top. The experiments from September 1995 to January 1996 to set the growth and exposure conditions included comparing the polyethylene trays with large glass Petri dishes (30 cm diameter) to investigate if the tray material influenced the results. No such influence was found.

Table 1. Dry Matter, Content of Soluble Lipids, and Mean Concentrations of α- and γ-HCH of Hydrated and Desiccated Moss (Hylocomium splendens) and Lichen (Cladina stellaris), with Standard Deviation (SD)a

Hylocomium splendens hydrated (n = 111) desiccated (n = 85) at start of experiment (desiccated, n = 6) Cladina stellaris hydrated (n = 35) desiccated (n = 24) at start of experiment (desiccated, n = 6)

dry matter % (SD)

soluble lipid % of dm (SD)

α-HCH ng/g dm (SD)

γ-HCH ng/g dm (SD)

9 (0.72) − −

7.3 (0.8) 6.9 (1.6) 6.7 (1.9)

184 (26) 58 (23) 46 (11)

393 (56) 103 (34) 89 (17)

5.3 (0.8) 5.1 (1.1) 5.1 (1.0)

344 (63) 96 (39) 82 (18)

414 (49) 117 (42) 99 (14)

14 (1.3) − −

The individual data points included in the 'hydrated” and “desiccated” groups are indicated in Tables S2−S4.

a

not have solved issues of swelling at hydration (see below, moss vs lichen). When quantifying hydrophobic compounds in biota with the intention to understand the environmental dynamics of POPs, it is customary to standardize the concentrations to a lipid mass basis,34 although the use of fresh mass may also be motivated. It should be noted though that, in most cases, the soluble lipid content as used in environmental chemistry is based on an operational definition of “lipid” as the part of the sample that is extracted with an organic solvent. This practice has been used also on plant material, but we have found no motivation, neither actual tests nor theoretical reasons, to motivate this on physiological grounds. Nor have we found any proof that it accounts for all hydrophobic constituents in the foliage. Parts of the plant “lipids” (e.g., cutin) are polymeric and will not dissolve, while other classes of endogenous hydrophobic constituents are too volatile to be accounted for by traditional lipid determination methods. Although, for example, most terpenoids will be readily extracted during lipid determination, many will evaporate together with the solvent.20 As there are a number of highly hydrophobic compound classes that will influence the partitioning of hydrophobic pollutants between the air and plant but that will not be included in the lipid determination, the “lipid” content will be systematically underestimated. The difficulties using lipid mass may be even worse for mosses as a group than for vascular plants. Among the mosses, different species have different lipid constituents.31,35−37 Some have polymerized lipids,35 some have an at least partially hydrophobic cuticle35,36 or surface waxes,37 and some use oil droplets to store energy,31,38 oil droplets that will vary with the physiological state of the moss. Moreover, at least some mosses produce semivolatile secondary metabolites39 that may evaporate together with the solvent. In different lichen species, the presence or absence of secondary metabolites, e.g., usnic acids, affects the determination of the “lipid content” in ways that are difficult to predict.24 The decision to choose C. stellaris instead of some other possible species was, inter alia, based on tests that showed that the former was easier to handle in the analytical procedure, as it contains little or no usnic acids. The amount of “lipid” in

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RESULTS AND DISCUSSION The concentrations of α- and γ-HCH in moss and lichen of different treatments are presented in Figure 1. The mean concentrations in hydrated and desiccated moss and lichen are given in Table 1. The hydration state clearly affects the HCH concentrations in the tissues of both moss and lichen. The concentrations were consistently higher in hydrated than in desiccated material. In addition, hydrated and desiccated material showed different selectivity for the two HCH isomers (see below). Basis of Calculation. The consequence of using different bases of calculation (surface area, fresh/dry mass, soluble lipid content) of POP concentrations in pine needles has been studied in depth,13,21,26 and similar investigations were performed for mosses and lichens in the feasibility study.24 As the uptake of contaminants from the air into “plants” is a surface process, the surface area is seemingly attractive as a basis of calculation. However, measuring the surface area of the three-dimensional structures of mosses and lichens was considered too complicated to be used in practice and would 10985



