Climate change impacts on population growth across a species' range differ due to nonlinear responses of populations to climate and variation in rates of climate change.

Impacts of climate change can differ substantially across species' geographic ranges, and impacts on a given population can be difficult to predict accurately. A commonly used approximation for the impacts of climate change on the population growth rate is the product of local changes in each climate variable (which may differ among populations) and the sensitivity (the derivative of the population growth rate with respect to that climate variable), summed across climate variables. However, this approximation may not be accurate for predicting changes in population growth rate across geographic ranges, because the sensitivities to climate variables or the rate of climate change may differ among populations. In addition, while this approximation assumes a linear response of population growth rate to climate, population growth rate is typically a nonlinear function of climate variables. Here, we use climate-driven integral projection models combined with projections of future climate to predict changes in population growth rate from 2008 to 2099 for an uncommon alpine plant species, Douglasia alaskana, in a rapidly warming location, southcentral Alaska USA. We dissect the causes of among-population variation in climate change impacts, including magnitude of climate change in each population and nonlinearities in population response to climate change. We show that much of the variation in climate change impacts across D. alaskana's range arises from nonlinearities in population response to climate. Our results highlight the critical role of nonlinear responses to climate change impacts, suggesting that current responses to increases in temperature or changes in precipitation may not continue indefinitely under continued changes in climate. Further, our results suggest the degree of nonlinearity in climate responses and the shape of responses (e.g., convex or concave) can differ substantially across populations, such that populations may differ dramatically in responses to future climate even when their current responses are quite similar.

Understanding microRNA Regulation Involved in the Metamorphosis of the Veined Rapa Whelk (Rapana venosa).

The veined rapa whelk (Rapana venosa) is widely consumed in China. Nevertheless, it preys on oceanic bivalves, thereby reducing this resource worldwide. Its larval metamorphosis comprises a transition from pelagic to benthic form, which involves considerable physiological and structural changes and has vital roles in its natural populations and commercial breeding. Thus, understanding the endogenous microRNAs (miRNAs) that drive metamorphosis is of great interest. This is the first study to use high-throughput sequencing to examine the alterations in miRNA expression that occur during metamorphosis in a marine gastropod. A total of 195 differentially expressed miRNAs were obtained. Sixty-five of these were expressed during the transition from precompetent to competent larvae. Thirty-three of these were upregulated and the others were downregulated. Another 123 miRNAs were expressed during the transition from competent to postlarvae. Ninety-six of these were upregulated and the remaining 27 were downregulated. The expression of miR-276-y, miR-100-x, miR-183-x, and miR-263-x showed a >100-fold change during development, while the miR-242-x and novel-m0052-3p expression levels changed over 3000-fold. Putative target gene coexpression, gene ontology, and pathway analyses suggest that these miRNAs play important parts in cell proliferation, migration, apoptosis, metabolic regulation, and energy absorption. Twenty miRNAs and their target genes involved in ingestion, digestion, cytoskeleton, cell adhesion, and apoptosis were identified. Nine of them were analyzed with real-time polymerase chain reaction (PCR), which showed an inverse correlation between the miRNAs and their relative expression levels. Our data elucidate the role of miRNAs in R. venosa metamorphic transition and serve as a solid basis for further investigations into regulatory mechanisms of gastropod metamorphosis.

Response of phenolic metabolism to cadmium and phenanthrene and its influence on pollutant translocations in the mangrove plant Aegiceras corniculatum (L.) Blanco (Ac).

