Long-term evaluation of the needle surface wax condition of Pinus sylvestris around different industries in Lithuania

The aim of our study was to evaluate the annual dynamics of needle surface wax erosion and wettability in Scots pines exposed to a gradient of industrial pollutants emitted from the main factories of Lithuania: a nitrogen fertilizer factory, an oil refinery and a cement factory. Decreased emissions (in the case of the oil refinery and the cement factory) were reflected in the increased structural surface area (SSA, i.e. area covered by tubular waxes) on the needles. The nearly constant amount of emissions from the nitrogen fertilizer factory within the 1994-2000 period corresponded to negligible annual differences in SSA. Annual changes in the hydrophobicity of needles on the investigated transects were small. Despite the decreased pollution within the 7-year period, industrial emissions are still causing significantly accelerated wax erosion and increased wettability in needles sampled from the stands most heavily affected by pollutants.     

Uptake,translocation and fate of trichloroacetic acid in a Norway spruce/soil system

Trichloroacetic acid (TCA) is a secondary atmospheric pollutant formed by photooxidation of chlorinated solvents in the troposphere--it has, however, recently been ranked among natural organohalogens. Its herbicidal properties might be one of the factors adversely affecting forest health. TCA accumulates rapidly in conifer needles and influences the detoxification capacity in the trees. The aim of the investigations--a survey of which is briefly given here--was to elucidate the uptake, distribution and fate of TCA in Norway spruce. For this purpose young nursery-grown plants of Norway spruce (Picea abies (L.) Karst.) were exposed to [1,2-14C]TCA and the fate of the compound was followed in needles, wood, roots, soil and air with appropriate radio-indicator methods. As shown by radioactivity monitoring, the uptake of TCA from soil by roots proceeded most rapidly into current needles at the beginning of the TCA treatment and was redistributed at later dates so that TCA content in older needles increased. The only product of TCA metabolism/biodegradation found in the plant/soil-system was CO(2) (and corresponding assimilates). TCA biodegradation in soil depends on TCA concentration, soil humidity and other factors.     

Investigation of uptake, translocation and fate of trichloroacetic acid in Norway spruce (Picea abies/L./Karst.) using 14C-labelling.

[1,2-14C]TCA of a high specific activity (3.7 GBq/mmol) and appropriate radioindicator techniques were used, to study the effect of trichloroacetic acid (TCA) on conifers. Easy uptake of TCA from soil through spruce roots and its further translocation by the transpiration stream up to the needles (where damage of the photosynthetic apparatus occurs) has been proved. During the growth period, after one-shot load of TCA, the uptake was most intensive in current-year needles at first; over an extended period a decrease in the level of [1,2-14C]TCA-derived radioactivity was found in the current-year needles while in older needles (C + 2), the level rose. Symptoms of TCA biodegradation and/or metabolism were found in the plant/soil system under study. During an eight-week exposure significant losses of radioactivity into the atmosphere were noticed, at least a part of them in the form of carbondioxide. The results of these more or less preliminary experiments demonstrated the suitability and advantages of the radioisotopic technique used.     

Trichloroacetic acid cycling in Sitka spruce saplings and effects on sapling health following long term exposure

Trichloroacetic acid (TCA, CCl(3)COOH) has been associated with forest damage but the source of TCA to trees is poorly characterised. To investigate the routes and effects of TCA uptake in conifers, 120 Sitka spruce (Picea sitchensis (Bong.) Carr) saplings were exposed to control, 10 or 100 microg l(-1) solutions of TCA applied twice weekly to foliage only or soil only over two consecutive 5-month growing seasons. At the end of each growing season similar elevated TCA concentrations (approximate range 200-300 ng g(-1) dwt) were detected in both foliage and soil-dosed saplings exposed to 100 microg l(-1) TCA solutions showing that TCA uptake can occur from both exposure routes. Higher TCA concentrations in branchwood of foliage-dosed saplings suggest that atmospheric TCA in solution is taken up indirectly into conifer needles via branch and stemwood. TCA concentrations in needles declined slowly by only 25-30% over 6 months of winter without dosing. No effect of TCA exposure on sapling growth was measured during the experiment. However at the end of the first growing season needles of saplings exposed to 10 or 100 microg l(-1) foliage-applied TCA showed significantly more visible damage, higher activities of some detoxifying enzymes, lower protein contents and poorer water control than needles of saplings dosed with the same TCA concentrations to the soil. At the end of each growing season the combined TCA storage in needles, stemwood, branchwood and soil of each sapling was <6% of TCA applied. Even with an estimated half-life of tens of days for within-sapling elimination of TCA during the growing season, this indicates that TCA is eliminated rapidly before uptake or accumulates in another compartment. Although TCA stored in sapling needles accounted for only a small proportion of TCA stored in the sapling/soil system it appears to significantly affect some measures of sapling health.     

