Long-term effects of amendment with liquid swine manure on proton binding behavior of soil humic substances

The acid-base properties of humic acids (HAs) and fulvic acids (FAs) isolated from liquid swine manure (LSM), soils amended with either 90 or 150 m(3)ha(-1)year(-1) of LSM for 7 years, and the corresponding unamended control soil were investigated by a current potentiometric titration method. The non-ideal competitive adsorption (NICA)-Donnan model for proton binding by two classes of binding sites (i.e., carboxylic- and phenolic-type groups) was fit to titration data, and a set of fitting parameters was obtained for each HA and FA sample. The NICA-Donnan model was shown to describe with a great degree of accuracy the behavior of experimental titration datasets, and highlighted important differences in the acid-base properties of the HAs and FAs examined. When compared to the unamended soil HA and FA, LSM-HA and LSM-FA, had smaller acidic functional group contents, larger proton binding affinities of both carboxylic- and phenolic-type groups, smaller heterogeneity of carboxylic-type groups, and smaller, in the case of HA, or similar, in the case of FA, heterogeneity of phenolic-type groups. Amendment with LSM caused a decrease of acidic functional group contents and a slight increase of proton binding affinities of carboxylic- and phenolic-type groups of soil HAs and FAs. Further, LSM application induced a decrease of the heterogeneity of carboxylic-type groups, whereas appeared not to affect substantially the heterogeneity of phenolic-type groups of LSM-amended soil HAs and FAs. These effects were more evident for HAs than for FAs and tended to slightly increase with increasing LSM amendment rate.     

Copper(II) complexation by humic and fulvic acids from pig slurry and amended and non-amended soils

The effect of the consecutive annual additions of pig slurry at rates of 0 (control), 90 and 150 m3 ha(-1) y(-1) over a 4-year period on the binding affinity for Cu(II) of soil humic acids (HAs) and fulvic acids (FAs) was investigated in a field plot experiment under semiarid conditions. A ligand potentiometric titration method and a single site model were used for determining the Cu(II) complexing capacities and the stability constants of Cu(II) complexes of HAs and FAs isolated from pig slurry and control and amended soils. The HAs complexing capacities and stability constants were larger than those of the corresponding FA fractions. With respect to the control soil HA, pig-slurry HA was characterized by a much smaller binding capacity and stability constant. Amendment with pig slurry decreased the binding affinity of soil HAs. Similar to the corresponding HAs, the binding affinity of pig-slurry FA was much smaller while that of amended-soil FAs were slightly smaller when compared to the control soil FA. The latter effect was, however, more evident with increasing the amount of pig slurry applied to soil per year and the number of years of pig slurry application.     

Detection of copper(II) and zinc(II) binding to humic acids from pig slurry and amended soils by fluorescence spectroscopy

The effect of the consecutive annual additions of pig slurry at rates of 0 (control), 90 and 150 m3 ha(-1) yr(-1) after a 7-year period on the Cu(II) and Zn(II) binding behavior of soil HAs was investigated in a field experiment. A fluorescence titration method and a single site model were used for determining metal ion complexing capacities and stability constants of metal ion complexes of HAs isolated from pig slurry and unamended and amended soils. With respect to control soil HA, pig-slurry HA featured much smaller Cu(II) and Zn(II) binding capacities and stability constants. Pig-slurry application to soil decreased Cu(II) and Zn(II) complexing capacities and binding affinities of soil HA. These effects increased with increasing the rate per year of PS application to soil, and are expected to have a large impact on bioavailability, mobilization, and transport of Cu(II) and Zn(II) ions in pig slurry-amended soils.     

Binding of polycyclic aromatic hydrocarbons by humic acids formed during composting

Binding of two model polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by humic acids (HAs) isolated from an organic substrate at different stages of composting and a soil was investigated using a batch fluorescence quenching method and the modified Freundlich model. With respect to soil HA, the organic substrate HA fractions were characterized by larger binding affinities for both phenanthrene and pyrene. Further, isotherm deviation from linearity was larger for soil HA than for organic substrate HAs, indicating a larger heterogeneity of binding sites in the former. The composting process decreased the binding affinity and increased the heterogeneity of binding sites of HAs. The changes undergone by the HA fraction during composting may be expected to contribute to facilitate microbial accessibility to PAHs. The results obtained also suggest that bioremediation of PAH-contaminated soils with matured compost, rather than with fresh organic amendments, may result in faster and more effective cleanup.     