Polyphenolic compounds are abundant in mangrove plants, playing a pivotal role in the detoxification of pollutants extruded from surrounding environments into plant tissues. The present study aimed to examine the variations of phenolic compounds, namely total polyphenolics, soluble tannins, condensed tannins and lignin, in the mangrove plant Aegiceras corniculatum (L.) due to the presence of exogenous cadmium and phenanthrene and to explore the influence of phenolic metabolism on biological translocation of these pollutants from roots to leaves. After a 6-week exposure to cadmium and phenanthrene, significant accumulations of both pollutants were observed. All determined phenolic compounds in both leaves and roots at high dosage levels were enhanced compared to the uncontaminated plant. Elevations of polyphenols in both treatments are possibly a result of stimulation in the activity of phenylalanine ammonia-lyase (PAL) and the enrichment of soluble sugar. Additionally, a significantly positive dosage relationship between polyphenolic metabolism intensity and phenanthrene contamination levels was found, while the trend observed in cadmium treatment was weak since cadmium at high levels inhibited phenolic production. The enrichment of polyphenols led to a decline in the biological translocation of these pollutants from roots to leaves. The immobilization of pollutants in the plant roots is possibly linked to the adsorption potential of polyphenols. These results will improve the understanding of the tolerance of mangrove plants to exogenous pollutants and will guide the selection of plants in phytoremediation because of the variability of polyphenol concentrations among species.

Shading and litter mediate the effects of soil fertility on the performance of an understorey herb.

BACKGROUND AND AIMS: Soil fertility and topographic microclimate are common determinants of plant species distributions. However, biotic conditions also vary along these abiotic gradients, and may mediate their effects on plants. In this study, we investigated whether soils and topographic microclimate acted directly on the performance of a focal understorey plant, or indirectly via changing biotic conditions. METHODS: We examined direct and indirect relationships between abiotic variables (soil fertility and topographic microclimate) and biotic factors (overstorey and understorey cover, litter depth and mycorrhizal colonization) and the occurrence, density and flowering of a common understorey herb, Trientalis latifolia, in the Klamath-Siskiyou Mountains, Oregon, USA. RESULTS: We found that the positive effects of soil fertility on Trientalis occurrence were mediated by greater overstorey shading and deeper litter. However, we did not find any effects of topographic microclimate on Trientalis distribution that were mediated by the biotic variables we measured. The predictive success of Trientalis species distribution models with soils and topographic microclimate increased by 12 % with the addition of the biotic variables. CONCLUSIONS: Our results reinforce the idea that species distributions are the outcome of interrelated abiotic gradients and biotic interactions, and suggest that biotic conditions, such as overstorey density, should be included in species distribution models if data are available.

Occurrence and distribution of the environmental pollutant antibiotics in Gaoqiao mangrove area, China.

Occurrence and distribution of 15 antibiotics belonging to families of sulfonamides, fluoroquinolones, tetracyclines and chloramphenicols were investigated in water and sediment in Gaoqiao mangrove area, China, using LC-MS-MS. The influence of tidal level and mangrove vegetation on antibiotic residues were examined. The levels of antibiotics were found to be ranged from 0.15 to 198 ng L(-1) in water and from 0.08 to 849 µg kg(-1) in sediment. No significant difference in concentrations of 15 different antibiotics from water and sediment samples was observed among the high, middle and low intertidal channel. The residues of SMZ, SMTZ, OFL, NOR, ENR, OXY and FLO were significantly higher in Aegiceras corniculatum assemblage than in Avicennia marina assemblage. Although no significant difference in tested antibiotics was found between the surface and bottom sediment, mangrove vegetation can to some extent reduce the accumulation for SMZ, SMTZ, OFL, NOR, CIP, OXY and TET in sediments relative to corresponding bare mudflats, implying that the environmental pollution from antibiotics may be mitigated by mangrove vegetation. Principal components analysis revealed that the terrestrial input and different habitats directly influenced the occurrence and distribution of antibiotics.

Contamination and distribution of heavy metals, polybrominated diphenyl ethers and alternative halogenated flame retardants in a pristine mangrove.

Owing to the expanding metal and electronics industries, pollution in the Pearl River Estuary needs special concern. Given the hydrodynamic effect, the pristine mangrove in Qi'ao Island would be contaminated by tidal flushing. Thus, we examined (1) the contamination of pollutants in this mangrove, including heavy metals, polybrominated diphenyl ethers (PBDEs) and alternative halogenated flame retardants (AHFRs), and (2) how habitat characteristics and sediment properties affect their distribution. Results showed that the sediment in Qi'ao mangrove had higher concentrations of heavy metals, PBDEs and AHFRs than that in other pristine mangroves, and similar concentrations to those mangroves impacted by point sources. Heavy metal concentrations were lower in the vegetated areas than mudflat, while the opposite was found for PBDEs and AHFRs. The findings imply that tidal flushing was an important pollution source, while mangrove plants have the capacity to minimize the impact of heavy metals, but not PBDEs and AHFRs.