Determination of trichloroacetic acid in environmental studies using carbon 14 and chlorine 36

Radioisotopes carbon 14 and chlorine 36 were used to elucidate the environmental role of trichloroacetic acid (TCA) formerly taken to be a herbicide and a secondary air pollutant with phytotoxic effects. However, use of ¹⁴C-labeling posed again known analytical problems, especially in TCA extraction from the sample matrix. Therefore—after evaluation of available methods—a new procedure using decarboxylation of [1,2-¹⁴C]TCA combined with extraction of the resultant ¹⁴C-chloroform with a non-polar solvent and its subsequent radiometric measurement was developed. The method solves previous difficulties and permits an easy determination of amounts between 0.4 and 20 kBq (10–500 ng g⁻¹) of carrier-less [1,2-¹⁴C]TCA in samples from environmental investigations. The procedure is, however, not suitable for direct [³⁶Cl]TCA determination in chlorination studies with ³⁶Cl. Because TCA might be microbially degraded in soil during extraction and sample storage and its extraction from soil or needles is never complete, the decarboxylation method—i.e. 2 h TCA decomposition to chloroform and CO₂ in aqueous solution or suspension in closed vial at 90 °C and pH 4.6 with subsequent CHCl₃ extraction—is recommended here, estimated V < 7%. Moreover, the influence of pH and temperature on the decarboxylation of TCA in aqueous solution was studied in a broad range and its environmental relevance is shown in the case of TCA decarboxylation in spruce needles which takes place also at ambient temperatures and might amount more than 10–20% after a growing season. A study of TCA distribution in spruce needles after below-ground uptake shows the highest uptake rate into current needles which have, however, a lower TCA content than older needle-year classes, TCA biodegradation in forest soil leads predominatingly to CO₂.     

Trichloroacetic acid in the environment

Suppositions that the trichloroacetic acid (TCA, CCl3C(O)OH) found in nature was a consequence solely of the use of chlorinated hydrocarbon solvents prompted this critical review of the literature on its environmental fluxes and occurrences. TCA is widely distributed in forest soils (where it was rarely used as an herbicide) and measurements suggest a soil flux of 160 000 tonnes yr(-1) in European forests alone. TCA is also produced during oxidative water treatment and the global flux could amount to 55 000 tonnes yr(-1) (from pulp and paper manufacture, potable water and cooling water treatments). By contrast, the yields of TCA from chlorinated hydrocarbon solvents are small: from tetrachloroethene 13 600 tonnes yr(-1) and from 1,1,1-trichloroethane 4300 tonnes yr(-1) on a global basis, at the atmospheric burdens and removal rates typical of the late 1990s. TCA is ubiquitous in rainwater and snow. Its concentrations are highly variable and the variations cannot be connected with location or date. However, there is no significant difference between the concentrations found in Chile and in eastern Canada (by the same analysts), or between Malawi and western Canada, or between Antarctica and Switzerland, nor any significant difference globally between the concentrations in cloud, rain and snow (although local enhancement in fog water has been shown). TCA is present in old ice and firn. At the deepest levels, the firn was deposited early in the 19th century, well before the possibility of contamination by industrial production of reactive chlorine, implying a non-industrial background. This proposition is supported by plume measurements from pulp mills in Finland. TCA is ubiquitous in soils; concentrations are very variable but there are some indications that soils under coniferous trees contain higher amounts. The concentrations of TCA found in plant tissue are region-specific and may also be plant-specific, to the extent that conifers seem to contain more than other species. TCA is removed from the environment naturally. There is abundant evidence that soil microorganisms dehalogenate TCA and it is lost from within spruce needles with a half-life of 10 days. There is also recent evidence of an abiotic aqueous decarboxylation mechanism with a half-life of 22 days. The supposedly widespread effects of TCA in conifer needles are not shown in controlled experiments. At concentrations in the needles of Scots pine similar to those observed in needles in forest trees, changes consequent on TCA treatment of field laboratory specimens were almost all insignificant.     