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Hydrated vs Desiccated Lichen. Similarly to the moss, the lichen C. stellaris had higher concentrations of HCHs when hydrated than when desiccated (Figure 1). Neither soluble lipid content nor dry matter (Table 1) could explain the differences between HCH levels in hydrated and desiccated material. The % soluble lipid change was not significant for the lichen (p = 0.51, decrease from 5.3% hydrated, to 5.1% desiccated; t test). Moss vs Lichen. C. stellaris had a percentage of dry matter significantly greater than that of H. splendens (p < 0.0001, t test, Table 1). For both hydrated and desiccated samples, H. splendens had a mean % soluble lipid significantly greater than that of C. stellaris (both p < 0.0001, t tests, Table 1). The steady-state concentrations of α-HCH in hydrated samples over the first 22 days differed significantly (moss mean α-HCH = 179, SD = 25; lichen mean α-HCH = 330; SD = 57; t test p < 0.0001). However, the steady-state concentration of γHCH did not differ significantly (moss mean γ-HCH = 410; SD = 57; lichen mean γ-HCH = 420; SD = 58; t test p = 0.35). Although moss and lichen samples were exposed to the same air concentrations, there was a substantial difference between the two species’ capacities to take up and retain α-HCH but not γ-HCH. For desiccated samples, the same pattern was found for αHCH (moss mean α-HCH = 45, SD = 12; lichen mean α-HCH = 88, SD = 12; t test p < 0.0001), while γ-HCH was virtually the same and not significantly different (moss mean γ-HCH = 92; SD = 14; lichen mean γ-HCH = 90; SD = 13; t test p = 0.61). The difference in the amount of extractable hydrophobic material (“soluble lipid”) in the hydrated and desiccated samples of H. splendens (Table 1) was not significant (p = 0.084, t test; decrease from mean 7.3% hydrated, to 6.9% desiccated) and too small to explain the concentration differences of the HCH isomers. Neither can the dry mass of the hydrated material add much understanding of the data. A tentative explanation is that hydration causes the constituent materials to swell, increasing the volume of the moss or lichen and thus exposing sorption sites. Because there were no changes in isomer concentrations in the indoor air over the duration of the experiment (Table S1), all the data sets of treatments 1 and 4 for moss were pooled, and pairwise α/γ ratios were calculated and compared (Table S5, Supporting Information). The difference in α/γ ratios in moss between the two treatments was significant (unpaired t test p = 0.029, using log-transformed ratios). Given that the α/γ ratio in air was 0.63 (Table S1), testing the respective α/γ treatment means against this mean using separate one-sample t tests shows that both treatments 1 and 4 α/γ ratios were also significantly different from the air ratio (p < 0.0001 and p = 0.0017, respectively). Because there were no treatment 1 or 4 with lichen, the α/γ ratios were calculated using the data from the first 22 days of treatments 2 and 3. The difference in α/γ ratios between the two treatments was also significant (unpaired t test p = 0.006, using untransformed ratios). Treatments 2 and 3 also differed from the air ratio (p < 0.0022 and p < 0.0001, respectively). Comparing the α/γ ratios between moss and lichen, the ratios were significantly different (both p < 0.0001, unpaired t tests, log-transformed data) for both hydrated and desiccated states. Therefore, moss and lichen, hydrated or desiccated, treat the isomers differently despite the constant isomer ratio in air. The most straightforward explanation is biological differences between the two species. At present, the exact nature of any such