[A Cellular Automata Model for a Community Comprising Two Plant Species of Different Growth Forms].

A cellular automata computer model for the interactions between two plant species of different growth forms--the lime hairgrass Deschampsia caespitosa (L.) P. Beauv., a sod cereal, and the moneywort Lysimachia nummularia L., a ground creeping perennial herb--is considered. Computer experiments on the self-maintenance of the populations of each species against the background of a gradual increase in the share of randomly eliminated individuals, coexistence of the populations of two species, and the effect of the phytogenous field have been conducted. As has been shown, all the studied factors determine the number of individuals and self-sustainability of the simulated populations by the degree of their impact. The limits of action have been determined for individual factors; within these limits, the specific features in plant reproduction and dispersal provide sustainable coexistence of the simulated populations. It has been demonstrated that the constructed model allows for studying the long-term developmental dynamics of the plants belonging to the selected growth forms.

[Histomorphological study on folk medicine Lysimachia fortunei].

To set standards for histomorphological studies on Lysimachia fortunei, an efficacious and widely applied folk medicine in this study, in order to develop its resources. Its species were identified by observing plant morphology and herbs appearance characters, preparing slices with routine methods and defining structural characters. According to the results of morphologic observation, leaves, stamen and pistil of this plant were different from the descriptions in Flora of China. The whole herb can be used in medicines, mainly including rhizomes, stems and leaves. According to the findings in the first study on microscopic structures, its rhizomes, stems and leaves were characteristic and worth identifying. The transaction tissue structures of rhizomes and stems were under developed and contained endodermis, secretory structures; Stems had sclerenchymata of different shapes of sclereids; Leaves were bifacial and had vascular bundles under midribs, which were surrounded by parenchymal sheathes. On the surface of leaves, stomata, glandular hairs and keratin lines were morphologically different in upper and lower epidermis. The herbal power had glandular hairs, sclereids and vessels. In conclusion, herbs of L. fortunei can be identified by the above histomorphological characteristics, which lays a foundation for further development and application of L. fortunei.

Physiological and biochemical response to drought stress in the leaves of Aegiceras corniculatum and Kandelia obovata.

Drought stress is one of the major abiotic stresses that affects plant growth and metabolism adversely around the world. According to this research, the effect of drought stress on the activity of antioxidative enzymes, soluble sugar, protein and lipid peroxidation were studied in leaves of two mangrove plants, Kandelia obovata and Aegiceras corniculatum. The result showed that superoxide dismutase (SOD) and peroxidase (POD) varied significantly between the leaves and roots studied. The activities increased in different stress levels. The production rate of O 2 (-·) changed with the activity of SOD and POD. Lipid peroxidation was enhanced and Glycine betaine (GB) could decrease the level of malonaldehyde in order to reduce the damage of membrane system. The content of soluble sugar and protein also increased under drought stress and GB helped to eliminate the accumulation of them which somehow enhance the ability of defensing the plants under drought stress. These results indicated that antioxidative activity may play an important role in A. corniculatum and K. obovata and that cell membrane in leaves of K. obovata had greater stability than those of A. corniculatum. Exogenous application of GB had positive effects on A. corniculatum and K. obovata under drought stress which could be products exogenously applied to mangrove plants in order to alleviates the adverse effects.

Contamination of polycyclic aromatic hydrocarbons (PAHs) in surface sediments and plants of mangrove swamps in Shenzhen, China.

