Climate and Environmental Variables Drive Stream Biofilm Bacterial and Fungal Diversity on Tropical Mountainsides.

High mountain freshwater systems are particularly sensitive to the impacts of global warming and relevant environmental changes. Microorganisms contribute substantially to biogeochemical processes, yet their distribution patterns and driving mechanism in alpine streams remain understudied. Here, we examined the bacterial and fungal community compositions in stream biofilm along the elevational gradient of 745-1874 m on Mt. Kilimanjaro and explored their alpha and beta diversity patterns and the underlying environmental drivers. We found that the species richness and evenness monotonically increased towards higher elevations for bacteria, while were non-significant for fungi. However, both bacterial and fungal communities showed consistent elevational distance-decay relationships, i.e., the dissimilarity of assemblage composition increased with greater elevational differences. Bacterial alpha diversity patterns were mainly affected by chemical variables such as total nitrogen and phosphorus, while fungi were affected by physical variables such as riparian shading and stream width. Notably, climatic variables such as mean annual temperature strongly affected the elevational succession of bacterial and fungal community compositions. Our study is the first exploration of microbial biodiversity and their underlying driving mechanisms for stream ecosystems in tropical alpine regions. Our findings provide insights on the response patterns of tropical aquatic microbial community composition and diversity under climate change.

Methane ebullition fluxes and temperature sensitivity in a shallow lake.

Inland waters are important sources of atmospheric methane (CH4), with a major contribution from the CH4 ebullition pathway. However, there is still a lack of CH4 ebullition flux (eFCH4) and their temperature sensitivity (Q10) in shallow lakes, which might lead to large uncertainties in CH4 emission response from aquatic to climate and environmental change. Herein, the magnitude and regulatory of two CH4 pathways (ebullition and diffusion) were studied in subtropical Lake Chaohu, China, using the real-time portable greenhouse gas (GHG) analyzer-floating chamber method at 18 sites over four seasons. eFCH4 (12.06 ± 4.10 nmol m-2 s-1) was the dominant contributing pathway (73.0 %) to the two CH4 emission pathways in Lake Chaohu. The whole-lake mass balance calculation demonstrated that 56.6 % of the CH4 emitted from the sediment escaped through the ebullition pathway. eFCH4 was significantly higher in the western (WL: 16.54 ± 22.22 nmol m-2 s-1) and eastern lake zones (EL: 11.89 ± 15.43 nmol m-2 s-1) than in the middle lake zone (ML: 8.86 ± 13.78 nmol m-2 s-1; p < 0.05) and were significantly higher in the nearshore lake zone (NL: 15.94 ± 19.58 nmol m-2 s-1) than in the pelagic lake zone (PL: 6.64 ± 12.37 nmol m-2 s-1; p < 0.05). eFCH4 was significantly higher in summer (32.12 ± 13.82 nmol m-2 s-1) than in other seasons (p < 0.05). eFCH4 had a strong temperature dependence. Sediment total organic carbon (STOC) is an important ecosystem level Q10 driver of eFCH4. The meta-analysis also verified that across ecosystems the ecosystem-level Q10 of eFCH4 was significantly positively correlated with STOC and latitude (p < 0.05). This study suggests that eFCH4 will become increasingly crucial in shallow lake ecosystems as climate change and human activities increase. The potential increase in ebullition fluxes in high-latitude lakes is of great importance.

Evaluating potential ecological risks of emerging toxic elements in lacustrine sediments: A case study in Lake Fuxian, China.

The fragile ecosystems of plateau lakes are in face of ecological risks from emerging toxic elements. Beryllium (Be) and thallium (Tl) have been considered priority control metals in recent years owing to their persistence, toxicity, and bioaccumulation. However, the toxic factors of Be and Tl are scarce and ecological risks of them in the aquatic environment were seldom investigated. Hence, this study developed a framework for calculating the potential ecological risk index (PERI) of Be and Tl in aquatic systems and used it to assess the ecological risks of Be and Tl in Lake Fuxian, a plateau lake in China. The toxicity factors of Be and Tl were calculated to be 40 and 5, respectively. In sediments of Lake Fuxian, the concentrations of Be and Tl were between 2.18 and 4.04 mg/kg and 0.72-0.94 mg/kg, respectively. The spatial distribution indicated that Be was more abundant in the eastern and southern regions, and Tl had higher concentrations near the northern and southern banks, consistent with the distribution of anthropogenic activities. The background values were calculated as 3.38 mg/kg and 0.89 mg/kg for Be and Tl, respectively. In comparison with Be, Tl was more enriched in Lake Fuxian. The increasing Tl enrichment has been attributed to anthropogenic activities (e.g., coal burning and non-ferrous metal production), especially since the 1980s. Generally, Be and Tl contamination has decreased over the past several decades, from moderate to low, since the 1980s. The ecological risk of Tl was low, whereas Be might have caused low to moderate ecological risks. In the future, the obtained toxic factors of Be and Tl in this study can be adopted in assessing the ecological risks of them in sediments. Moreover, the framework can be employed for the ecological risk assessment of other newly emerging toxic elements in the aquatic environment.