Review of concentrations and chemistry of trichloroacetate in the environment

This paper reviews the concentrations of trichloroacetate (TCA) in the atmosphere-plant-soil system. Data originate mainly from Europe. The median TCA concentration in rainwater and canopy drip decreased until 1995. From then the median TCA concentration in rainwater remains rather constant while for canopy drip later data are not available. The same seems to hold for concentrations in air although a very limited data set is available. The median concentrations in coniferous needles and groundwater are constant for the period observed. The median TCA concentrations in soil decreased until 1992 and then remained constant.The TCA formation from chlorinated solvents in the atmosphere may explain a substantial percentage of the TCA amount in the atmosphere. The TCA concentrations in rainwater and canopy drip indicate that there will be other sources contributing to 10-50%. Waste incineration, biomass burning and natural formation in the marine boundary layer are potential candidate sources of TCA, but nothing can be said as yet on their TCA emission rates. Anthropogenic emissions of chlorine could also be a source.TCA can be formed from chlorinated solvents by biota. However, for coniferous trees the uptake of TCA from soil may be the predominant route. Biotic and abiotic reactions can cause to formation of TCA in soil, but also formation of TCA from chlorinated solvents by biota that excrete TCA, may contribute. Mass balance calculations of the bioactive soil top layer show that the production rate of TCA in certain soil types could be substantial. The mass balance calculations could not distinguish between natural and anthropogenic sources in soil.     

Haloacetic acids,phytotoxic secondary air pollutants

Haloacetic acids are atmospheric oxidation products of airborne C₂-halocarbons which are important solvents and propellants. Levels of trichloroacetate (TCA) in conifer needles from mountain ranges in Germany (Black Forest, Erzgebirge) and from two sites in Finland are compared; TCA is present in conifer needles at concentrations up to 0.7 μmol/kg, MCA up to 0.2 μmol/kg. At the Finnish sites, TCA-concentrations and branch degeneration symptoms of Scots pine are correlated. Monochloroacetate (MCA) has been determined in needle samples from Southern Germany in concentrations exceeding its phytotoxicity threshold towards photoautotrophic organisms. Data on atmospheric chloroacetate levels in Germany are also given; ambient air levels of chloroacetic acids range from about 2 pmol/m³ (TCA) to 390 pmol/m³ (MCA). TCA and dichloroacetic acid (DCA) arise from atmospheric oxidation of airborne C₂-chlorocarbons, while the source of MCA is not yet known; several tentative pathways are suggested.     

Development of a comprehensive analytical method for the determination of chlorinated paraffins in spruce needles applied in passive air sampling

Conifer needles are used for the monitoring of atmospheric persistent organic pollutants. The objective of the present study was to develop a method for the detection of airborne chlorinated paraffins (CPs) using spruce needles as a passive sampler. The method is based on liquid extraction of the cuticular wax layer followed by chromatographic fractionation and detection of CPs using two different GCMS techniques. Total CP concentrations (sum of short (SCCP), medium (MCCP) and long chain CPs (LCCP)) were determined by EI-MS/MS. SCCP and MCCP levels as well as congener group patterns (n-alkane chain length, chlorine content) could be evaluated using ECNI-LRMS. For the first time, data on environmental airborne CPs on spruce needles taken within the Monitoring Network in the Alpine Region for Persistent and other Organic Pollutants (MONARPOP) are presented providing evidence that spruce needles are a suitable passive sampling system for the monitoring of atmospheric CPs.     

