Plasma membrane intrinsic protein OsPIP2;6 is involved in root-to-shoot arsenic translocation in rice (Oryza sativa L.).

KEY MESSAGE: This study demonstrates the crucial role of OsPIP2;6 for translocation of arsenic from roots to shoots, which can decrease arsenic accumulation in rice for improved food safety. Arsenic (As) contamination in food and water, primarily through rice consumption, poses a significant health risk due to its natural tendency to accumulate inorganic arsenic (iAs). Understanding As transport mechanisms is vital for producing As-free rice. This study investigates the role of rice plasma membrane intrinsic protein, OsPIP2;6, for AsIII tolerance and accumulation. RNAi-mediated suppression of OsPIP2;6 expression resulted in a substantial (35-65%) reduction in As accumulation in rice shoots, while root arsenic levels remained largely unaffected. Conversely, OsPIP2;6 overexpression led to 15-76% higher arsenic accumulation in shoots, with no significant change in root As content. In mature plants, RNAi suppression caused (19-26%) decrease in shoot As, with flag leaves and grains showing a 16% reduction. OsPIP2;6 expression was detected in both roots and shoots, with higher transcript levels in shoots. Localization studies revealed its presence in vascular tissues of both roots and shoots. Overall, our findings highlight OsPIP2;6's role in root-to-shoot As translocation, attributed to its specific localization in the vascular tissue of roots and leaves. This knowledge can facilitate the development of breeding programs to mitigate As accumulation in rice and other food crops for improved food safety and increasing productivity on As-contaminated soils.

Nanoscale Sulfur Improves Plant Growth and Reduces Arsenic Toxicity and Accumulation in Rice (Oryza sativa L.).

Rice is known to accumulate arsenic (As) in its grains, posing serious health concerns for billions of people globally. We studied the effect of nanoscale sulfur (NS) on rice seedlings and mature plants under As stress. NS application caused a 40% increase in seedling biomass and a 26% increase in seed yield of mature plants compared to untreated control plants. AsIII exposure caused severe toxicity to rice; however, coexposure of plants to AsIII and NS alleviated As toxicity, and growth was significantly improved. Rice seedlings treated with AsIII + NS produced 159 and 248% more shoot and root biomass, respectively, compared to plants exposed to AsIII alone. Further, AsIII + NS-treated seedlings accumulated 32 and 11% less As in root and shoot tissues, respectively, than the AsIII-alone treatment. Mature plants treated with AsIII + NS produced 76, 110, and 108% more dry shoot biomass, seed number, and seed yield, respectively, and accumulated 69, 38, 18, and 54% less total As in the root, shoot, flag leaves, and grains, respectively, compared to AsIII-alone-treated plants. A similar trend was observed in seedlings treated with AsV and NS. The ability of sulfur (S) to alleviate As toxicity and accumulation is clearly size dependent as NS could effectively reduce bioavailability and accumulation of As in rice via modulating the gene expression activity of As transport, S assimilatory, and glutathione synthesis pathways to facilitate AsIII detoxification. These results have significant environmental implications as NS application in agriculture has the potential to decrease As in the food chain and simultaneously enable crops to grow and produce higher yields on marginal and contaminated lands.

New insight into the mechanism of graphene oxide-enhanced phytotoxicity of arsenic species.

Graphene oxide (GO) has exhibited significant potential to improve crop cultivation and yield. The application of GO in agriculture will inevitably result in interactions with conventional contaminants, causing potential changes to environmental behavior and toxicity of conventional contaminants. This study explored the joint phytotoxicity of GO and arsenic species (arsenite [As (III)], arsenate [As (V)]) to monocot (Triticum aestivum L.) and dicot (Solamun lycopersicum) plant species. Under the environmentally relevant concentrations, GO (1 mg/L) significantly increased the phytotoxicity of As (III) and As (V) (1 mg/L), with effects being both As- and plant species-specific. One mechanism of enhanced arsenic phytotoxicity could be GO-induced up-regulation of the aquaporin and phosphate transporter related genes expression, which would lead to the increased accumulation of As (III) and As (V) in plants. In addition, co-exposure with GO resulted in more severe oxidative stress than single As exposure, which could subsequently induce damage in root plasma membranes and compromise key arsenic detoxification pathways such as complexation with glutathione and efflux. Co-exposure to GO and As also led to more significant reduction in macro- and micronutrient content. The provided data highlight the high-impact of nanomaterials on the environmental risk of As in agricultural systems.