In-lake processing counteracts the effect of allochthonous input on the composition of color dissolved organic matter in a deep lake.

Color dissolved organic matter (CDOM) plays a key role in lacustrine ecosystems and its composition is commonly mediated by the allochthonous input and autochthonous production. Deep lakes have a strong in-lake processing, which highly affects the sources, composition and cycle of CDOM. Here, the second deepest lake (Lake Fuxian) in China was selected to investigate the effects of allochthonous input and in-lake processing on lacustrine CDOM in deep lakes. Firstly, a detailed survey on CDOM composition across Lake Fuxian in the top water layer and inflowing rivers was carried out in the wet season representing the allochthonous input. In addition, CDOM in Lake Fuxian was compared with those in other lakes with distinct catchment characteristics and lake morphology. The results showed that compared to lacustrine CDOM in Lake Fuxian, the riverine CDOM contained much more humic-like substances, resulting in the humic-like fluorescence intensity peaked at the confluence of rivers into Lake Fuxian. In contrast, CDOM in Lake Fuxian was dominated by the protein-like substance. Comparison of CDOM composition among Lake Fuxian (well-vegetated catchment, deep lakes) with other diverse lakes in China (shallow/deep lakes with poor-vegetated catchment, and shallow lakes with well-vegetated catchment) showed similar CDOM quality in all type lakes, which were dominated by non-humified and autochthonous CDOM. Yet, CDOM quantity increased as the orders of deep lakes within poor-vegetated (Tibetan deep lakes) < the deep lake within well-vegetated catchment (Lake Fuxian) < shallow lakes within poorly-vegetated catchment (Tibetan shallow lakes) < shallow lakes within well-vegetated catchment (lakes along the middle and lower reaches of Yangtze River). Our results evidenced that the effect of allochthonous input on CDOM composition could be counteracted by in-lake processing in deep lakes. For deep lakes, a comprehensive understanding of in-lake processing of CDOM is critical for predicting lacustrine DOM composition and cycle.

Algal blooms modulate organic matter remineralization in freshwater sediments: A new insight on priming effect.

This study provides a novel insight into the degradation of sediment organic matter (SOM) regulated by algae-derived organic matter (AOM) based on priming effect. We tracked the dynamics of SOM mineralization products and pathways, together with priming effects (PE) using the compound-specific stable isotope (δ13C) technique following addition of low- and high-density algal debris in sediments. We found that algal debris increased the total carbon oxidation rate, and resulted in denitrification and methanogenesis-dominated SOM mineralization. While iron reduction and sulphate reduction played important roles in the early period of algal accumulation. Total carbon oxidation rate and anaerobic rates (Ranaerobic) were higher in the amended treatments compared with that in the control. Analysis indicated that algal debris had a positive PE on SOM mineralization, which caused an intensified mineralization in the initial phase with over 80% of dissolved inorganic carbon deriving from SOM degradation. Total carbon oxidation rate of SOM deduced from priming effect (RTCOR-PE) was similar to Ranaerobic, further indicating SOM mineralization was a critical source of the end products. These findings deviate the causal focus from the decomposition of AOM, and confirm the accumulation of AOM as the facilitator of SOM mineralization. Our study offers empirical evidences to advance the traditional view on the effect of AOM on SOM mineralization.

Abundant sediment organic matter potentially facilitates chemical iron reduction and surface water blackness in a Chinese deep lake.