Scots pine needle surfaces on radial transects across the north boreal area of Finnish Lapland and the Kola Peninsula of Russia

To gain an understanding of the characteristics of the needle surfaces of naturally regenerated Scots pine (Pinus sylvestris L.) and their geographical distribution, eight physicochemical variables were investigated within the north boreal forest area. The visibly undamaged needles were collected in autumn 1990 from 114 plots (3-5 pines per plot) along radial transects from the Monchegorsk and Nikel smelters, emitting SO2 and heavy metals, on the Kola Peninsula, Russia, to Finnish Lapland. The needles were examined by scanning electron microscopy (SEM) and measured for surface wettability using the droplet contact angle (DCA) method. Significant geographical south-north and west-east patterns could be observed in needle surface characteristics, correlating with emissions from the smelters and to climate. Despite the slightly higher initial DCAs (61.4-87.6 degrees) towards the north, the pine needle surfaces of the northernmost transects exhibited higher annual needle wettability change (ANWC) and wax erosion rate (AWER), expressed as the reduction in DCAs and epistomatal wax tube distribution (WTD) during one year, respectively, but a lower occurrence of particles and fungal hyphae than those of more southern transects. The higher ANWC was related to higher atmospheric SO2 concentration, and to the lower long-term temperature sum, but not clearly to annual precipitation. In the Monchegorsk smelter area, the current needles exhibited, on average, a 15% higher WTD and seven degree larger DCA, resulting in more hydrophobic needle surfaces than in Finnish Lapland, but during their first year, both the AWER and ANWC, were greatly increased. In Finnish Lapland, 30%, of all the epistomatal wax tubes disappeared from the needle surface during the first year, the value being 70% for the pines located 8 km from the smelter. The mineral composition of the particles deposited on the needle surfaces mirrored that of the minerals being produced by the smelters (e.g. FexSx, CuFeS2, NixSx, FexOx). Stomatal densities were in the range of 71.1-141.7 stomata mm(-2). The lower densities of stomata on needles close to the smelters correlated with reduced number of needle age classes of the pines, higher dry weights, higher pollutant accumulation and lower Mn and Zn concentrations in the needles. The AWER and ANWC were able to indicate the most seriously deteriorated needle surfaces on a regional scale, e.g. including the surroundings of both smelters, although the plot-specific relationships between 'needle surface variables' and 'pollution variables' were generally weak. The present study showed that the exposure of pine needles to the combined effects of ambient pollutants and harsh climate led to a deterioration in the physicochemical characteristics of the epicuticular wax, which may have serious ecophysiological consequences in the long term.     

Environmental risk assessment of airborne trichloroacetic acid--a contribution to the discussion on the significance of anthropogenic and natural sources

In environmental risk assessments the question has to be answered, whether risk reduction measures are necessary in order to protect the environment. If the combination of natural and anthropogenic sources of a chemical substance leads to an unacceptable risk, the man-made emissions have to be reduced. In this case the proportions of the anthropogenic and natural emissions have to be quantified. Difficulties and possible solutions are discussed in the scope of the OECD- and EU-risk assessments of trichloroacetic acid (TCA) and tetrachloroethylene.In the atmosphere, TCA is formed by photo-oxidative degradation of tetrachloroethylene (PER) and 1,1,1-trichloroethane. The available data on atmospheric chemistry indicate that tetrachloroethylene is the more important pre-cursor. With its high water solubility and low volatility, TCA is adsorbed onto aerosol particles and precipitated during rainfalls. Extended monitoring in rainwater confirmed the global distribution of airborne TCA. TCA reaches soils by dry and wet deposition. In addition formation of TCA from tetrachloroethylene in plants was observed. Consequently, high concentrations were detected in needles, leaves and in forest soil especially in mountain regions.The effect assessment revealed that plants exposed via soil are the most sensitive species compared to other terrestrial organisms. A PNECsoil of 2.4 microg/kg dw was derived from a long-term study with pine and spruce seedlings. When this PNEC is compared with the measured concentrations of TCA in soil, in certain regions a PEC/PNEC ratio >1 is obtained. This clearly indicates a risk to the terrestrial ecosystem, with the consequence that risk reduction measures are deemed necessary.To quantify the causes of the high levels of TCA in certain soils, and to investigate the geographical extent of the problem, intensive and widespread monitoring of soil, air and rainwater for TCA and tetrachloroethylene would be necessary to be able to perform a full mass balance study at an appropriate number of sites. In addition, measurements of the 14C content in TCA isolated from soil could clarify whether a significant proportion of the TCA occurs from natural sources. The possible formation of TCA in soil can also be tested by incubation of isotope enriched inorganic chloride with subsequent mass spectrometry of TCA.     