Thermodynamics of metal cation binding by a solid soil derived humic acid. 2. Binding of Mn(II), Co(NH3)6aq3+ and Hg(II)

Metal binding is an important function of humic acids (HAs) in soils, sediments and waters. At pH 2.0, Mn(II) and Co(NH3)6aq3+ bind tightly in one step labeled A to a solid humic acid NHA isolated from a New Hampshire soil. Two consecutive steps are observed for Hg(II) binding. All the binding isotherms fit the Langmuir model in the temperature range 10.0-50.0 degrees C. Stoichiometric site capacities indicate predominant binding by charge-neutralizing HA carboxylate groups for Mn(II) and the second step A of Hg(II) binding. The binding affinity order in step A is Co(NH3)(6)3+>Hg(II)>Mn(II). Metal binding enthalpy and entropy changes fit the linear correlation found previously for binding of other metal cations by solid HAs. Free energy buffering from cooperative enthalpy and entropy changes and lower enthalpies for metal-HA interactions in solution suggest that desolvation of the cations and HA binding sites as well as HA conformational changes to allow for inner-sphere complexation predominate metal binding by hydrated solid HAs.     

Effects of sewage sludge amendment on heavy metal accumulation and consequent responses of Beta vulgaris plants

Use of sewage sludge, a biological residue produced from sewage treatment processes in agriculture is an alternative disposal technique of waste. To study the usefulness of sewage sludge amendment for palak (Beta vulgaris var. Allgreen H-1), a leafy vegetable and consequent heavy metal contamination, a pot experiment was conducted by mixing sewage sludge at 20% and 40% (w/w) amendment ratios to the agricultural soil. Soil pH decreased whereas electrical conductance, organic carbon, total N, available P and exchangeable Na, K and Ca increased in soil amended with sewage sludge in comparison to unamended soil. Sewage sludge amendment led to significant increase in Pb, Cr, Cd, Cu, Zn and Ni concentrations of soil. Cd concentration in soil was found above the Indian permissible limit in soil at both the amendment ratios.

The increased concentration of heavy metals in soil due to sewage sludge amendment led to increases in heavy metal uptake and shoot and root concentrations of Ni, Cd, Cu, Cr, Pb and Zn in plants as compared to those grown on unamended soil. Accumulation was more in roots than shoots for most of the heavy metals. Concentrations of Cd, Ni and Zn were more than the permissible limits of Indian standard in the edible portion of palak grown on different sewage sludge amendments ratios. Sewage sludge amendment in soil decreased root length, leaf area and root biomass of palak at both the amendment ratios, whereas shoot biomass and yield decreased significantly at 40% sludge amendment. Rate of photosynthesis, stomatal conductance and chlorophyll content decreased whereas lipid peroxidation, peroxidase activity and protein and proline contents, increased in plants grown in sewage sludge-amended soil as compared to those grown in unamended soil.

The study clearly shows that increase in heavy metal concentration in foliage of plants grown in sewage sludge-amended soil caused unfavorable changes in physiological and biochemical characteristics of plants leading to reductions in morphological characteristics, biomass accumulation and yield. The study concludes that sewage sludge amendment in soil for growing palak may not be a good option due to risk of contamination of Cd, Ni and Zn and also due to lowering of yield at higher mixing ratio.     

Pyrolysis of humic acids from digested and composted sewage sludge

Humic acids (HAs) were extracted from four digested sewage sludge samples composted for four months, one, two and four years. HAs were pyrolyzed at three different temperatures applying both conventional and in situ methylation (ISM) pyrolysis. The pyrolysates were analyzed using gas chromatography-mass spectrometry (GC/MS). Derivatization (ISM) and pyrolysis temperature had dramatic effects on the composition and relative amounts of the pyrolysates. Among the derivatized HA fragments aliphatic compounds prevailed under all the pyrolysis conditions tested. Aromatic substances consisting mainly of guaiacyl-type compounds were detected in higher abundances only at elevated temperatures. Without ISM the contribution of aromatic structures to the total pyrogram was considerably greater than that of the aliphatics. Increase of the pyrolysis temperature from 450 degrees C to 600 degrees C had smaller effect on the proportions and composition of the compounds studied than increase from 350 degrees C to 450 degrees C.     