Black bloom has become an increasingly severe environmental and ecological problem in lots of lakes. Ferrous monosulfide (FeS), which is closely related to chemical iron reduction (CIR), is considered the major cause for black water in shallow lakes, but few studies focus on the effect of organic matters (OM) content on iron and sulfate reduction and its contribution to the black bloom in deep lakes. Here, in Lake Fuxian, a Chinese deep lake which has also suffered from black bloom, FeS was identified responsible for the surface water blackness by using multiple microscopy and element analyses. Dissolved oxygen (DO) penetrated 1.6-4.2 mm in all sediment sites, further indicating FeS formed in the sediments instead of the permanently oxic water column. Geochemical characteristics revealed by diffusive gradients in thin films (DGT) showed that DGT-Fe2+ concentration was 57.6-1919.4 times higher than the DGT-S2- concentration and both were positively correlated with DGT-PO43-. Combining DGT profiles and anaerobic OM remineralization rate according to bag incubation, iron reduction is more effective than sulfate reduction although the two processes coexisted. Moreover, correlation of DGT-Fe2+ and DGT-PO43- was better than that of DGT-PO43- and DGT-S2- at OM-depleted sites but opposite at OM-rich sites. In addition, total organic carbon (TOC) was significantly positively related to acid volatile sulfide (AVS). We therefore conclude that abundant OM potentially exacerbate chemical iron reduction and further lead to surface water blackness. Our study revealed the mechanisms behind the black bloom and gives credence to the management strategy of reducing OM loading to protect water quality in deep lakes.

Comparison of copper binding properties of DOM derived from fresh and pyrolyzed biomaterials: Insights from multi-spectroscopic investigation.

The binding of dissolved organic matter (DOM) with metals affects the latter's biogeochemical processing in the environment. This study used multi-spectroscopic analyses to compare the heterogeneities of the Cu(II) binding properties of DOM derived from fresh and pyrolyzed biomaterials. The results showed that the DOM derived from fresh macrophyte (MDOM) and their corresponding biochar (BDOM) consisted mostly of protein-like and humic-like substances, respectively. The stability constant (log KM) of protein-like matter in the MDOM was 5.27, and the values of humic-like components in the BDOM were 4.32-5.15. Compared with the MDOM, the BDOM exhibited lower affinities and active binding sites for Cu(II). In addition, the BDOM contents decreased after pyrolysis. Therefore, the pyrolysis of fresh biomaterials into biochar is a promising method for reducing the potential migration risk posed by Cu(II) due to the MDOM being a positive carrier for Cu(II) contamination. Polysaccharide was the only functional group that participated in the binding of Cu(II) in both MDOM and BDOM. Aliphatic groups and amides associated with protein-like matter were responsible for the Cu(II) binding to MDOM, whereas phenolic and aromatic groups mainly participated in the complexation of BDOM-Cu(II). The CO group of amide I in the MDOM, and polysaccharide in the BDOM, showed the fastest response to Cu(II). This study was helpful for elucidating the effects of fresh and pyrolyzed biomaterials (biochars) on the environmental behavior of Cu(II) at the molecular level.

Spatial and Historical Occurrence, Sources, and Potential Toxicological Risk of Polycyclic Aromatic Hydrocarbons in Sediments of the Largest Chinese Deep Lake.

Lake sediments are important reservoirs for polycyclic aromatic hydrocarbons (PAHs) in catchments. Knowledge of occurrence, sources, and toxicological risk of PAHs is crucial to abate their pollution and risk. We investigated the spatial and temporal occurrence, sources, and potential toxicological risks of 12 PAHs in the surface sediments and one sediment core of the largest deep lake (Lake Fuxian) of China. Our results indicated the average ΣPAH12 in the surface sediments of this lake was 1550.6 ± 231.4 ng g dw -1 , much higher than those of most Chinese shallow lakes. The average ΣPAH12 in the lake area was higher than that in the estuaries. The average ΣPAH12 in the estuaries of influent rivers was higher than that of the outlet river. Coal combustion, gasoline combustion, and diesel combustion were the major sources, which contributed 68.5%, 19.8%, and 11.8% to the ΣPAH12. The average total benzo[a]pyrene toxic equivalent concentration (TEQcarc) of the six most carcinogenic PAHs was 317.1 ± 86.3 ngTEQcarc g-1 in the surface sediments. The ΣPAH12 increased from 301.7 to 1964.4 ng g dw -1 from 1945 to 2011 and significantly increased with the GDP and population of the catchment. The contribution of coal combustion to the concentrations of PAHs increased gradually with time. The total TEQcarc, and the percentage of ΣPAHcarc to ΣPAH12 in the sediment core increased from 5.0 to 84.6 ngTEQcarc g-1 and from 5.7 to 23.3%, respectively. Our study highlights the importance of such deep waters in burying PAHs and the increasing risk of PAHs from human activities.