Effects of trichloroacetic acid on the nitrogen metabolism of Pinus sylvestris--a 13C/15N tracer study

Trichloroacetic acid (TCA) can be found in various environmental compartments like air, rain and plants all over the world. It is assumed that TCA is an atmospheric degradation product of volatile chloroorganic hydrocarbons. The herbicide effect of TCA in higher concentrations is well known, but not much is known about the phytotoxic effects in environmentally relevant concentrations. It can be shown in this study by using the 13C/15N stable isotope tracer technique that [13C]TCA is taken up by roots of two-year-old seedlings of Pinus sylvestris L. and transported into the needles. At the same time the effect of the substance on nitrogen metabolism can be analyzed by measuring the incorporation of 15NO3- into different nitrogen fractions of the plant. The more [13C]TCA incorporation, the higher the synthesis of 15N labelled amino acids and proteins is. These effects on the nitrogen metabolism are probably based on the activation of stress- and detoxification metabolism. It has to be assumed that there is an influence on N metabolism of Pinus sylvestris caused by the deposition of environmentally relevant TCA concentrations.     

Responses of spruce seedlings (Picea abies) to exhaust gas under laboratory conditions--II. Ultrastructural changes and stomatal behaviour

This study examines the effects of exhaust gas exposure on the epistomatal wax structure and mesophyll ultrastructure in needles of Norway spruce (Picea abies (L.) Karst.) seedlings. Stomatal diffusive resistance was also measured. Two independent exhaust gas fumigations were performed: 100 and 200 ppb measured as NO(x), for 10 days and 50, 100 and 200 ppb NO(x) for 19 days. The obstructive effect of exhaust gas exposure on epistomatal wax tubules was apparent. The stomata became covered by flat and solid wax resulting from the structural degradation of the wax crystalloids. Increasing the exhaust gas concentration in the chamber atmosphere exacerbated the degradation of the wax structure. Exhaust gas exposure induced aggregation and electron translucence of plastoglobuli, swelling of thylakoids, increase of cytoplasmic lipids and slight increase of vesiculation of cytoplasm in mesophyll cells of current and previous year needles. These changes were exemplified in current year needles. Damage to the epicuticular waxes and mesophyll ultrastructure of spruce needles most likely reflects the NO(x) and volatile hydrocarbon fraction. The alterations in epicuticular waxes and mesophyll ultrastructure can be related to accelerated senescence of the youngest, photosynthetically active, needle generation. The exhaust gas also resulted in decreased diffusive stomatal resistance at night which indicates that the exhaust gas exposure disturbed the gas exchange of spruce seedlings. The results show that even relatively short-term exposure to realistic concentrations of exhaust gas in the atmosphere can induce rather severe injuries to the needle surface structure as well as ultrastructure at the cellular level.     

The annual course of TCA formation in the lower troposphere: a modeling study

We present a modeling study investigating the influence of climate conditions and solar radiation intensity on gas-phase trichloroacetic acid (TCA) formation. As part of the ECCA-project (Ecotoxicological Risk in the Caspian Catchment Area), this modeling study uses climate data specific for the two individual climate regimes, namely "Kalmykia" and "Kola Peninsula". A third regime has also been included in this study, namely "Central Europe", which serves as a reference to somehow more moderate climate conditions. The simulations have been performed with a box modeling package (SBOX, photoRACM), which uses Regional Atmospheric Chemistry Mechanism (RACM) as its chemistry scheme. For this model a mechanism supplement has been developed including the reaction pathways of methyl chloroform photooxidation. The investigations are completed by a detailed sensitivity study addressing the impact of temperature and relative humidity. Atmospheric OH and HO2 concentrations and the NOx/HO2 ratio were identified as the governing quantities controlling the TCA formation trough methyl chloroform oxidation in the gas phase. Model calculations show a TCA production rate ranging between almost zero and 6.5 x 10(3) molecules cm(-3) day(-1) depending on location and season. In the Kalmykia regime the model predicts mean TCA production rates of 1.3 x 10(-4) and 5.4 x 10(-5) microg m(-3) year(-1) for the urban and rural environment, respectively. From the comparison of model calculations with measured TCA burdens in the soil ranging between 130 g m(-3) and 1750 g m(-3) we conclude that TCA formation through methyl chloroform photooxidation in the gas-phase is probably not the principal atmospheric TCA source in this region.     