Long-term effect of sewage sludge application on soil humic acids

Sewage sludges are used in agriculture because they act as a fertilizer. Long-term studies are needed to evaluate the effect of sewage sludge on soil properties by paying particular attention to the soil organic matter. Soil plots were amended for 10 years with 1Mg dry matter ha(-1)year(-1) of sewage sludge. Chemical parameters such as total organic carbon (TOC), N, C/N ratio and CEC were determined when this period ended. Moreover, TOC was fractionated into humified and non-humified fractions. Humic acids (HA) were isolated and studied by elemental analysis, DRIFT, (1)H NMR and CPMAS 13C NMR spectroscopies. At the end of the tests, compared to the control soil, the sludge-amended soil did not exhibit change in total organic C and related humified fractions. However, the HA composition of the soil treated with sludge had developed an HA composition closer to that of the HA-sludge as a result of the enrichment of recalcitrant fractions contained in the sludge.     

2-Undecanone and 2-tridecanone in field-grown onion

A field study was conducted to investigate the impact of soil amendments on concentrations of two volatile organic compounds, 2-undecanone and 2-tridecanone, in onion bulbs. The soil in five plots was mixed with sewage sludge, five plots were mixed with yard waste compost, five plots were mixed with laying hen manure each at 15 t acre^?1, and five unamended plots that never received soil amendments were used for comparison purposes. Plots (n = 20) were planted with onion, Allium cepa L. var. Super Star-F1 bulbs. Gas chromatographic/mass spetrometric (GC/MS) analyses of mature onion bulbs crude extracts revealed the presence of two major fragment ions that correspond to 2-undecanone and 2-tridecanone. Soil amended with yard waste compost enhanced 2-undecanone and 2-tridecanone production by 31 and 59%, respectively. Soil amended with chicken manure enhanced 2-undecanone and 2-tridecanone production by 28 and 43%, respectively. Concentrations of 2-undecanone and 2-tridecanone were lowest in onion bulbs of plants grown in sewage sludge and unamended soil, respectively. The increased concentrations of 2-undecanone and 2-tridecanone in onion bulbs may provide a protective character against insect and spider mite attack in field grown onions.     

Effect of organic amendments on the mobility of trace elements in phytoremediated techno-soils: role of the humic substances

The efficiency of aided phytostabilization using organic amendments such as ramial chipped wood (RCW) and composted sewage sludge (CSS) was studied on contaminated techno-soils, on nine experimental plots. The objective was to characterize the role of fulvic (FA) and humic acids (HA) on the mobilization of trace elements, specifically As, Cu, Mo, Pb and Zn. Results showed that the addition of CSS increased the total organic carbon and nitrogen content more than with RCW and as a result, the C/N ratio in the CSS soil was higher than in the RCW and non-amended (NE) soil, reflecting the high decomposition of soil organic matter in the CSS soil compared with the other soils. The RCW and CSS amendments increased the hydrogen index (HI) values and the oxygen index (OI) values compared with the NE soil, especially for the soil treated with CSS which contained more aliphatic than aromatic compounds. The addition of CSS to the techno-soil significantly increased the percentage of C _org associated with the HA fractions compared with the RCW and NE soils. The soil amended with CSS showed the highest E ₄/E ₆ ratio and the lowest E ₂/E ₃ ratio of FA. Zn and As were more abundant in the FA fraction than in the HA fraction, whereas Pb, Cu and Mo were more associated to HA than to FA in the treated and untreated soils, which may explain the difference in their mobility and availability.     

Effects of sewage sludge fertilizer on heavy metal accumulation and consequent responses of sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>)

Use of sewage sludge, a biological residue produced from sewage treatment processes in agriculture, is an alternative disposal technique of waste. To study the usefulness of sewage sludge amendment for Helianthus annuus, a pot experiment was conducted by mixing sewage sludge at 2.5, 5, and 7.5 % (w/w) amendment ratios to the agricultural soil. Soil pH decreased whereas electrical conductivity, organic matter, total N, available P, and exchangeable Na, K, and Ca increased in soil amended with sewage sludge in comparison to unamended soil. Sewage sludge amendment led to significant increase in Pb, Ni, Cu, Cr, and Zn concentrations of soil. The increased concentration of heavy metals in soil due to sewage sludge amendment led to increases in shoot and root concentrations of Cr, Cu, Ni, and Zn in plant as compared to those grown on unamended soil. Accumulation was more in roots than shoots for most of the heavy metals. Moreover, high metal removal for the harvestable parts of the crops was recorded. Sewage sludge amendment increased root and shoot length, leaves number, biomass, and antioxidant activities of sunflower. Significant increases in the activities of antioxidant enzymes and in the glutathione, proline, and soluble sugar content in response to amendment with sewage sludge may be defense mechanisms induced in response to heavy metal stress.

