Speciation of rare earth elements in soil and accumulation by wheat with rare earth fertilizer application

A greenhouse study was conducted to investigate the accumulation of rare earth elements (REEs), La, Ce, Pr and Nd, in winter wheat (Triticum aestivum L.), and the speciation of these elements in soil following the application of REE-based fertilizers. Improved crop yield was confirmed by the experiment. The accumulation behavior of La, Ce, Pr and Nd in wheat varied depending on the concentration of REE fertilizer application, i.e. increased with increasing REE concentration at low fertilization application, constant over the medium REE range, and decreased with increasing REE concentration at high fertilizer application. Significant negative correlation was obtained between REE contents in roots and soil pH (r = -0.5787 to -0.8442 for La). REEs in both the fertilized and unfertilized soils were fractionated by a three-stage sequential extraction procedure into three chemically distinct fractions: water soluble, exchangeable and carbonate bound (B1), Fe-Mn oxide bound (B2), and organic and sulfide bound (B3). REEs in fertilized soils were found mainly in the B2 and B3 fractions, with only a small amount in the B1 fraction. REEs in B1 and B2 fractions were negatively correlated with soil pH (r = -0.6892 to -0.8927 and -0.7462 to -0.9482). Significant correlation was obtained between REEs in B1 fraction and REE contents in root. The correlation coefficients ranged from 0.6159 to 0.7410 when fertilizer application was lower than 20.0 mg/kg soil. No acceptable relationship was observed between REE contents in shoot and any of the extractable fractions in soils.     

Bioaccumulation of lanthanum and cerium and their effects on the growth of wheat (Triticum aestivum L.) seedlings

Through short-term exposure (7-d exposure), long-term exposure (16-d exposure) and exposure-recovery (7-d exposure + 9-d recovery), the bioaccumulation and distribution of La and Ce and their effects on growth of wheat seedlings were studied. Addition of La (0.5-25 mg/l) and Ce (0.5-25 mg/l) to the culture medium individually and in combination inhibited primary root elongation, reduced the dry weight of roots and shoots and the content of mineral elements (Ca, Mg, K, Cu, Zn). The damage increased with an increase in the concentrations of La and Ce in culture medium. Relative damage ratio increased with an increase in concentrations of La and Ce in the culture medium and with exposure time. Comparing exposure-recovery groups with long-term exposure groups, primary root lengths, dry weight of roots and shoots and the content of five mineral elements were higher. The accumulation of La and Ce in the seedlings was positively correlated with the concentrations of La and Ce in the culture medium and with exposure time. Bioaccumulation factors of La and Ce in roots were much higher than those of shoots. The uptake rates of La and Ce by the plants were much higher than the translocation rates from roots to shoots. The accumulation and distribution of La and Ce in the seedlings in exposure-recovery groups showed that there was very little excretion through metabolism during the recovery period, but redistribution occurred throughout the whole plant. No apparent selective uptake was found between La and Ce by the plants when they were applied in combination.     

Application of microwave extraction for the evaluation of bioavailability of rare earth elements in soils

A single microwave extraction procedure was conducted to investigate the bioavailability of rare earth elements (REEs) by using different extractants of 0.05 M EDTA, 0.10 M CH3COOH, 0.10 M HCl, and 0.05 M CaCl2. The experimental conditions of heating time and microwave power were optimized. A microwave power of 60% and extraction time of 30 min were adopted. Compared to the conventional single extraction schemes reported in the literature, the recommended technique shortened the operational time, simplified the experimental task, improved the precision, and obtained consistent results with those obtained by using the conventional methods. In addition, the suggested microwave extraction method has been successfully used to evaluate the bioavailability of REEs in soils. Soil samples collected from 15 sites in China were extracted by four different extractants with microwave ancillary. Extractable REEs from soils were well correlated with REEs contents in shoots of wheat (Troticum aestivum L.) under the greenhouse conditions. The correlation coefficients were 0.6514-0.8996, 0.4522-0.7783, 0.6506-0.8671, 0.4869-0.7014 between the extractable REEs in soils and their concentrations in wheat shoots for the extractants of CaCl2, EDTA, CH3COOH and HCl, respectively.     

Toxicity of arsenate and arsenite on germination, seedling growth and amylolytic activity of wheat

Effects of different concentrations of arsenite and arsenate (0-16 mg/l) on seed germination, relative root length and shoot height, arsenic accumulation in young seedlings, alpha-amylase, beta-amylase and total amylolytic activity in wheat were investigated in order to elucidate the toxicity of arsenic in the early developmental stage. Germination percentages of different wheat varieties had different responses to arsenic species and decreased significantly with increasing arsenic concentrations except Duokang 1. Relative root length (RRL) and relative shoot height (RSH) of wheat seedlings decreased with increasing concentrations of arsenite and arsenate. The relative root lengths were correlated with the relative shoot heights for arsenite (r2 = 0.79) and arsenate (r2 = 0.77). Arsenic uptake by seedlings increased with the increasing concentrations of arsenite or arsenate and followed the Michaelis-Menten kinetics function. The average total amylolytic activity and beta-amylase activity had no significant difference comparable to that of controls at the concentration 2 mg/l arsenite or arsenate, but decreased apparently when the concentration was higher than 2 mg/l. Whereas the alpha-amylase activity decreased with increasing concentrations of arsenite or arsenate over the whole concentration range. Arsenite decreased all the endpoints more remarkably than arsenate. In comparison, shoot height and root length were more sensitive to arsenic than other endpoints and might be used as indicators for arsenic toxicity.     