Trichloroacetic acid in Norway spruce/soil-system. II. Distribution and degradation in the plant

Independently from its origin, trichloroacetic acid (TCA) as a phytotoxic substance affects coniferous trees. Its uptake, distribution and degradation were thus investigated in the Norway spruce/soil-system using 14C labeling. TCA is distributed in the tree mainly by the transpiration stream. As in soil, TCA seems to be degraded microbially, presumably by phyllosphere microorganisms in spruce needles. Indication of TCA biodegradation in trees is shown using both antibiotics and axenic plants.     

Regional aspects and statistical characterisation of the load with semivolatile organic compounds at remote Austrian forest sites

Spruce needles and humus layer of 25 remote forest sites spread all over Austria were investigated for their concentrations of PCDD/F, PCB, HCH, HCB, PCP, DDX and PAH. Influences of the sites' location on the detected concentrations have been identified: The north of Austria was characterised by a comparably higher overall pollutant load. In addition, altitudinal aspects were addressed. Between several compounds significant positive correlations have been identified, which were more pronounced for compounds with a stronger causal relationship. Pattern analyses allowed to identify--even for the remote sites--differences between the relative PCDD/F, HCH, DDX and PAH patterns of the sites. Partly, these different patterns were related to significantly higher or lower corresponding absolute concentrations of the sites.     

Persistent effects of ozone on needle water loss and wettability in Norway spruce

Four-year-old, seed-grown trees of Norway spruce (Picea abies (L.) Karst.) were exposed in open-top chambers to charcoal-filtered air (8 h daily mean 54 microg O(3) m(-3)) over three consecutive summers (1986-1988). In mid-May 1988, before the third season of fumigation and more than 7 months after exposure to ozone the previous summer had terminated, daily rates of transpiration from intact shoots and water loss from excised needles were measured together with the amount of wax on the needle surface. In mid-July, 92 days after the beginning of the third year of exposure, the wettability of needles was assessed by measuring the contact angle of water droplets on the surface of needles. Exposure to 156 microg O(3) m(-3) resulted in a 16% increase in daily transpiration in current year's needles and a 28% increase in 1-year old needles. These effects were associated with slower stomatal closure in response to increasing water deficit in the needles previously exposed to 156 microg m(-3) ozone. The long-lasting nature of such ozone-induced effects could predispose trees to drought and winter desiccation. No significant effects of ozone were found on the amount of wax covering the needle surface, but a marked increase in the wettability of needles exposed to ozone was observed. The far reaching physiological consequences of these effects in the field and the possibility that similar disturbances may contribute to the decline of high-altitude forests of Norway spruce in Europe are discussed.     

Surface structures and epicuticular wax composition of spruce needles after long-term treatment with ozone and acid mist

Epicuticular wax structures and epicuticular wax composition were studied in needles of Picea abies (L.) Karst. plants after a long-term fumigation experiment. SEM observations showed aggregated as well as undamaged waxes without any relation to treatments. The chemical wax analysis revealed clone specific differences, but only one compound, the nonacosan-10-ol (C(29)-ol), was found enriched in ozone/acid mist treatments.     

Effects of the ultraviolet-B radiation (UV-B) on conifers: a review

The current knowledge on conifer responses to enhanced ultraviolet-B (UV-B) radiation is mainly based on greenhouse or growth chamber experiments of one growing season in duration. However, the biomass losses observed in greenhouses do not occur in field-grown trees in their natural habitats. Moreover, the majority of the 20 conifer species studied have been 1-year-old seedlings, and no studies have been undertaken on mature trees. Fully grown needles, with their glaucous waxy surfaces and thick epidermal cells with both soluble and wall-bound UV-B screening metabolites, are well protected against UV-B radiation. However, it is not known whether these are sufficient protectants in young emerging needles or during the early spring period of high UV-B levels reflected from snow. In order to understand all the mechanisms that result in the protection of conifer needles against UV-B radiation, future research should focus on the epidermal layer, separating the waxes, cuticle and epidermal and hypodermal cells. Parallel studies should consist of wall-bound and soluble secondary metabolite analysis, antioxidant measurements and microscopic observations.