Co-Occurrence of Microcystins and Taste-and-Odor Compounds in Drinking Water Source and Their Removal in a Full-Scale Drinking Water Treatment Plant.

The co-occurrence of cyanotoxins and taste-and-odor compounds are a growing concern for drinking water treatment plants (DWTPs) suffering cyanobacteria in water resources. The dissolved and cell-bound forms of three microcystin (MC) congeners (MC-LR, MC-RR and MC-YR) and four taste-and-odor compounds (geosmin, 2-methyl isoborneol, ß-cyclocitral and ß-ionone) were investigated monthly from August 2011 to July 2012 in the eastern drinking water source of Lake Chaohu. The total concentrations of microcystins and taste-and-odor compounds reached 8.86 µg/L and 250.7 ng/L, respectively. The seasonal trends of microcystins were not consistent with those of the taste-and-odor compounds, which were accompanied by dominant species Microcystis and Dolichospermum. The fate of the cyanobacteria and metabolites were determined simultaneously after the processes of coagulation/flocculation, sedimentation, filtration and chlorination in the associated full-scale DWTP. The dissolved fractions with elevated concentrations were detected after some steps and the breakthrough of cyanobacteria and metabolites were even observed in finished water. Chlorophyll-a limits at intake were established for the drinking water source based on our investigation of multiple metabolites, seasonal variations and their elimination rates in the DWTP. Not only microcystins but also taste-and-odor compounds should be taken into account to guide the management in source water and in DWTPs.

Comparison of bacterial growth in response to photodegraded terrestrial chromophoric dissolved organic matter in two lakes.

Terrestrial chromophoric dissolved organic matter (CDOM) could subsidize lake food webs. Trophic state and altitude have a pronounced influence on the CDOM concentration and composition of a lake. The impact of future changes in solar radiation on high-altitude lakes is particularly alarming because these aquatic ecosystems experience the most pronounced radiation variation worldwide. Photodegradation experiments were conducted on terrestrial CDOM samples from oligotrophic alpine Lake Tiancai and low-altitude eutrophic Lake Xiaohu to investigate the response of bacterial growth to photodegraded CDOM. During the photo-irradiation process, the fluorescent CDOM intensity evidently decreased in an inflowing stream of Lake Tiancai, with the predominance of humic-like fluorescence. By contrast, minimal changes were observed in the riverine CDOM of Lake Xiaohu, with the predominance of protein-like fluorescence. The kinetic constants of photodegradation indicated that the degradation rate of terrestrial (soil) humic acid in Lake Tiancai was significantly higher than that in Lake Xiaohu (p<0.001). Soil humic and fulvic acids irradiated in the simulated experiment were applied to incubated bacteria. The specific growth rate of bacteria incubated with soil humic substances was significantly higher in Lake Tiancai than in Lake Xiaohu (p<0.05). Furthermore, the utilizing rate of dissolved oxygen (DO) confirmed that the DO consumption by bacteria incubated with terrestrial CDOM in Lake Tiancai was significantly greater than that in Lake Xiaohu (p<0.05). In summary, the exposure of terrestrial CDOM to light significantly enhances its availability to heterotrophic bacteria in Lake Tiancai, an oligotrophic alpine lake, which is of importance in understanding bacterial growth in response to photodegraded terrestrial CDOM for different types of lakes.

The establishment of preliminary safety threshold values for cyanobacteria based on periodic variations in different microcystin congeners in Lake Chaohu, China.