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Graphical abstract Origin, fate and behavior of sewage sludge fertilizer

     

Influence of cadmium on carbon and nitrogen mineralization in sewage sludge amended soils

The effect of cadmium on C and N mineralization in sewage sludge amended and unamended sandy loam, loam and clay loam soils was studied during 2 months incubation at 30+/-1 degrees C. The sludge amendment caused 15-39% increase in microbial respiration, with the maximum C mineralization in sandy loam and the minimum in loam soil. The addition of 10 microg Cd g(-1) soil had no remarkable effect on C and N mineralization and microbial biomass; whereas significant decreases in the above parameters were observed at 25 and 50 microg Cd g(-1) soil, irrespective of the sludge addition. Less NO3(-)-N accumulated at higher Cd concentration. Cd recovery was high in sandy loam and low in clay loam soil. DTPA extractable Cd exhibited a significant negative correlation with microbial biomass (r=-0.58* to -0.86*; p < 0.05).     

Heavy metal mobility in runoff water and absorption by eggplant fruits from sludge treated soil

Sewage sludge addition to agricultural lands requires judicious management to avoid environmental risks arising from heavy metal and nitrate contamination of surface water and accumulation in edible plants. A field study was conducted on a silty-loam soil of 10% slope at Kentucky State University Research Farm. Eighteen plots of 22 x 3.7 m each were separated using metal borders and the soil in six plots was mixed with sewage sludge and yard waste compost mix (SS-YW) at 15 t acre(-1), six plots were mixed with sewage sludge (SS) at 15 t acre(-1), and six unamended plots that never received sludge were used for comparison purposes. Plots were planted with eggplant, Solanum melongena L. as the test plant. The objectives of this investigation were to: 1) assess the effect of soil amendments on the transport of NO3, NH4, and heavy metals (Cd, Cr, Ni, Pb, Zn, Cu, and Mo) into surface water; 2) investigate the effect of soil amendments on heavy metal bioavailability in eggplant fruits at harvest; and 3) assess chemical and physical properties of soil following addition of soil amendments and their impact on the yield and quality of eggplant fruit. SS-YW treatments reduced runoff water by 63% while plots incorporated with sewage sludge alone reduced runoff water by 37% compared to control treatment. The SS-YW treatments transported more mineral nitrogen (NO3-N and NH4-N) in runoff water than SS treatments. Total marketable yield (lbs acre(-1)) and number of eggplant fruits were greatest in SS-YW treatments. This response may be due to improved soil porosity, water, and nutrient retention of the soil amended with SS-YW mixture. Concentrations of heavy metals in soil amended with sludge were below the U.S. Environmental Protection Agency (USEPA) limits. Chromium, Ni, Zn, and Cu were taken up by eggplant fruits but their concentrations were below the Codex Commission allowable levels.     

The influence of application rate on the bacterial mutagenicity of soil amended with municipal sewage sludge

The mutagenic potential of two soils amended with a municipal sewage sludge at two application rates was monitored over a 2-year period using Salmonella/microsome mutagenicity assay. Samples were collected from undisturbed monolith lysimeters of Weswood sandy clay (Fluventic Ustochrept) and Padina sandy loam (Grossarenic Paleustalf) amended with dried sewage sludge at 50 and 100 Mg/ha. Soil samples were collected and sequentially extracted with methylene chloride and methanol. The residues from these extracts were tested for mutagenicity at five doses with and without metabolic activation in Salmonella strain TA98. In general, the mutagenic potential of the amended soils of both application rates for the first 8 weeks following sludge application increased and then slowly decreased. The maximum mutagenic response observed in the soil extracts was 222 revertants at a dose of 10 mg of residue. This response was induced by the methanol extract from the Weswood soil collected 56 days after the application of 50 Mg/ha sewage sludge as compared to the 100 Mg/ha application which induced 202 revertants/mg. The mutagenicity of all fractions extracted from the sludge-amended soil at both application rates collected 717 days after application were not appreciably different from extracts from the unamended soils. The data indicate that chemicals that were mutagenic in bacteria persist in the soil and that at the higher application rates, as much as 2 years may be required for the mutagenic potential of the soil to return to background levels.     

