Assessment of estrogenic and androgenic activity in PM10 air samples from an urban, industrial and rural area in Flanders (Belgium) using the CALUX bioassay.

BACKGROUND: Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. OBJECTIVES: The objective of this study was to investigate and compare the overall estrogen and androgen activating potential of PM10 air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay. METHODS: PM10 samples were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen- or androgen responsive human carcinoma cell line. Results were expressed as bioanalytical equivalents (BEQs) per cubic meter of air. RESULTS: High fluctuations in estrogenic activity were observed during the entire sampling period, with median BEQs of 32.1, 35.9 and 31.1 fg E2-Eq m(-)³ in the industrial, urban and rural background area, respectively. Estrogenic activity was measured in 70% of the samples, while no androgenic activity was observed in any of the samples. The estrogenic activity in the industrial area was positively correlated with the airborne concentration of the sum of the non-carcinogenic PAHs pyrene and fluoranthene (rho=0.48; p<0.01) and the sum of the carcinogenic PAHs (rho=0.36; p=0.05). CONCLUSIONS: This study showed that no androgenic activity was present in PM10 and that although the median estrogenic activity was rather low and comparable in the three locations, high fluctuations in estrogenic response exist over time. While atmospheric PAHs contributed to the observed estrogenic response, especially in the industrial area, the chemicals responsible for the majority of estrogenic activity remain to be identified.

Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses. This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses.

Plant movements and climate warming: intraspecific variation in growth responses to nonlocal soils.

Most range shift predictions focus on the dispersal phase of the colonization process. Because moving populations experience increasingly dissimilar nonclimatic environmental conditions as they track climate warming, it is also critical to test how individuals originating from contrasting thermal environments can establish in nonlocal sites. We assess the intraspecific variation in growth responses to nonlocal soils by planting a widespread grass of deciduous forests (Milium effusum) into an experimental common garden using combinations of seeds and soil sampled in 22 sites across its distributional range, and reflecting movement scenarios of up to 1600 km. Furthermore, to determine temperature and forest-structural effects, the plants and soils were experimentally warmed and shaded. We found significantly positive effects of the difference between the temperature of the sites of seed and soil collection on growth and seedling emergence rates. Migrant plants might thus encounter increasingly favourable soil conditions while tracking the isotherms towards currently 'colder' soils. These effects persisted under experimental warming. Rising temperatures and light availability generally enhanced plant performance. Our results suggest that abiotic and biotic soil characteristics can shape climate change-driven plant movements by affecting growth of nonlocal migrants, a mechanism which should be integrated into predictions of future range shifts.

Multilayered modeling of particulate matter removal by a growing forest over time, from plant surface deposition to washoff via rainfall.

Airborne fine particulate matter (PM) is responsible for the most severe health effects induced by air pollution in Europe. Vegetation, and forests in particular, can play a role in mitigating this pollution since they have a large surface area to filter PM out of the air. Many studies have solely focused on dry deposition of PM onto the tree surface, but deposited PM can be resuspended to the air or may be washed off by precipitation dripping from the plants to the soil. It is only the latter process that represents a net-removal from the atmosphere. To quantify this removal all these processes should be accounted for, which is the case in our modeling framework. Practically, a multilayered PM removal model for forest canopies is developed. In addition, the framework has been integrated into an existing forest growth model in order to account for changes in PM removal efficiency during forest growth. A case study was performed on a Scots pine stand in Belgium (Europe), resulting for 2010 in a dry deposition of 31 kg PM2.5 (PM < 2.5 µm) ha(-1) yr(-1) from which 76% was resuspended and 24% washed off. For different future emission reduction scenarios from 2010 to 2030, with altering PM2.5 air concentration, the avoided health costs due to PM2.5 removal was estimated to range from 915 to 1075 euro ha(-1) yr(-1). The presented model could even be used to predict nutrient input via particulate matter though further research is needed to improve and better validate the model.

Nitrogen saturation and net ecosystem production.

Magnani et al. found that net carbon (C) sequestration of temperate and boreal forests is clearly driven by nitrogen (N) deposition. From the positive relationship between average net ecosystem production (NEP) and wet N deposition, the authors further conclude that "no signs of N saturation were apparent" in the studied forests and that this is "casting doubts on the risk of widespread ecosystem nitrogen saturation". Nitrogen additions can clearly alter net ecosystem production, but net ecosystem production cannot be used as an indicator of N saturation.

Effects of two contrasting hemiparasitic plant species on biomass production and nitrogen availability.