Interaction of graphene oxide with co-existing arsenite and arsenate: Adsorption, transformation and combined toxicity.

The outstanding commercial application potential of graphene oxide (GO) will inevitably lead to its increasing release into the environment, and then affect the environmental behavior and toxicity of conventional pollutants. Interactions between arsenite [As (III)]/arsenate [As (V)] with GO and their combined toxicity to Chlorella pyrenoidosa were investigated. Under abiotic conditions, approximately 42% of the adsorbed As (III) was oxidized by GO with simulated sunlight illumination, which was induced by electron-hole pairs on the surface of GO. Co-exposure with GO greatly enhanced the toxicity of As (III, V) to alga. When adding 10 mg/L GO, the 72 h median effect concentration of As (III) and As (V) to C. pyrendoidosa decreased to 12.7 and 9.4 mg/L from 30.1 and 16.3 mg/L in the As alone treatment, respectively. One possible mechanism by which GO enhanced As toxicity could be that GO decreased the phosphate concentration in the algal medium, and then increased the accumulation of As (V) in algae. In addition, transmission electron microscope (TEM) images demonstrated that GO acted as a carrier for As (III) and As (V) transport into the algal cells. Also, GO induced severe oxidative stress, which could have subsequently compromised important detoxification pathways (e.g., As complexation with glutathione, As methylation, and intracellular As efflux) in the algal cells. Our findings highlight the significant impact of GO on the fate and toxicity of As in the aquatic environment.

Bioavailability of cerium oxide nanoparticles to Raphanus sativus L. in two soils.

Cerium oxide nanoparticles (CeO2 NP) are a common component of many commercial products. Due to the general concerns over the potential toxicity of engineered nanoparticles (ENPs), the phytotoxicity and in planta accumulation of CeO2 NPs have been broadly investigated. However, most previous studies were conducted in hydroponic systems and with grain crops. For a few studies performed with soil grown plants, the impact of soil properties on the fate and transport of CeO2 NPs was generally ignored even though numerous previous studies indicate that soil properties play a critical role in the fate and transport of environmental pollutants. The objectives of this study were to evaluate the soil fractionation and bioavailability of CeO2 NPs to Raphanus sativus L (radish) in two soil types. Our results showed that the silty loam contained slightly higher exchangeable fraction (F1) of cerium element than did loamy sand soil, but significantly lower reducible (F2) and oxidizable (F3) fractions as CeO2 NPs concentration increased. CeO2 NPs associated with silicate minerals or the residue fraction (F4) dominated in both soils. The cerium concentration in radish storage root showed linear correlation with the sum of the first three fractions (r2 = 0.98 and 0.78 for loamy sand and silty loam respectively). However, the cerium content in radish shoots only exhibited strong correlations with F1 (r2 = 0.97 and 0.89 for loamy sand and silty loam respectively). Overall, the results demonstrated that soil properties are important factors governing the distribution of CeO2 NPs in soil and subsequent bioavailability to plants.

Exposure of agricultural crops to nanoparticle CeO2 in biochar-amended soil.

Biochar is seeing increased usage as an amendment in agricultural soils but the significance of nanoscale interactions between this additive and engineered nanoparticles (ENP) remains unknown. Corn, lettuce, soybean and zucchini were grown for 28 d in two different soils (agricultural, residential) amended with 0-2000 mg engineered nanoparticle (ENP) CeO2 kg-1 and biochar (350 °C or 600 °C) at application rates of 0-5% (w/w). At harvest, plants were analyzed for biomass, Ce content, chlorophyll and lipid peroxidation. Biomass from the four species grown in residential soil varied with species and biochar type. However, biomass in the agricultural soil amended with biochar 600 °C was largely unaffected. Biochar co-exposure had minimal impact on Ce accumulation, with reduced or increased Ce content occurring at the highest (5%) biochar level. Soil-specific and biochar-specific effects on Ce accumulation were observed in the four species. For example, zucchini grown in agricultural soil with 2000 mg CeO2 kg-1 and 350 °C biochar (0.5-5%) accumulated greater Ce than the control. However, for the 600 °C biochar, the opposite effect was evident, with decreased Ce content as biochar increased. A principal component analysis showed that biochar type accounted for 56-99% of the variance in chlorophyll and lipid peroxidation across the plants. SEM and µ-XRF showed Ce association with specific biochar and soil components, while µ-XANES analysis confirmed that after 28 d in soil, the Ce remained largely as CeO2. The current study demonstrates that biochar synthesis conditions significantly impact interactions with ENP, with subsequent effects on particle fate and effects.