lichens also varies with physiological status and may be affected by seasonal factors, elevation, and changes in hydration.40,41 As a consequence of the above-mentioned studies on vascular plants, mosses, and lichens, lipid mass was ruled out as a basis of calculation of POP concentrations, because an operationally defined soluble lipid fraction will not include all hydrophobic compartments of “plants”. The water relations in mosses are complex, as they have three different water compartments, external capillary water, apoplast water in the cell walls, and symplast water within the cells,28,42 complicating the use of fresh mass. Depending on turgor, the moss will have different volume and surface area. Although the water compartmentalization of lichens may not be quite as complex as in mosses,7 hydration will affect the volume and surface area as in mosses. Thus, difficulties determining the hydration state of field-collected samples rule out wet mass as a basis of calculation and leaves the dry mass as the best and most straightforward alternative. However, as the hydration state also affects the concentrations of the most volatile POPs in mosses and lichens, drying the samples before extraction will affect the concentrations. The extraction method used in this study, placing the sample material in the extraction solvent without drying, was designed to minimize evaporative losses of the analytes during drying of hydrated samples. Moss: Hydrated vs Desiccated at Steady State. There were no significant differences in mean α- or γ-HCH levels in moss kept hydrated or desiccated during the entire 56 days (treatments 1 and 4, respectively) when the mean concentration for days 1−22 and days 24−56 were compared (t tests within treatments p > 0.05). This indicates that the levels of HCHs in moss during the experiment were constant. Mean αHCH levels in treatments 1 and 2 for the first 22 days (both hydrated) were not significantly different (t test p > 0.05). The mean α-HCH levels for treatments 3 (mean 58 ng/g, SD = 16) and 4 (mean 45 ng/g, SD = 12) for the first 22 days were, however, significantly different (t test p = 0.014). But as these concentrations were the lowest measured in desiccated moss (compare first 22 days of treatments 3 and 4 in Figure 1), this difference at the beginning of the experiment was deemed not to influence further interpretation. Linear regression analysis of the time trends for α- and γHCH in treatments 1 and 4 (constantly hydrated or desiccated over 56 days) shows all slopes equal (p = 0.058), not different from horizontal (all p > 0.7), and linear (all runs tests shows all lines not significantly nonlinear, all p > 0.7). Hydrated vs Desiccated Moss. The concentrations of HCHs in H. splendens clearly depend on a dynamic uptake− revolatilization process tied to the hydration state (Figure 1). Moss kept hydrated during the whole exposure period (treatment 1) had concentrations of HCHs significantly higher than that of moss kept desiccated (treatment 4), which had consistently low concentrations (treatment 1 α-HCH mean = 180, SD = 26; treatment 4 α-HCH mean = 50, SD = 14; p < 0.0001, t test: treatment 1 γ-HCH mean = 400, SD = 61; treatment 4 γ-HCH mean = 93, SD = 14; p < 0.0001, t test). Reflecting the importance of the hydration state, desiccated moss that was hydrated (treatment 3) attained α- and γ-HCH levels equal and not significantly different from treatment 1 based on data pooled between days 24−56 (t tests p > 0.1). Treatment 2 moss that was desiccated from day 22 onward, however, only reached α- and γ-HCH levels equal to treatment 4 from day 42 onward based on pooled data (t tests p > 0.05). 10986



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Figure 2. Four-parameter logistic regressions of α- and γ-HCH in moss (M) and lichen (L); revolatilization at desiccation (treatment 2) and accumulation at hydration (treatment 3). Day 22 is indicated with a vertical dotted line. Shared symbols (* or #) indicate where a combination of Hill slope and half-time differ significantly. Parameters are provided in Table S6.

biological differences is unknown, but they may include, for example, the degree of swelling, the type of constituent molecules that swell upon wetting, the structural differences and availability of sorption sites, and the differences in the presence and composition of hydrophobic or amphoteric constituents. Kinetics of Change. Figure 2 shows the nonlinear fits of the changes in HCH isomers when desiccated (treatments 2) or hydrated (treatments 3), with additional data in Table S6, Supporting Information. R2 were all >0.8, and none of the curves deviated significantly from the model (runs test, p > 0.5). Some Hill slopes and half-times differed (Figure 2, Table S6) and were tested in two-way comparisons between the different isomers within the same organism and between the same isomers between organisms. While there was no significant difference in the revolatilization kinetics between α-HCH and γ-HCH from the moss, the revolatilization kinetics of the two compounds from the lichen were significantly different (p = 0.021). Between lichen and moss, only the revolatilization kinetics of γ-HCH was significantly different (p = 0.039). It seems that revolatilization of γ-HCH from lichen starts sooner after the start of desiccation than for α-HCH, and sooner than both isomers from moss (Figure 2, Table S6). After water sprinkling ceases, there seems to be a lag period before α-HCH from lichen, and α- and γ-HCH from moss starts to revolatilize. In contrast, there was no lag period for γHCH from lichen. On the other hand, despite the shallower slope of α-HCH revolatilizing from lichen (Table S6), the revolatilization reached bottom after day 30, while for the other three cases, bottom is reached only at or after day 40 (Figure 2). Because the top and bottom parameters were tested as described above, they were not further tested for differences in this regression. In contrast to the revolatilization, there were no differences in the accumulation kinetics for the two isomers within each species (Figure 2, Table S6) but a significant difference between moss and lichen for both isomers (α-HCH p < 0.0001, γ-HCH p < 0.008). The accumulation and revolatilization processes proceed via different pathways. While the uptake that occurs on the hydration of desiccated moss proceeds very rapidly, the revolatilization upon desiccation is slower. Treatment 2 samples were totally desiccated in about two weeks, while the HCH concentrations took another two weeks before reaching the same concentrations as in treatment 4. Tentatively, a moss at hydration has to gain full biological activity as quickly as