Hemiparasitic plants can substantially change plant community structure; the drainage of host resources has a direct negative effect on host biomass and, as a consequence, promotes non-host biomass production (parasitism pathway); on the other hand, hemiparasitic litter inputs can enhance nutrient cycling which may have an indirect positive effect on both host and non-host biomass production (litter pathway). We evaluated the net effect of both pathways on total shoot biomass (with and without the hemiparasite) and shoot biomass of graminoids, forbs and ericaceous shrubs using a removal experiment in three sites infested with the annual Rhinanthus angustifolius, and three sites infested with the biennial Pedicularis sylvatica. We addressed the potential importance of litter effects by determination of litter quantity and quality, as well as modeling N release during decomposition. In the second year after removing the hemiparasites, total plant biomass at Rhinanthus sites was 24 % higher in weeded plots than in control plots, while weeding had no significant effect at Pedicularis sites. The increase in total biomass following Rhinanthus removal was mainly due to a higher biomass of graminoids. The amount of litter produced by Rhinanthus was only half of that produced by Pedicularis; N contents were similar. The amount of N in the litter was 9 and 30 % of the amount removed by mowing for Rhinanthus and Pedicularis sites, respectively. Within 2 months, about 45 % of the N in both hemiparasitic litter types was released by decomposition. Our results suggest that in addition to the suppression of host biomass due to parasitism, positive litter feedbacks on host and non-host biomass-via an increase in nutrient availability-also affect plant community structure. We propose that, depending on the particular hemiparasite and/or site conditions, these positive litter feedbacks on shoot biomass can compensate for the negative effect of parasitism.

Four decades of post-agricultural forest development have caused major redistributions of soil phosphorus fractions.

Fertilisation of agricultural land causes an accumulation of nutrients in the top soil layer, among which phosphorus (P) is particularly persistent. Changing land use from farmland to forest affects soil properties, but changes in P pools have rarely been studied despite their importance to forest ecosystem development. Here, we describe the redistributions of the P pools in a four-decadal chronosequence of post-agricultural common oak (Quercus robur L.) forests in Belgium and Denmark. The aim was to assess whether forest age causes a repartitioning of P throughout the various soil P pools (labile P, slowly cycling P and occluded P); in particular, we addressed the time-related alterations in the inorganic versus organic P fractions. In less than 40 years of oak forest development, significant redistributions have occurred between different P fractions. While both the labile and the slowly cycling inorganic P fractions significantly decreased with forest age, the organic fractions significantly increased. The labile P pool (inorganic + organic), which is considered to be the pool of P most likely to contribute to plant-available P, significantly decreased with forest age (from >20 to <10% of total P), except in the 0-5 cm of topsoil, where labile P remained persistently high. The shift from inorganic to organic P and the shifts between the different inorganic P fractions are driven by biological processes and also by physicochemical changes related to forest development. It is concluded that the organic labile P fraction, which is readily mineralisable, should be taken into account when studying the bioavailable P pool in forest ecosystems.

Advances in 15N-tracing experiments: new labelling and data analysis approaches.

To obtain an in-depth understanding of soil nitrogen dynamics, it is necessary to quantify a variety of simultaneously occurring gross nitrogen transformation processes. In order to do so, most studies apply 15N in a disturbed soil-microbial-root system and quantify gross rates based on the principles of 15N isotope dilution. However, this approach has several shortcomings. First, studying disturbed soil provides only limited information on in situ soil nitrogen dynamics. Secondly, the analytical data analysis allows the quantification of total production and consumption rates of the labelled pool, but does not provide information on process-specific transformation rates. Combining in situ 15N isotope labelling over 1-2 weeks with numerical data analysis allows determining process-specific gross nitrogen transformations in undisturbed soils under field conditions in the presence of live roots and their associated microbial communities. This has the potential to increase our understanding of nitrogen dynamics in the soil environment.

Toxicity of Urban PM10 and Relation with Tracers of Biomass Burning.