The concentrations of 16 individual and total polycyclic aromatic hydrocarbons (∑PAHs) in sediments, roots and leaves of three mangrove swamps in Shenzhen, China, namely Futian, Baguang and Waterlands, were determined. The mean concentration of ∑PAHs in Futian (4480 ng g(-1)) was significantly higher than that in Baguang (1262 ng g(-1)) and Waterlands (2711 ng g(-1)). Among the 16 PAHs, the concentration of naphthalene was the highest. Based on the ratios of phenanthrene/anthracene and fluoranthene/pyrene, PAHs in Futian and Waterlands came from petrogenic and pyrolytic sources, while Baguang was mainly from pyrolytic. More PAHs were accumulated in leaves, as reflected by its higher mean concentration of ∑PAHs (3697 ng g(-1)) and bioconcentration factor of PAHs (BCF) (>1.5) than that in roots. The BCF values in plants collected from Futian were significantly higher than that from Waterlands. These results indicated that more attention should be paid to the PAH contamination in Futian.

Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata.

OBJECTIVE: Labisia pumila var. alata, commonly known as 'Kacip Fatimah' or 'Selusuh Fatimah' in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. METHODS: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. RESULTS: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34 ± 19.55)% and (70.40 ± 14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00 ± 7.07)% and (77.78 ± 16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5 ± 5.0) and (30.0 ± 8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00 ± 0.00)%) that was obtained in 1 mg/L zeatin after (11.0 ± 2.8) d of culture. CONCLUSIONS: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs.

Primary, secondary metabolites, photosynthetic capacity and antioxidant activity of the Malaysian Herb Kacip Fatimah (Labisia Pumila Benth) exposed to potassium fertilization under greenhouse conditions.

A randomized complete block design was used to characterize the relationship between production of total phenolics, flavonoids, ascorbic acid, carbohydrate content, leaf gas exchange, phenylalanine ammonia-lyase (PAL), soluble protein, invertase and antioxidant enzyme activities (ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) in Labisia pumila Benth var. alata under four levels of potassium fertilization experiments (0, 90, 180 and 270 kg K/ha) conducted for 12 weeks. It was found that the production of total phenolics, flavonoids, ascorbic acid and carbohydrate content was affected by the interaction between potassium fertilization and plant parts. As the potassium fertilization levels increased from 0 to 270 kg K/ha, the production of soluble protein and PAL activity increased steadily. At the highest potassium fertilization (270 kg K/ha) L. pumila exhibited significantly higher net photosynthesis (A), stomatal conductance (g(s)), intercellular CO(2) (C(i)), apparent quantum yield (ξ) and lower dark respiration rates (R(d)), compared to the other treatments. It was found that the production of total phenolics, flavonoids and ascorbic acid are also higher under 270 kg K/ha compared to 180, 90 and 0 kg K/ha. Furthermore, from the present study, the invertase activity was also found to be higher in 270 kg K/ha treatment. The antioxidant enzyme activities (APX, CAT and SOD) were lower under high potassium fertilization (270 kg K/ha) and have a significant negative correlation with total phenolics and flavonoid production. From this study, it was observed that the up-regulation of leaf gas exchange and downregulation of APX, CAT and SOD activities under high supplementation of potassium fertilizer enhanced the carbohydrate content that simultaneously increased the production of L. pumila secondary metabolites, thus increasing the health promoting effects of this plant.

[Secondary productivity of macrobenthos in mangrove and salt marsh in Gaoqiao of Zhanjiang, Guangdong Province of South China].

In order to understand the secondary productivity of macrobenthos in different botanic habitats in intertidal zone in Gaoqiao of Zhanjiang, the Brey's empirical formula was applied to calculate the secondary productivity based on the investigations in the habitats of Sonneratia apetala, Aegiceras corniculatum, Sporobolus virginicus, and Bruguiera conjugate in four seasons, 2010. The secondary productivity of the macrobenthos in the habitats in four seasons was averagely 11.77 g AFDM x m(-2) x a(-1), being the highest in S. apetala habitat (18.16 g AFDM x m(-2) x a(-1)), followed by in A. corniculatum habitat (17.67 g AFDM x m(-2) x a(-1)), S. virginicus habitat (8.34 g AFDM x m(-2) x a(-1)), and B. conjugate habitat (2.92 g AFDM x m(-2) x a(-1)). The P/B ratio of the macrobenthos was the highest in B. conjugate habitat (2.38), followed by in S. virginicus, S. virginicus, and A. corniculatum habitats, with the values of 1.23, 0.99, and 0.48, respectively. The differences in the secondary productivity and P/B ratio of the macrobenthos among the four botanic habitats were mainly related to the sediment total organic carbon, food type, and macrobenthos individual size.