possible, starting the metabolic processes essentially instantly.7,28 The accumulation of HCHs proceeds rapidly probably because the total distance from the surface to interior sorption sites in a moss is small. On the other hand, during desiccation, metabolism is shut down and deswelling of the constituent materials occurs successively. The difference in timing of full desiccation and attaining the HCH concentrations typical of treatment 4 may be due to differences in the diffusion rates of water and the HCHs. It may also be due to the presence of some organic compound, less volatile than water but too volatile to affect the measured “soluble lipid” content that can retain some of the HCH in the tissues, as previously shown for vascular plants.19 Vascular Plants vs Moss and Lichen. The uptake behavior shown by H. splendens and C. stellaris stands in stark contrast to the uptake behavior of Scots pine needles19,21 in which airborne HCHs accumulate more during hot and dry than during cold and damp conditions. In all likelihood, this is due to life-history differences between homoihydric and poikilohydric “plants”. The homoihydric pines produce more and increase their pool of hydrophobic oils under hot and dry conditions,20 in part to protect their photosynthetic apparatus43 but presumably also to reduce water loss.19 In contrast, we have no indication that the pool of hydrophobic material increases with hydration in the tested poikilohydric moss or lichen. Instead, it seems as if processes induced by hydration, e.g., increased surface area and swelling of constituent molecules, facilitate increased uptake of HCHs from the gas phase. Laboratory vs Field Studies. The difference between the HCH concentrations in hydrated and desiccated moss and lichen was smaller in this laboratory study than in field samples collected during the feasibility study.24 Samples taken in weeks with overcast sky and especially during or after rain invariably had concentrations higher than those of samples taken on sunny days; substantial variations between different species of mosses and lichens were also observed. In an extreme case, the moss Pleurozium schreiberi had up to 15 times higher HCH concentrations during a cool, rainy week than those during the subsequent hot and sunny week. No air sampling was performed during the feasibility study, but variations of HCH air concentration in the range of 1 order of magnitude were observed during the preceding years.44 Thus, the larger variation among field samples than among laboratory samples reflects the larger variation in exposure conditions, including air 10987