Tannic acid alleviates bulk and nanoparticle Nd2O3 toxicity in pumpkin: a physiological and molecular response.

The effect of dissolved organic matter (DOM) on nanoparticle toxicity to plants is poorly understood. In this study, tannic acid (TA) was selected as a DOM surrogate to explore the mechanisms of neodymium oxide NPs (Nd2O3 NPs) phytotoxicity to pumpkin (Cucurbita maxima). The results from the tested concentrations showed that 100 mg L(-1) Nd2O3 NPs were significantly toxic to pumpkin in term of fresh biomass, and the similar results from the bulk particles and the ionic treatments were also evident. Exposure to 100 mg L(-1) of Nd2O3 NPs and BPs in 1/5 strength Hoagland's solution not only significantly inhibited pumpkin growth, but also decreased the S, Ca, K and Mg levels in plant tissues. However, 60 mg L(-1) TA significantly moderated the observed phytotoxicity, decreased Nd accumulation in the roots, and notably restored S, Ca, K and Mg levels in NPs and BPs treated pumpkin. TA at 60 mg L(-1) increased superoxide dismutase (SOD) activity in both roots (17.5%) and leaves (42.9%), and catalase (CAT) activity (243.1%) in the roots exposed to Nd2O3 NPs. This finding was confirmed by the observed up-regulation of transcript levels of SOD and CAT in Nd2O3 NPs treated pumpkin analyzed by quantitative reverse transcription polymerase chain reaction. These results suggest that TA alleviates Nd2O3 BPs/NPs toxicity through alteration of the particle surface charge, thus reducing the contact and uptake of NPs by pumpkin. In addition, TA promotes antioxidant enzymatic activity by elevating the transcript levels of genes involved in ROS scavenging. Our results shed light on the mechanisms underlying the influence of DOM on the bioavailability and toxicity of NPs to terrestrial plants.

Enhanced Boron Tolerance in Plants Mediated by Bidirectional Transport Through Plasma Membrane Intrinsic Proteins.

High boron (B) concentration is toxic to plants that limit plant productivity. Recent studies have shown the involvement of the members of major intrinsic protein (MIP) family in controlling B transport. Here, we have provided experimental evidences showing the bidirectional transport activity of rice OsPIP1;3 and OsPIP2;6. Boron transport ability of OsPIP1;3 and OsPIP2;6 were displayed in yeast HD9 mutant strain (∆fps1∆acr3∆ycf1) as a result of increased B sensitivity, influx and accumulation by OsPIP1;3, and rapid efflux activity by OsPIP2;6. RT-PCR analysis showed strong upregulation of OsPIP1;3 and OsPIP2;6 transcripts in roots by B toxicity. Transgenic Arabidopsis lines overexpressing OsPIP1;3 and OsPIP2;6 exhibited enhanced tolerance to B toxicity. Furthermore, B concentration was significantly increased after 2 and 3 hours of tracer boron ((10)B) treatment. Interestingly, a rapid efflux of (10)B from the roots of the transgenic plants was observed within 1 h of (10)B treatment. Boron tolerance in OsPIP1;3 and OsPIP2;6 lines was inhibited by aquaporin inhibitors, silver nitrate and sodium azide. Our data proved that OsPIP1;3 and OsPIP2;6 are indeed involved in both influx and efflux of boron transport. Manipulation of these PIPs could be highly useful in improving B tolerance in crops grown in high B containing soils.

Reduced Silver Nanoparticle Phytotoxicity in Crambe abyssinica with Enhanced Glutathione Production by Overexpressing Bacterial γ-Glutamylcysteine Synthase.