The effect of mangrove reforestation on the accumulation of PCBs in sediment from different habitats in Guangdong, China.

To investigate the influence of mangrove reforestation on the accumulation of PCBs, the concentrations and homologue patterns of polychlorinated biphenyls in surface sediments from different mangrove forests and their adjacent mud flats in Guangdong Province were determined. The total PCB concentrations in the sediments ranged from 3.03 to 46.62 ng g⁻¹ (dry weight). Differences in the accumulation and distribution of PCBs were found between the mangrove sites and the mud flats. Furthermore, the natural forests and restored mangrove forests of native species showed slight PCB contamination, whereas the exotic species Sonneratia apetala exacerbated the PCB pollution at certain sites. It was suggested that the native mangrove species Kandelia candel and Aegiceras corniculatum could represent good choices for the phytoremediation of PCB contamination.

[Eco-physiological responses of high-latitude transplanted Aegiceras corniculatum seedlings to NaCl stress].

A sand culture pot experiment was conducted to study the eco-physiological responses of high-latitude transplanted mangrove Aegiceras corniculatum seedlings to varying concentration of NaCl (0, 100, 200, 300, and 400 mmol x L(-1)). Under the stress of 100 mmol NaCl x L(-1), the seedling growth was slightly promoted; whereas at 300 mmol NaCl x L(-1), the plant height, stem basal diameter, fresh and dry mass, and root/shoot ratio were decreased significantly. High salt stress inhibited the leaf superoxide dismutase (SOD) and peroxidase (POD) activities, increased the leaf malondialdehyde (MDA) content, and decreased the leaf chlorophyll and carotenoids contents as well as the total soluble sugar and free amino acid contents in different organs. Under the stress of different concentration NaCl, the Na+ contents in leaf, stem, and root increased rapidly while the K+ contents had a relatively decrease, resulting in a rapid decrease of K+/Na+ ratio and an imbalance between K+ and Na+ in A. corniculatum vegetative organs. When the NaCl concentration in the medium was higher than 300 mmol x L(-1), the C and N metabolism of A. corniculatum vegetative organs was maladjusted, which inhibited the normal growth of the seedlings, resulting in a significant decrease in the plant height and fresh and dry mass.

The role of radial oxygen loss and root anatomy on zinc uptake and tolerance in mangrove seedlings.

Root anatomy, radial oxygen loss (ROL) and zinc (Zn) uptake and tolerance in mangrove plants were investigated using seedlings of Aegiceras corniculatum, Bruguiera gymnorrhiza and Rhizophora stylosa. The results revealed that B. gymnorrhiza, which possessed the 'tightest barrier' in ROL spatial patterns among the three species studied, took up the least Zn and showed the highest Zn tolerance. Furthermore, zinc significantly decreased the ROL of all three plants by inhibition of root permeability, which included an obvious thickening of outer cortex and significant increases of lignification in cell walls. The results of SEM X-ray microanalysis further confirmed that such an inducible, low permeability of roots was likely an adaptive strategy to metal stress by direct prevention of excessive Zn entering into the root. The present study proposes new evidence of structural adaptive strategy on metal tolerance by mangrove seedlings.

[Spatial patterns of and specific correlations between dominant tree species in a karst evergreen and deciduous broadleaved mixed forest in Mulun Karst National Nature Reserve].