Biodegradation of phthalate esters in polluted soil by using organic amendment

This study investigated the biodegradation of the phthalate esters (PAEs) di-n-butyl phthalate (DBP) and di-(2-ethyl hexyl) phthalate (DEHP) in sludge and sludge-amended soil. DBP (100 mg kg(-1)) and DEHP (100 mg kg(-1)) were added to sewage sludge, which was subsequently added to soil. The results showed that sewage sludge can degrade PAEs and the addition of sewage sludge to soil enhanced PAE degradation. Sludge samples were separated into fractions with various particle size ranges, which spanned 0.1-0.45 μm to 500-2000 μm. The sludge fractions with smaller particle sizes demonstrated higher PAE degradation rates. However, when the different sludge fractions were added to soil, particle size had no significant effect on the rate of PAE degradation. The results from this study showed that microbial strains F4 (Rhodococcus sp.) and F8 (Microbacterium sp.) were constantly dominant in the mixtures of soil and sludge.     

XAFS studies of cobalt(II) binding by solid peat and soil-derived humic acids and plant-derived humic acid-like substances

This work has examined cobalt(II) binding by a variety of solid humic acids (HAs) isolated from peat, plant and soil sources at temperatures down to 60K. The results confirm that X-ray absorption near-edge spectroscopy (XANES) measurements cannot distinguish between aquo and carboxylato ligands in the inner coordination sphere of Co(II). However, between 1 and 2 inner-sphere carboxylato ligands can be detected in all the peat, plant and soil-derived HA samples by extended X-ray absorption fine structure (EXAFS) measurements, indicating inner-sphere coordination of HA-bound Co(II). The precision of C(carboxylate) detection is limited by the extent and quality of the data and the contribution from inner-sphere O to the Fourier transformed peaks used to detect carbon. Putative chelate ring formation is consistent with a relatively negative entropy change in step A, the stronger Co(II) binding step by HA functional groups, and could relate to 'non-exchangeable' metal binding by HSs.     

Determination of chemical availability of cadmium and zinc in soils using inert soil moisture samplers

A rapid method for extracting soil solutions using porous plastic soil-moisture samplers was combined with a cation resin equilibration based speciation technique to look at the chemical availability of metals in soil. Industrially polluted, metal sulphate amended and sewage sludge treated soils were used in our study. Cadmium sulphate amended and industrially contaminated soils all had > 65% of the total soil solution Cd present as free Cd2+. However, increasing total soil Cd concentrations by adding CdSO4 resulted in smaller total soil solution Cd. Consequently, the free Cd2+ concentrations in soil solutions extracted from these soils were smaller than in the same soil contaminated by sewage sludge addition. Amendment with ZnSO4 gave much greater concentrations of free Zn2+ in soil solutions compared with the same soil after long-term Zn contamination via sewage sludge additions. Our results demonstrate the difficulty in comparing total soil solution and free metal ion concentrations for soils from different areas with different physiochemical properties and sources of contamination. However, when comparing the same Woburn soil, Cd was much less available as Cd2+ in soil solution from the CdSO4 amended soils compared with soil contaminated by about 36 years of sewage sludge additions. In contrast, much more Zn was available in soil solution as free Zn2+ in the ZnSO4 amended soils compared with the sewage sludge treated soils.     

Heavy metal mobility in runoff water and absorption by eggplant fruits from sludge treated soil

Sewage sludge addition to agricultural lands requires judicious management to avoid environmental risks arising from heavy metal and nitrate contamination of surface water and accumulation in edible plants. A field study was conducted on a silty-loam soil of 10% slope at Kentucky State University Research Farm. Eighteen plots of 22 × 3.7 m each were separated using metal borders and the soil in six plots was mixed with sewage sludge and yard waste compost mix (SS-YW) at 15 t acre^?1, six plots were mixed with sewage sludge (SS) at 15 t acre^?1, and six unamended plots that never received sludge were used for comparison purposes. Plots were planted with eggplant, Solanum melongena L. as the test plant. The objectives of this investigation were to: 1) assess the effect of soil amendments on the transport of NO₃, NH₄, and heavy metals (Cd, Cr, Ni, Pb, Zn, Cu, and Mo) into surface water; 2) investigate the effect of soil amendments on heavy metal bioavailability in eggplant fruits at harvest; and 3) assess chemical and physical properties of soil following addition of soil amendments and their impact on the yield and quality of eggplant fruit. SS-YW treatments reduced runoff water by 63% while plots incorporated with sewage sludge alone reduced runoff water by 37% compared to control treatment. The SS-YW treatments transported more mineral nitrogen (NO₃-N and NH₄-N) in runoff water than SS treatments. Total marketable yield (lbs acre^?1) and number of eggplant fruits were greatest in SS-YW treatments. This response may be due to improved soil porosity, water, and nutrient retention of the soil amended with SS-YW mixture. Concentrations of heavy metals in soil amended with sludge were below the U.S. Environmental Protection Agency (USEPA) limits. Chromium, Ni, Zn, and Cu were taken up by eggplant fruits but their concentrations were below the Codex Commission allowable levels.     