The chemical composition of particles varies with space and time and depends on emission sources, atmospheric chemistry and weather conditions. Evidence suggesting that particles differ in toxicity depending on their chemical composition is growing. This in vitro study investigated the biological effects of PM10 in relation to PM-associated chemicals. PM10 was sampled in ambient air at an urban traffic site (Borgerhout) and a rural background location (Houtem) in Flanders (Belgium). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) were exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) and the induction of interleukin-8 (IL-8). The mutagenic capacity was assessed using the Ames II Mutagenicity Test. The endotoxin levels in the collected samples were analyzed and the oxidative potential (OP) of PM10 particles was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 included tracers for biomass burning (levoglucosan, mannosan and galactosan), elemental and organic carbon (EC/OC) and polycyclic aromatic hydrocarbons (PAHs). Most samples displayed dose-dependent cytotoxicity and IL-8 induction. Spatial and temporal differences in PM10 toxicity were seen. PM10 collected at the urban site was characterized by increased pro-inflammatory and mutagenic activity as well as higher OP and elevated endotoxin levels compared to the background area. Reduced cell viability (-0.46 < rs < -0.35, p < 0.01) and IL-8 induction (-0.62 < rs < -0.67, p < 0.01) were associated with all markers for biomass burning, levoglucosan, mannosan and galactosan. Furthermore, direct and indirect mutagenicity were associated with tracers for biomass burning, OC, EC and PAHs. Multiple regression analyses showed levoglucosan to explain 16% and 28% of the variance in direct and indirect mutagenicity, respectively. Markers for biomass burning were associated with altered cellular responses and increased mutagenic activity. These findings may indicate a role of biomass burning in the observed adverse health effect of particulate matter.

Spatial variability and temporal stability of throughfall deposition under beech (Fagus sylvatica L.) in relationship to canopy structure.

Although the spatial variability of throughfall (TF) in forest ecosystems can have important ecological implications, little is known about the driving factors of within-stand TF variability, particularly in deciduous forests. While the spatial variability of TF water amount and H+ deposition under a dominant beech (Fagus sylvatica L.) tree was significantly higher in the leafed period than in the leafless period, the spatial TF deposition patterns of most major ions were similar in both periods. The semiannual TF depositions of all ions other than H+ were significantly positively correlated (r=0.68-0.90, p<0.05) with canopy structure above sample locations throughout the entire year. The amounts of TF water and H+ deposition during the leafed period were negatively correlated with branch cover. We conclude that the spatial heterogeneity of ion deposition under beech was significantly affected by leaves in the growing period and by branches in non-foliated conditions.

Acidification of forested podzols in North Belgium during the period 1950-2000.

Acidification of forest soils in Europe and North America has been an important concern over the last decades. The last area-covering survey of forest soil acidification in Flanders (North Belgium) goes back to 1985 [Ronse A, De Temmerman L, Guns M, De Borger R. Evolution of acidity, organic matter content, and CEC in uncultivated soils of North Belgium during the past 25 years. Soil Sci; 146, (1988), 453-460] and highlighted a significant acidification of the upper layer (0.3-0.4 m) of forested podzols during the period 1950-1985. The present study aimed to assess (1) to what extent further acidification of forested podzols occurred during the period 1985-2000 at different depths and (2) whether the average annual acidification rate accelerated or slowed down between 1985 and 2000 compared to the period 1950-1985. Average soil pH-KCl values of podzols in northern Belgium dropped during the period 1985-2000. This decline extends to a depth of about 50 cm but was most pronounced and significant in the A horizon. In the A(0), A(1) and A(2) horizons, average pH dropped with 0.2, 0.3 and 0.1 units, and in the B(ir) and C horizons with 0.1 units. No change in average pH value occurred in the B(h) horizon. Average annual acidification rate of the A(1) horizon was significantly higher in the period 1985-2000 than in the period 1950-1985. Changes in pH occurred in the entire soil profile during the period 1950/67-1985 likely because sulphate was the major form of acid deposition before 1985. After 1985, acid sulphur deposition decreased with more than 50% in North Belgium. In contrast, ammonium deposition almost doubled between 1950 and 1980, which may explain why soil acidification between 1985 and 2000 has been restricted to the upper soil horizons.

Assessment of estrogenic activity in PM10 air samples with the ERE-CALUX bioassay: Method optimization and implementation at an urban location in Flanders (Belgium).

Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. The objective of this study was to investigate the overall estrogen activating potential of PM10 air samples at an urban location with high traffic incidence in Flanders, using a human in vitro cell bioassay. PM10 samples (n = 36) were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen-responsive human ovarian carcinoma (BG1Luc4E2) cell line. In addition, several samples and procedural blanks were extracted with ultra-pure ethanol or acetonitrile to compare extraction efficiencies. Results were expressed as bioanalytical equivalents (BEQs) in femtogram 17ß-estradiol equivalent (fg E2-Eq) per cubic meter of air. High fluctuations in estrogenic activity were observed during the entire sampling period, with mean and median BEQs of 50.7 and 35.9 fg E2-Eq m(-)(3), respectively. Estrogenic activity was measured in more than 70% of the samples and several sample extracts showed both high BEQs and high cytotoxicity, which could not be related to black carbon, PM10 or heavy metal concentrations. At this moment, it remains unclear which substances cause this toxicity, but comparison of results obtained with different extraction solvents indicated that acetone/hexane extracts contained more compounds that were cytotoxic and suppressive of responses than those extracted using ultra-pure ethanol. Although more research is needed, the use of a more polar extraction solvent seems to be advisable.