In order to understand the biological characteristics and specific correlations of dominant tree species in a karst characteristic evergreen and deciduous broad-leaved mixed forest in Mulun National Nature Reserve of Guangxi, a point pattern analysis was made on the spatial distribution patterns and inter- and intraspecific correlations of four dominant species in a one-hectare plot. Among the four species, Boniodendron minius dominated in tree sublayer I, while Ligustrum japonicum, Sinosideroxylon wightianum, and Rapanea kwangsiensis dominated in tree sublayers II and III. All the four species had a clumped distribution at scale <10 m, a transition from clumped to random distribution at scale 10-25 m, and a random or regular distribution at scale >25 m. The critical scale from clumped to random distribution varied with species. No significant correlations were observed between the B. minius in sublayer I and the dominant species in sublayer II. The correlations of B. minius with the dominant species in sublayers II and III showed greater fluctuation, with significant positive correlation for L. japonicum at scale <50 m, no significant correlation for S. wightianum, and no significant correlation for R. kwangsiensis at scale <20 m but significant negative correlation at scale 20-50 m.

Mixed heavy metals tolerance and radial oxygen loss in mangrove seedlings.

The effects of a mixture of heavy metals (Pb, Zn and Cu) on growth, radial oxygen loss (ROL) and the spatial pattern of ROL were investigated in mangrove seedlings of three species: Aegiceras corniculatum, Avicennia marina and Bruguiera gymnorrhiza. Heavy metals inhibited the growth of seedlings and led to decreased ROL and changes in the "tight" barrier spatial pattern of ROL. There was a significant positive correlation between the amount of ROL from the roots of seedlings and metal tolerance. The species with the highest ROL amount, B. gymnorrhiza, were also the most tolerant to heavy metals. The "tight" barrier spatial ROL pattern was also related to metal tolerance in the seedlings. Therefore, we conclude that both ROL amount and "tight" barrier spatial ROL pattern in the roots of the mangrove seedlings play an important role in resistance to heavy metal toxicity.

Effects of salinity on treatment of municipal wastewater by constructed mangrove wetland microcosms.

The effects of salinity on the removal of dissolved organic carbon and nutrients from municipal wastewater by constructed mangrove microcosms planted with Aegiceras corniculatum were investigated. During the four-month wastewater treatment, the treatment efficiency was reduced by high salinity, and the removal percentages of dissolved organic carbon, ammonia-N and inorganic N dropped from 91% to 71%, from 98% to 83% and from 78% to 56%, respectively, with salinity increasing from 0 to 30 parts per thousands (ppt). In spite of such inhibition at high salinity, 100% of the effluents discharge from the constructed mangrove microcosms still complied with the discharge standards set by the Hong Kong Government for Coastal Water Control Zones. These results suggested that constructed mangrove wetland treatment systems were promising to effectively treat municipal wastewater, even those with high salinity. In addition, the denitrification potential in soil was found to be retarded by the high salinity while mangrove plants grew best at 15 ppt salinity condition.

Effectiveness of bacterial inoculum and mangrove plants on remediation of sediment contaminated with polycyclic aromatic hydrocarbons.

The remediation of mangrove sediment contaminated with mixed polycyclic aromatic hydrocarbons (PAHs) having 3-, 4- and 5-rings by natural attenuation, bioaugmentation, phytoremediation and its combination was compared by greenhouse microcosm studies. At Days 90 and 154, the decreases of PAHs in contaminated mangrove sediment by phytoremediation, planted with one-year old Aegiceras corniculatum, and bioaugmentation, the inoculation of PAH-degrading bacterial strains isolated from mangrove sediment, either SCSH (Mycobacterium parafortuitum) or SAFY (Sphingobium yanoikuyae), were not better than that by natural attenuation (the non-vegetated and un-inoculated microcosms). The populations of SCSH and SAFY in sediment could not be maintained even with repeated inoculation, suggesting that the two isolates were not able to compete with the indigenous microbes and had little enhancement effect. Although some PAHs were accumulated in roots, root uptake only accounted for <15% of the spiked PAHs and the effect of plants on remediation were also insignificant. At the end of the 154-day experiment, the mass balance calculation revealed that the overall losses of PAHs by phytoremediation were comparable to that by bioaugmentation but were lower than that by natural attenuation, especially for the high molecular weight PAHs. Under natural attenuation, around 90% fluorene, 80% phenanthrene, 70% fluoranthene, 68% pyrene and 32% benzo[a]pyrene in contaminated sediment were removed. These results demonstrated that the mangrove sediment itself had sufficient indigenous microorganisms capable of naturally remedying PAH contamination.