Silver nanoparticles (Ag NPs) are widely used in consumer products, and their release has raised serious concerns about the risk of their exposure to the environment and to human health. However, biochemical mechanisms by which plants counteract NP toxicity are largely unknown. We have previously engineered Crambe abyssinica plants expressing the bacterial γ-glutamylecysteine synthase (γ-ECS) for enhancing glutathione (GSH) levels. In this study, we investigated if enhanced levels of GSH and its derivatives can protect plants from Ag NPs and AgNO3 (Ag(+) ions). Our results showed that transgenic lines, when exposed to Ag NPs and Ag(+) ions, were significantly more tolerant, attaining a 28%-46% higher biomass and 34-49% more chlorophyll content, as well as maintaining 35-46% higher transpiration rates as compared to those of wild type (WT) plants. Transgenic γ-ECS lines showed 2-6-fold Ag accumulation in shoot tissue and slightly lower or no difference in root tissue relative to levels in WT plants. The levels of malondialdehyde (MDA) in γ-ECS lines were also 27.3-32.5% lower than those in WT Crambe. These results indicate that GSH and related peptides protect plants from Ag nanotoxicity. To our knowledge, this is the first direct report of Ag NP detoxification by GSH in transgenic plants, and these results will be highly useful in developing strategies to counteract the phytotoxicty of metal-based nanoparticles in crop plants.

Analysis of silver nanoparticles in antimicrobial products using surface-enhanced Raman spectroscopy (SERS).

Silver nanoparticles (AgNPs) are the most commonly used nanoparticles in consumer products. Concerns over human exposure to and risk from these particles have resulted in increased interest in novel strategies to detect AgNPs. This study investigated the feasibility of surface-enhanced Raman spectroscopy (SERS) as a method for the detection and quantification of AgNPs in antimicrobial products. By using ferbam (ferric dimethyl-dithiocarbamate) as an indicator molecule that binds strongly onto the nanoparticles, AgNPs detection and discrimination were achieved based on the signature SERS response of AgNPs-ferbam complexes. SERS response with ferbam was distinct for silver ions, silver chloride, silver bulk particles, and AgNPs. Two types of AgNPs with different coatings, citrate and polyvinylpirrolidone (PVP), both showed strong interactions with ferbam and induced strong SERS signals. SERS was effectively applicable for detecting Ag particles ranging from 20 to 200 nm, with the highest signal intensity in the 60-100 nm range. A linear relationship (R(2) = 0.9804) between Raman intensity and citrate-AgNPs concentrations (60 nm; 0-20 mg/L) indicates the potential for particle quantification. We also evaluated SERS detection of AgNPs in four commercially available antimicrobial products. Combined with ICP-MS and TEM data, the results indicated that the SERS response is primarily dependent on size, but also affected by AgNPs concentration. The findings demonstrate that SERS is a promising analytical platform for studying environmentally relevant levels of AgNPs in consumer products and related matrices.

Uptake and accumulation of bulk and nanosized cerium oxide particles and ionic cerium by radish (Raphanus sativus L.).

The potential toxicity and accumulation of engineered nanomaterials (ENMs) in agricultural crops has become an area of great concern and intense investigation. Interestingly, although below-ground vegetables are most likely to accumulate the highest concentrations of ENMs, little work has been done investigating the potential uptake and accumulation of ENMs for this plant group. The overall objective of this study was to evaluate how different forms of cerium (bulk cerium oxide, cerium oxide nanoparticles, and the cerium ion) affected the growth of radish (Raphanus sativus L.) and accumulation of cerium in radish tissues. Ionic cerium (Ce(3+)) had a negative effect on radish growth at 10 mg CeCl3/L, whereas bulk cerium oxide (CeO2) enhanced plant biomass at the same concentration. Treatment with 10 mg/L cerium oxide nanoparticles (CeO2 NPs) had no significant effect on radish growth. Exposure to all forms of cerium resulted in the accumulation of this element in radish tissues, including the edible storage root. However, the accumulation patterns and their effect on plant growth and physiological processes varied with the characteristics of cerium. This study provides a critical frame of reference on the effects of CeO2 NPs versus their bulk and ionic counterparts on radish growth.

Two rice plasma membrane intrinsic proteins, OsPIP2;4 and OsPIP2;7, are involved in transport and providing tolerance to boron toxicity.