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(7) Nash, T. H., III, Ed. Lichen Biology, 2nd ed.; Cambridge University Press: New York, 2008. (8) Boethling, R. S.; Mackay, D., Eds. Handbook of property estimation methods for chemicals: environmental and health sciences; Lewis Publishers: Boca Raton, 2000. (9) Davidson, D. A.; Wilkinson, A. C.; Blais, J. M.; Kimpe, L. E.; McDonald, K. M.; Schindler, D. W. Orographic cold-trapping of persistent organic pollutants by vegetation in mountains of western Canada. Environ. Sci. Technol. 2003, 37, 209−215. (10) Calamari, D.; Bacci, E.; Focardi, S.; Gaggi, C.; Morosini, M.; Vighi, M. Role of plant biomass in the global environmental partitioning of chlorinated hydrocarbons. Environ. Sci. Technol. 1991, 25, 1489−1495. (11) Bidleman, T. F. Atmospheric processes − Wet and dry deposition of organic compounds are controlled by their vapor-particle partitioning. Environ. Sci. Technol. 1988, 22, 361−367. (12) Stockholm Convention, www.pops.int, viewed April 24, 2010. (13) Kylin, H. Airborne lipophilic pollutants in pine needles. Environ. Sci. Pollut. Res. 1996, 3, 218−223. (14) Carlberg, G. E.; Baumann Ofstad, E.; Drangholt, H.; Steinnes, E. Atmospheric deposition of organic micropollutants in Norway studied by means of moss and lichen analysis. Chemosphere 1983, 12, 341− 356. (15) Lead, W.; Steinnes, E.; Jones, K. C. Atmospheric deposition of PCBs to moss (Hylocomium splendens) in Norway between 1977 and 1990. Environ. Sci. Technol. 1996, 30, 524−530. (16) Villeneuve, J.-P.; Fogelqvist, E.; Cattini, C. Lichens as bioindicators for atmospheric pollution by chlorinated hydrocarbons. Chemosphere 1988, 17, 399−403. (17) Augusto, S.; Máguas, C.; Banquinho, C. Understanding the performance of different lichen species as biomonitors of atmospheric dioxins and furans: potential for intercalibration. Ecotoxicology 2009, 18, 1036−1042. (18) Augusto, S.; Máguas, C.; Matos, J.; Pereira, M. J.; Branquinho, C. Lichen as an integrating tool for monitoring PAH atmospheric deposition: A comparison with soil, air and pine needles. Environ. Pollut. 2010, 158, 483−489. (19) Hellström, A. Uptake of Airborne Organic Pollutants in Pine Needles: Geographical and Seasonal Variations. Ph.D. Dissertation, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2003. (20) Kylin, H.; Söderkvist, K.; Undeman, A.; Franich, R. Seasonal variation of the terpene content, an overlooked factor in the determination of environmental pollutants in pine needles. Bull. Environ. Contam. Toxicol. 2002, 68, 155−160. (21) Kylin, H.; Sjödin, A. Accumulation of airborne hexachlorocyclohexanes and DDT in pine needles. Environ. Sci. Technol. 2003, 37, 2350−2355. (22) Rühling, Å.; Tyler, G. Changes in atmospheric deposition rates of heavy metals in Sweden: A summary of nationwide Swedish Surveys in 1968/70−1995. Water Air Soil Pollut.: Focus 2001, 1, 311−323. (23) Bringmark, L. Discussion document 1985−12−19: monitoring of organic contaminants with mosses and lichens (in Swedish); Swedish Environmental Protection Agency, Stockholm, Sweden, 1985. (24) Jensen, S. Use of moss and/or lichen to monitor airborne organic pollutants (in Swedish); report to the Swedish Environmental Protection Agency, Stockholm, Sweden, 1990. (25) Eriksson, G.; Jensen, S.; Kylin, H.; Strachan., W. M. J. The pine needle as a monitor of atmospheric pollution. Nature 1989, 341, 42− 44. (26) Strachan, W. M. J.; Eriksson, G.; Jensen, S.; Kylin, H. Organochlorine compounds in pine needles: Methods and trends. Environ. Chem. Toxicol. 1994, 13, 443−451. (27) Busby, J.-R.; Bliss, L. C.; Hamilton, C. D. Microclimate control of growth rates and habitats of the boreal forest mosses, Tomenthypnum nitens and Hylocomium splendens. Ecol. Monogr. 1978, 48, 95−110. (28) Goffinet, G.; Shaw, A. J., Eds. Bryophote Biology, 2nd ed.; Cambridge University Press: New York, 2008.

concentrations, temperature, and insolation, something that will always complicate field monitoring programs. Consequences for Sampling and Modeling. On the basis of the information obtained in this study, we cannot recommend using mosses or lichens for the biomonitoring of airborne organic contaminants present in the gas phase. Experience shows that differences in field samples are larger those than found under laboratory conditions.24 This is likely due to larger variations of temperature, air concentrations of HCHs, rain events, insolation, etc. Therefore, it is advisible to minimize differences by adapting stringent sampling protocols as to species, weather, and sun exposure conditions if mosses or lichens are to be used. Together with previous studies,19−21 this study demonstrates that physiological adaptations and life histories will affect the uptake of POPs not only in animals but also among the Linnaean “plants”. Clearly, the uptake mechanisms of airborne POPs in poikilohydric and homoihydric “plants” are fundamentally different. On the basis of this study in combination with recent phylogenetic and ecological knowledge that updates a 300-year old division of the living world, we recommend that data from vascular plants, mosses, and lichens be considered separately to build valid models of the behavior of volatile POPs in the environment.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1 with growth pattern of H. splendens, Table S1 with air concentrations, Tables S2−S4 with the concentrations in individual samples, Table S5 with α-HCH/γ-HCH ratios in moss and lichen in the different types of treatments, and Table S6 with kinetic data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +46 13282278; fax: +46 13149403; e-mail: henrik.kylin@ liu.se. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Anders Bengtsson assisted in the cultivation and laboratory sampling. The criticisms of four reviewers improved the manuscript substantially.

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REFERENCES

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Hydration State of the Moss Hylocomium splendens and the Lichen

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