As harmful cyanobacterial proliferation threatens the safety of drinking water supplies worldwide, it is essential to establish a safety threshold (ST) for cyanobacteria to control cyanobacterial density effectively in water sources. For this purpose, cyanobacterial abundance, microcystin (MC) production, and environmental parameters were monitored monthly from September 2011 to August 2012 in one drinking water source of Lake Chaohu. The cyanobacterial density ranged from 1400 to 220 000 cells per mL with the succession of two dominant species Microcystis and Dolichospermum, which was determined by water temperature and nutrient loading. The MC concentrations were correlated significantly with the cyanobacterial density and they varied between 0.28 and 8.86 µg L(-1). Therefore, the characteristics of MC cell quotas were classified according to four stages of the development of cyanobacteria, namely: recruitment, multiplication, decline and dormancy. The ST for cyanobacteria was established for different periods based on the MC cell quota and its guideline wherein three commonly monitored MC congeners (MC-LR, -RR and -YR) were considered in the present study. Its reliability was verified in the water source using the data collected between June 2013 and May 2014. The results highlighted the necessity to classify the ST-values in different periods referring to the main MC congeners rather than MC-LR, which will facilitate the management and control of toxic cyanobacterial proliferation in drinking water sources.

Polycyclic aromatic hydrocarbons in surface sediments from drinking water sources of Taihu Lake, China: sources, partitioning and toxicological risk.

Sources, partitioning and toxicological risk of 15 priority polycyclic aromatic hydrocarbons (PAHs) in surface sediments from drinking water sources of Taihu Lake, with an area of 2428 km(2) located in the most developed and populated area of China, were studied, and the results were compared with those in other lakes of China and the USA. Concentrations of the 15 PAHs in sediments ranged from 436.6 to 1334.9 ng g(-1) (dw). Gasoline combustion, coal combustion, diesel combustion from shipping and spillage of petroleum were apportioned to be the main sources of PAHs in this area by principal component analysis, which contributed 35.19%, 26.43%, 25.41% and 12.97% to the PAH sources estimated by further multiple linear regression. Levels of PAHs in sediments were negatively correlated with contents of clay and fine silt (<16 µm), while positively with contents of medium silt, coarse silt and sand (>16 µm). Humin with size larger than 16 µm contained the largest part of the burden of PAHs in sediments, but the specific partitioning domain (bound humic acid, lipid or insoluble residue) depended on properties of organic matter reflected by optical absorbance at 465 and 665 nm. Total toxic benzo[a]pyrene equivalent (TEQ(carc)) of the carcinogenic PAHs in sediments varied from 31.8 to 209.3 ngTEQ(carc) g(-1). Benzo[a]pyrene and dibenzo[a,h]anthracene contributed 45.36 and 25.31% to total TEQ(carc), posing high toxicological risk to this area.

Effects of copper, lead, and cadmium on the sorption of 2,4,6-trichlorophenol onto and desorption from wheat ash and two commercial humic acids.

The effects of copper (Cu2+), lead (Pb2+), and cadmium (Cd2+) on the sorption of 2,4,6-trichlorophenol (TCP) to and desorption from wheat ash and two commercial humic acids were studied. Copper and Pb2+ diminished the sorption of TCP onto all adsorbents, and made desorption of TCP less hysteretic from ash and German humic acids (GeHA), but more hysteretic from Tianjin humic acids (TJHA). Cadmium had little effect on TCP sorption and desorption. Fourier-transform infra red (FTIR) and X-ray absorption spectroscopy (XAS) in conjunction with fluorescence quenching studies provided insights into the mechanisms of TCP sorption and desorption as affected by Cu2+ and Pb2+, indicating that complexation of Cu2+ and Pb2+ was likely via carboxylic, hydroxylic and phenolic groups of ash, TJHA and GeHA, and that theses same functional groups also reacted with TCP during sorption. In contrast, Cd, a "soft acid", had no effect on the adsorption of TCP. Hydration shells of dense water around adsorbed Cu2+ and Pb2+ ions may also compete with TCP for available surface area. Fluorescence quenching of pyrene verified that for TJHA, Cu2+ and Pb2+ promoted the formation of supramolecular associations with interior hydrophobic regions separated from aqueous surroundings by exterior hydrophilic layers.

Adsorption of arsenate on soils. Part 2: modeling the relationship between adsorption capacity and soil physiochemical properties using 16 Chinese soils.