Boron (B) toxicity is responsible for low cereal crop production in a number of regions worldwide. In this report, we characterized two rice genes, OsPIP2;4 and OsPIP2;7, for their involvement in B permeability and tolerance. Transcript analysis demonstrated that the expression of OsPIP2;4 and OsPIP2;7 were downregulated in shoots and strongly upregulated in rice roots by high B treatment. Expression of both OsPIP2;4 and OsPIP2;7 in yeast HD9 strain lacking Fps1, ACR3, and Ycf1 resulted in an increased B sensitivity. Furthermore, yeast HD9 strain expressing OsPIP2;4 and OsPIP2;7 accumulated significantly higher B as compared to empty vector control, which suggests their involvement in B transport. Overexpression of OsPIP2;4 and OsPIP2;7 in Arabidopsis imparted higher tolerance under B toxicity. Arabidopsis lines overexpressing OsPIP2;4 and OsPIP2;7 showed significantly higher biomass production and greater root length, however there was no difference in B accumulation in long term uptake assay. Short-term uptake assay using tracer B (¹°B) in shoots and roots demonstrated increased ¹°B accumulation in Arabidopsis lines expressing OsPIP2;4 and OsPIP2;7, compare to wild type control plants. Efflux assay of B in the roots showed that ¹°B was effluxed from the Arabidopsis transgenic plants overexpressing OsPIP2;4 or OsPIP2;7 during the initial 1-h of assay. These data indicate that OsPIP2;4 and OsPIP2;7 are involved in mediating B transport in rice and provide tolerance via efflux of excess B from roots and shoot tissues. These genes will be highly useful in developing B tolerant crops for enhanced yield in the areas affected by high B toxicity.

Impact of Ag nanoparticle exposure on p,p'-DDE bioaccumulation by Cucurbita pepo (zucchini) and Glycine max (soybean).

The effect of nanoparticle (NP), bulk, or ionic Ag exposure on dichlorodiphenyldichloroethylene (p,p'-DDE; DDT metabolite) accumulation by Glycine max L. (soybean) and Cucurbita pepo L. (zucchini) was investigated. The plants were grown in 125-mL jars of vermiculite amended with 500 or 2000 mg/L of bulk or NP Ag; ion controls at 5 and 20 mg/L were established. During 19 d of growth, plants were amended with solution containing 100 ng/mL of p,p'-DDE. Total shoot p,p'-DDE levels in non-Ag exposed G. max and C. pepo were 500 and 970 ng, respectively; total root DDE content was 13,700 and 20,300 ng, respectively. Ag decreased the p,p'-DDE content of G. max tissues by up to 40%, with NP exposure resulting in less contaminant uptake than bulk Ag. Total Ag content of exposed G. max ranged from 50.5 to 373 µg; NP-exposed plants had 1.9-2.2 times greater overall Ag than corresponding bulk particle treatments and also significantly greater relative Ag transport to shoot tissues. Bulk and NP Ag at 500 mg/L suppressed DDE uptake by C. pepo by 21-29%, although Ag exposure at 2000 mg/L had no impact on contaminant uptake. Similar to G. max , C. pepo whole plant Ag content ranged from 50.5 to 182 µg, with tissue element content generally being greater for NP exposed plants. These findings show that the Ag may significantly alter the accumulation and translocation of cocontaminants in agricultural systems. Notably, the cocontaminant interactions vary both with Ag particle size (NP vs bulk) and plant species. Future investigations will be needed to clarify the mechanisms responsible for the cocontaminant interactions and assess the impact on overall exposure and risk.

Toxicity of silver and copper to Cucurbita pepo: differential effects of nano and bulk-size particles.

The phytotoxicity of bulk and nanoparticle Cu and Ag was directly compared. NP Ag reduced biomass and transpiration by 66-84% when compared with bulk Ag. The Ag ion concentration was 4.4-10-times greater in NP than bulk particle solutions. The Cu ion concentration was 1.4-4.4-times greater in bulk than NP amended solutions. Humic acid (50 mg/L) decreased the ion content of bulk Cu solution by 38-42% but increased ion Cu content of NP solutions by 1.4-2.9 times. Bulk and NP Cu were highly phytotoxic; growth and transpiration were reduced by 60-70% relative to untreated controls. NP Cu phytotoxicity was unaffected by solution type, but humic acid (50 mg/L) completely alleviated phytotoxicity caused by bulk Cu. The data demonstrate differential toxicity of Ag NP relative to bulk Ag. The finding that humic acid and solution chemistry differentially impact bulk and NP behavior highlights the importance of evaluating nanoparticles under environmentally relevant conditions.