Contributing factors in foliar uptake of dissolved inorganic nitrogen at leaf level.

We investigated the influence of leaf traits, rainwater chemistry, and pedospheric nitrogen (N) fertilisation on the aqueous uptake of inorganic N by physiologically active tree leaves. Leaves of juvenile silver birch and European beech trees, supplied with NH4NO3 to the soil at rates from 0 to 200 kg N ha(-1)y(-1), were individually exposed to 100 µl of artificial rainwater containing (15)NH4(+) or (15)NO3(-) at two concentration levels for one hour. In the next vegetative period, the experiment was repeated with NH4(+) at the highest concentration only. The N form and the N concentration in the applied rainwater and, to a lesser extent, the pedospheric N treatment and the leaf traits affected the aqueous foliar N uptake. The foliar uptake of NH4(+) by birch increased when leaves were more wettable. High leaf N concentration and leaf mass per area enhanced the foliar N uptake, and NO3(-) uptake in particular, by birch. Variation in the foliar N uptake by the beech trees could not be explained by the leaf traits considered. In the first experiment, N fertilisation stimulated the foliar N uptake in both species, which was on average 1.42-1.78 times higher at the highest soil N dose than at the zero dose. However, data variability was high and the effect was not appreciable in the second experiment. Our data suggest that next to rainwater chemistry (N form and concentration) also forest N status could play a role in the partitioning of N entering the ecosystem through the soil and the canopy. Models of canopy uptake of aqueous N at the leaf level should take account of leaf traits such as wettability and N concentration.

The response of the foliar antioxidant system and stable isotopes (δ(13)C and δ(15)N) of white willow to low-level air pollution.

In this study we aimed to determine and elucidate the effect of ambient air pollution on the foliar antioxidant system and stable carbon and nitrogen isotopes of white willow (Salix alba L.). We grew white willow in uniform potting soil in the near vicinity of sixteen air quality monitoring stations in Belgium where nitrogen dioxide (NO2), ozone, sulfur dioxide and particulate matter concentrations were continuously measured. The trees were exposed to ambient air during six months (April-September 2011), and, thereafter, the degree of lipid peroxidation and foliar content of antioxidant molecules (ascorbate, glutathione, polyphenols, flavonoids), antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, peroxidase) and foliar stable carbon (δ(13)C) and nitrogen (δ(15)N) isotopes were measured. We found that lipid peroxidation was caused by air pollution stress, arising from high ambient NO2 concentrations, as shown by an increased amount of malondialdehyde. The antioxidant system was activated by increasing the amount of polyphenols at monitoring stations with a high atmospheric NO2 and low O3 concentration, while no increase of key enzymes (e.g., ascorbate, glutathione) was observed. The δ(13)C also decreased with increasing NO2 concentrations and decreasing O3 concentrations, probably reflecting a decreased net photosynthesis and/or a concomitant decrease of (13)CO2 in the atmosphere. Shade also influenced foliar δ(13)C and the content of leaf ascorbate and glutathione.

Throughfall deposition and canopy exchange processes along a vertical gradient within the canopy of beech (Fagus sylvatica L.) and Norway spruce (Picea abies (L.) Karst).

To assess the impact of air pollution on forest ecosystems, the canopy is usually considered as a constant single layer in interaction with the atmosphere and incident rain, which could influence the measurement accuracy. In this study the variation of througfall deposition and derived dry deposition and canopy exchange were studied along a vertical gradient in the canopy of one European beech (Fagus sylvatica L.) tree and two Norway spruce (Picea abies (L.) Karst) trees. Throughfall and net throughfall deposition of all ions other than H(+) increased significantly with canopy depth in the middle and lower canopy of the beech tree and in the whole canopy of the spruce trees. Moreover, throughfall and net throughfall of all ions in the spruce canopy decreased with increasing distance to the trunk. Dry deposition occurred mainly in the upper canopy and was highest during the growing season for H(+), NH(4)(+), NO(3)(-) and highest during the dormant season for Na(+), Cl(-), SO(4)(2-) (beech and spruce) and K(+), Ca(2+) and Mg(2+) (spruce only). Canopy leaching of K(+), Ca(2+) and Mg(2+) was observed at all canopy levels and was higher for the beech tree compared to the spruce trees. Canopy uptake of inorganic nitrogen and H(+) occurred mainly in the upper canopy, although significant canopy uptake was found in the middle canopy as well. Canopy exchange was always higher during the growing season compared to the dormant season. This spatial and temporal variation indicates that biogeochemical deposition models would benefit from a multilayer approach for shade-tolerant tree species such as beech and spruce.