Uptake and translocation of metals in Spinacia oleracea L. grown on tannery sludge-amended and contaminated soils: effect on lipid peroxidation, morpho-anatomical changes and antioxidants

The plants of Spinacia oleracea L. grown on contaminated soil (CS) and different amendments of tannery sludge (TS) have shown high accumulation of metals in its edible part. The accumulation of toxic metal (Cr) in the leaves of the plants grown on CS was recorded as 40.67 microgg(-1)dw. However, the leaves of the plants grown on 100% TS have accumulated about two times (70.80 microgg(-1)dw) higher Cr than the 10% TS (31.21 microgg(-1)dw). Among growth parameters, the root length was more affected at 90 d than the shoot length, number of leaves and leaf area. The study of scanning electron micrographs showed 29.31% increase in stomatal length in the leaves of the plants grown on CS as compared to garden soil (GS), which served as control, however it decreased in the plants grown on higher amendments of TS. The decrease in MDA content at initial period of exposure and lower amendment was recorded in the leaves, whereas, significant increase (>10% TS onward) was observed with increase in tannery sludge ratio at 90 d as compared to GS. A coordinated increase in all the studied antioxidants (cysteine, non-protein thiol, ascorbic acid, carotenoid contents) was found up to 75 d of growth. At 90 d, most of the antioxidant decreased as compared to 75 d causing oxidative stress as evidenced by increased level of lipid peroxidation and decreased chlorophyll and protein contents. Maximum increase of 181.43% in MDA content and maximum decrease of 53.69% in total chlorophyll content was recorded in the leaves of the plants grown on 100% TS after 90 d of growth. The plants grown on CS have shown an increase in shoot length, number of leaves, leaf area, photosynthetic pigments and protein contents and in all the studied antioxidants. Thus, these plants are able to combat stress involving defense mechanism, resulting in healthy growth of the plants. The results are well coordinated as there is no change in the MDA content as compared to the plants grown on GS. In view of high Cr accumulation in edible part of S. oleracea grown on CS after irrigation with tap water, it is not advisable to use these plants for edible purposes. Summing up, it is recommended that the level of metals in the edible part should be checked instead of healthy growth as deciding parameter for consumption. It is demonstrated through this study that metal enriched plants have detoxification mechanism and grow well on organic matter enriched contaminated soil.     

Binding characteristics of Cu2+ to natural humic acid fractions sequentially extracted from the lake sediments

Humic acids (HAs) determine the distribution, toxicity, bioavailability, and ultimate fate of heavy metals in the environment. In this work, ten HA fractions (F1–F10) were used as adsorbent, which were sequentially extracted from natural sediments of Lake Wuliangsuhai, to investigate the binding characteristics of Cu²⁺ to HA. On the basis of the characterization results, differences were found between the ten extracted HA fractions responding to their elemental compositions and acidic functional groups. The characterization results reveal that the responses of ten extracted HA fractions to their elemental compositions and acidic functional groups were different. The O/C and (O + N)/C ratio of F1–F8 approximately ranged from 0.66 to 0.53 and from 0.72 to 0.61, respectively; the measured results showed that the contents of phenolic groups and carboxyl groups decreased from 4.46 to 2.60 mmol/g and 1.60 to 0.58 mmol/g, respectively. The binding characteristics of Cu²⁺ to the ten HA fractions were well modeled by the bi-Langmuir model; the binding behavior of Cu²⁺ to all the ten HA fractions were strongly impacted by pH and ionic strength. The FTIR and SEM-EDX image of HA fractions (pre- and post-adsorption) revealed that carboxyl and phenolic groups were responsible for the Cu²⁺ sorption on the ten sequentially extracted HA fractions process, which is the same with the analysis of the ligand binding and bi-Langmuir models Accordingly, the adsorption capacity of the former HA fractions on Cu²⁺ were higher than the latter ones, which may be attributed to the difference of carboxyl and phenolic group contents between the former and latter extracted HA fractions. Additionally, the functional groups with N and S should not be neglected. This work is hopeful to understand the environmental effect of humic substances, environmental geochemical behavior, and bioavailability of heavy metals in lakes.