Accumulation and phytotoxicity of engineered nanoparticles to Cucurbita pepo.

The effect of bulk and engineered nanoparticle (NP) Ag, Au, Cu, Si, and C at 250 and 750 mg/L on zucchini biomass, transpiration, and element content was determined. The pH of bulk and NP solutions prior to plant growth frequently differed. Nanoparticle Cu solution pH was significantly higher than bulk Cu, whereas for Ag and C, the NPs had significantly lower pH. Plants were unaffected by Au, regardless of particle size or concentration. NP Ag reduced plant biomass and transpiration by 49-91% compared to equivalent bulk Ag. NP Si at 750 mg/L reduced plant growth and transpiration by 30-51% relative to bulk Si. Bulk and NP Cu were phytotoxic but much of the effect was alleviated by humic acid. The shoot Ag and Cu content did not differ based on particle size or concentration. The accumulation of bulk Au was greater than the NP, but humic acid increased the accumulation of NP and bulk Au by 5.6-fold and 80%, respectively. The uptake of NP Si was 5.6-6.5-fold greater than observed with the bulk element. These findings show that the NPs may have unique phytotoxicity or accumulation patterns and that solution properties can significantly impact particle fate and effects.

Members of rice plasma membrane intrinsic proteins subfamily are involved in arsenite permeability and tolerance in plants.

Rice accumulates high level of arsenic (As) in its edible parts and thus plays an important role in the transfer of As into the food chain. However, the mechanisms of As uptake and its detoxification in rice are not well understood. Recently, members of the Nodulin 26-like intrinsic protein (NIP) subfamily of plant aquaporins were shown to transport arsenite in rice and Arabidopsis. Here we report that members of the rice plasma membrane intrinsic protein (PIP) subfamily are also involved in As tolerance and transport. Based on the homology search with the mammalian AQP9 and yeast Fps1 arsenite transporters, we identified and cloned five rice PIP gene subfamily members. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to arsenite treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis oocytes significantly increased the uptake of arsenite. Overexpression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis yielded enhanced arsenite tolerance and higher biomass accumulation. Further, these transgenic plants showed no significant accumulation of As in shoot and root tissues in long term uptake assays. Whereas, short duration exposure to arsenite caused both active influx and efflux of As in the roots. The data suggests a bidirectional arsenite permeability of rice PIPs in plants. These rice PIPs genes will be highly useful for engineering important food and biofuel crops for enhanced crop productivity on contaminated soils without increasing the accumulation of toxic As in the biomass or edible tissues.

Characterization of substances released from crumb rubber material used on artificial turf fields.

Crumb rubber material (CRM) used as infill on artificial turf fields can be the source of a variety of substances released to the environment and to living organisms in the vicinity of the CRM. To assess potential risks of major volatilized and leached substances derived from CRM, methods were developed to identify organic compounds and elements, either in the vapor phase and/or the leachate from CRM. A qualitative method based on solid phase micro-extraction (SPME) coupled with gas chromatography/mass spectrometry (GC-MS) was developed to identify the major volatile and semi-volatile organic compounds out-gassing from CRM samples under defined laboratory conditions. Direct vapor phase injection into the GC-MS was applied for the quantitative analysis. Ten organic compounds were identified in the vapor phase by the SPME method. Volatile benzothiazole (BT) was detected at the highest level in all commercial CRM samples, in the range 8.2-69 ng g(-1) CRM. Other volatile PAHs and antioxidants were quantified in the vapor phase as well. A decrease of volatile compounds was noted in the headspace over CRM samples from 2-years-old fields when compared with the virgin CRM used at installation. An outdoor experiment under natural weathering conditions showed a significant reduction of out-gassing organic compounds from the CRM in the first 14 d; thereafter, values remained consistent up to 70 d of observation. Zinc was the most abundant element in the acidified leachate (220-13000 microg g(-1)), while leachable BT was detected at relatively low amounts.