An attempt has been made to elucidate the effects of soil properties on arsenate adsorption by modeling the relationships between adsorption capacity and the properties of 16 Chinese soils. The model produced was validated against three Australian and three American soils. The results showed that nearly 93.8% of the variability in arsenate adsorption on the low-energy surface could be described by citrate-dithionite extractable Fe (Fe(CD)), clay content, organic matter content (OM) and dissolved organic carbon (DOC); nearly 87.6% of the variability in arsenate adsorption on the high-energy surface could be described by Fe(CD), DOC and total arsenic in soils. Fe(CD) exhibited the most important positive influence on arsenate adsorption. Oxalate extractable Al (Al(OX)), citrate-dithionite extractable Al (Al(CD)), extractable P and soil pH appeared relatively unimportant for adsorption of arsenate by soils.

Adsorption of arsenate on soils. Part 1: laboratory batch experiments using 16 Chinese soils with different physiochemical properties.

Laboratory batch experiments were carried out to study the adsorption of arsenate on 16 Chinese soils with different physicochemical properties. Wide differences in arsenate adsorption were observed, and the Jiangxi and Hubei soils were more effective sorbents for arsenate than other soils. The Langmuir one-surface and two-surface equations were used to model the arsenate adsorption data. Except for the Jiangxi and Hubei soils, the Langmuir one-surface equation gave reasonably good fits to the arsenate adsorption data. However, the Langmuir two-surface equation generally provided a better fit than the Langmuir one-surface equation. For soils with relative high organic matter (OM), dissolved organic carbon (DOC) or extractable phosphate, the Langmuir one-surface and two-surface equations described the adsorption isotherms similarly. In contrast, for soils with relatively low contents of OM, DOC or extractable phosphate, the Langmuir two-surface equation gave the better fit to the arsenate adsorption data.

A comparison of the rhizosphere-based method with DTPA, EDTA, CaCl2, and NaNO3 extraction methods for prediction of bioavailability of metals in soil to barley.

A rhizosphere-based method was compared with DTPA, EDTA, CaCl2, and NaNO3 extraction methods for the evaluation of bioavailability of heavy metals in soil to barley. The extractable amounts of Cr, Cu, Zn and Cd analyzed by the rhizosphere-based method correlated significantly with the metal content of barley roots. The extractable metals identified by DTPA, EDTA, CaCl2 and NaNO3 methods exhibited relatively poor or no correlation with the metal content of barley roots. The stepwise multiple regression equation of the rhizosphere-based method was the simplest one, as no soil properties needed to be entered, whereas the equations for the DTPA, EDTA, CaCl2 and NaNO3 extraction methods always require those variables. The most distinct feature of the rhizosphere-based method was that the proposed method was suitable for acidic, neutral and near alkaline soils. In contrast, the other extraction methods were restricted to soil types. In summary, the rhizosphere-based method is the most robust approach for evaluation of bioavailability of metals in soil to barley.

Comparison of a rhizosphere-based method with other one-step extraction methods for assessing the bioavailability of soil metals to wheat.

There is no method recognized as a universal approach for evaluation of bioavailability of heavy metals in soil. Based on the simulation of the rhizosphere soil conditions and integration of the combined effects of root-soil interactions as a whole, a rhizosphere-based method has been proposed. Wet fresh rhizosphere soil was extracted by low-molecular-weight organic acids (LMWOAs) to fractionate metal fractions of soil pools, which were then correlated with the metal contents of wheat roots and shoots. The rhizosphere-based method was compared with other one-step extraction methods using DTPA, EDTA, CaCl2, and NaNO3 as extractants and the first step of the Community Bureau of Reference (BCR) method. Simple correlation and stepwise multiple regression analysis were used for the comparison. Simple correlation indicated that the extractable Cu, Zn, Cr, and Cd of soils by the rhizosphere-based method were significantly correlated with the metal contents of wheat roots. For DTPA, BCR1 and EDTA methods there was a relatively poor correlation between the extractable Cu, Zn and Cd of soil and metal contents of wheat roots. Stepwise multiple regression analysis revealed that the equation of the rhizosphere-based method was the simplest one, and no soil properties variables needed to be added. In contrast, the equations of other one-step extraction methods were more complicated, and soil properties variables needed to be entered. The most distinct feature of the rhizosphere-based method was that the recommended method was suitable for acidic, neutral and near alkaline soils. However, the DTPA and EDTA extraction methods were suitable for calcareous soils only-or-only for acidic soils. The CaCl2, and NaNO3 extraction methods were only suitable for exchangeable metals. In short, the rhizosphere-based method was the most robust approach for evaluation of bioavailability of heavy metals in soils to wheat.