Comparison of forest edge effects on throughfall deposition in different forest types.

This study examined the influence of distance to the forest edge, forest type, and time on Cl-, SO4(2-), NO3(-), and NH4+ throughfall deposition in forest edges. The forests were dominated by pedunculate oak, silver birch, or Corsican/Austrian pine, and were situated in two regions of Flanders (Belgium). Along transects, throughfall deposition was monitored at distances of 0-128 m from the forest edge. A repeated-measures analysis demonstrated that time, forest type, and distance to the forest edge significantly influenced throughfall deposition of the ions studied. The effect of distance to the forest edge depended significantly on forest type in the deposition of Cl-, SO4(2-), and NO3(-): the edge effect was significantly greater in pine stands than in deciduous birch and oak stands. This finding supports the possibility of converting pine plantations into oak or birch forests in order to mitigate the input of nitrogen and potentially acidifying deposition.

The effect of forest type on throughfall deposition and seepage flux: a review.

Converting deciduous forests to coniferous plantations and vice versa causes environmental changes, but till now insight into the overall effect is lacking. This review, based on 38 case studies, aims to find out how coniferous and deciduous forests differ in terms of throughfall (+stemflow) deposition and seepage flux to groundwater. From the comparison of coniferous and deciduous stands at comparable sites, it can be inferred that deciduous forests receive less N and S via throughfall (+stemflow) deposition on the forest floor. In regions with relatively low open field deposition of atmospheric N (<10 kg N ha(-1) year(-1)), lower NH(4)(+) mean throughfall (+stemflow) deposition was, however, reported under conifers compared to deciduous forest, while in regions with high atmospheric N pollution (>10 kg N ha(-1) year(-1)), the opposite could be concluded. The higher the open field deposition of NH(4)(+), the bigger the difference between the coniferous and deciduous throughfall (+stemflow) deposition. Furthermore, it can be concluded that canopy exchange of K(+), Ca(2+) and Mg(2+) is on average higher in deciduous stands. The significantly higher stand deposition flux of N and S in coniferous forests is reflected in a higher soil seepage flux of NO(3)(-), SO(4)(2-), K(+), Ca(2+), Mg(2+) and Al(III). Considering a subset of papers for which all necessary data were available, a close relationship between throughfall (+stemflow) deposition and seepage was found for N, irrespective of the forest type, while this was not the case for S. This review shows that the higher input flux of N and S in coniferous forests clearly involves a higher seepage of NO(3)(-) and SO(4)(2-) and accompanying cations K(+), Ca(2+), Mg(2+) and Al(III) into the groundwater, making this forest type more vulnerable to acidification and eutrophication compared to the deciduous forest type.

Does the commonly used estimator of nutrient resorption in tree foliage actually measure what it claims to?

A descriptive temporal model is considered to be the best available estimator for accretion, resorption and proportional nutrient resorption. However, ecological studies rarely collect sufficient data for applying such a model. A less-demanding and commonly used estimator for proportional resorption (PR) calculates PR as the percentage of the nutrient pool that is withdrawn from mature foliage before leaf abscission. Data from an intensive sampling campaign of the aboveground nutrient pools and fluxes of two Betula pendula Roth. stands were used. We showed that the commonly used estimator is not an accurate estimator for accretion, resorption and proportional resorption. The commonly used estimator underestimated the proportional resorption of N on the average by 3-10%, and the proportional resorption of P by 20-25%. The low accuracy of the estimations was shown to be caused by a lack of selectiveness of the commonly used estimator. In other words, the commonly used estimator does not measure the underlying processes in specific nutrient accretion and resorption at the stand level. However, when a sufficiently high sampling density with several samples at a given point in time is used, then the commonly used estimator preserves the ranking relationship between the PR of different sites for N in 97% of the cases and for P in 71%. The commonly used estimator can thus be used in comparative studies as an index for proportional nutrient resorption only. The quantitative results should not be taken literally, as they are based on only two sets of observations. However, the results show that the commonly used estimator should no longer be used as a measure for accretion, resorption or PR whenever the plant accretes nutrients in the foliage as a compensation for nutrient losses due to foliar leaching and litterfall during the growing season.