Characterization of a simple bacterial consortium for effective treatment of wastewaters with reactive dyes and Cr(VI)

A microbial consortia consisting of three bacteria isolated from tanning and textile wastewaters revealed high capacity to simultaneously bioaccumulate dye and Cr(VI). The identity of the bacteria were determined by 16S rRNA gene analysis to be closely related to Ochrobactrium sp., Salmonella enterica and Pseudomonas aeruginosa. Dependence of initial pH values and range of concentrations of the dye Reactive Black B (33.2-103.1 mg l(-1)) and Cr(VI) (19.9-127.6 mg l(-1)) were examined to find the effect of pH on the dye and Cr(VI) bioaccumulation. Optimal pH for growth of the consortia in media containing 35 mg l(-1) dye and 50 mg l(-1) Cr(VI) was determined to be around 8. The Cr(VI) bioaccumulation by the consortia was rapid in media containing molasses with or without reactive dye with a maximum Cr(VI) bioaccumulation yield ranging from 90% to 99% within a 2-4d period. A slightly lower yield for the dye bioaccumulation was measured with a maximum dye bioaccumulation of 80% at 59.3 mg l(-1) dye and 69.8 mg l(-1) Cr(VI). The highest specific Cr uptake value was obtained as 76.7 mg g(-1) at 117.1 mg l(-1) Cr(VI) and 50.8 mg l(-1) dye concentration. This ability to bioaccumulate dye and Cr(VI) was more efficient than the enriched sludge from which they were isolated.     

Comparative performance evaluation of Aspergillus lentulus for dye removal through bioaccumulation and biosorption

Dyes used in various industries are discharged into the environment and pose major environmental concern. In the present study, fungal isolate Aspergillus lentulus was utilized for the treatment of various dyes, dye mixtures and dye containing effluent in dual modes, bioaccumulation (employing growing biomass) and biosorption (employing pre-cultivated biomass). The effect of dye toxicity on the growth of the fungal isolate was studied through phase contrast and scanning electron microscopy. Dye biosorption was studied using first and second-order kinetic models. Effects of factors influencing adsorption and isotherm studies were also conducted. During bioaccumulation, good removal was obtained for anionic dyes (100 mg/l), viz. Acid Navy Blue, Fast Red A and Orange-HF dye (99.4 %, 98.8 % and 98.7 %, respectively) in 48 h. Cationic dyes (10 mg/l), viz. Rhodamine B and Methylene Blue, had low removal efficiency (80.3 % [48 h] and 92.7 % [144 h], respectively) as compared to anionic dyes. In addition to this, fungal isolate showed toxicity response towards Methylene Blue by producing larger aggregates of fungal pellets. To overcome the limitations of bioaccumulation, dye removal in biosorption mode was studied. In this mode, significant removal was observed for anionic (96.7–94.3 %) and cationic (35.4–90.9 %) dyes in 24 h. The removal of three anionic dyes and Rhodamine B followed first-order kinetic model whereas removal of Methylene Blue followed second-order kinetic model. Overall, fungal isolate could remove more than 90 % dye from different dye mixtures in bioaccumulation mode and more than 70 % dye in biosorption mode. Moreover, significant color removal from handmade paper unit effluent in bioaccumulation mode (86.4 %) as well as in biosorption mode (77.1 %) was obtained within 24 h. This study validates the potential of fungal isolate, A. lentulus, to be used as the primary organism for treating dye containing wastewater.     

Molluscicidal activity of Ferula asafoetida, Syzygium aromaticum and Carum carvi and their active components against the snail Lymnaea acuminata

The molluscicidal activity of dried root latex powder of Ferula asafoetida, flower-bud powder of Syzygium aromaticum and seed powder of Carum carvi against the snail Lymnaea acuminata was studied. The molluscicidal activity of all the three plant products was found to be both time and concentration dependent. The toxicity of S. aromaticum flower-bud powder (96 h LC(50):51.98 mg/l) was more pronounced than that of root latex powder of F. asafoetida (96 h LC(50):82.71 mg/l) and seed powder of C. carvi (96 h LC(50):140.58 mg/l). Ethanol extract was more toxic than other organic extracts. The ethanol extract of S. aromaticum (24h LC(50):83.53 mg/l) was more effective than that of F. asafoetida (24h LC(50):132.31 mg/l) and C. carvi (24h LC(50):130.61 mg/l) in killing the test animals. The 96 h LC(50) of column purified fraction of seed powder of C. carvi was 5.40 mg/l whereas those of flower-bud powder of S. aromaticum and dried root latex powder of F. asafoetida were 7.87 and 9.67 mg/l, respectively. The product of F. asafoetida, S. aromaticum and C. carvi may be used as potent molluscicides.     

Chronic copper exposure and fatty acid composition of the amphipod Dikerogammarus villosus: results from a field study

Field study allows assessment of long-term effects on fatty acid (FA) composition of organisms under chronic exposure to metals. One expected effect of copper is peroxidation of lipids and essentially polyunsaturated fatty acids (PUFA). FA analysis was established for the amphipod Dikerogammarus villosus subjected to different degrees of copper exposure (4–40 μg Cu L⁻¹). A previous study in our team showed that this species regulates its body Cu concentration (106–135 mg Cu kg⁻¹ dry weight). Despite the high capacity of bioaccumulation, the absence of a correlation between copper concentration in D. villosus and water prevents its use as bioindicator of copper pollution. Both sexes from the most polluted site showed the lowest total FA content, but the highest PUFA percent, mainly of the long-chained variety (C20–C22). Mechanisms leading to the prevention of lipid peroxidation in this species were discussed (metallothioneins and intracellular granules) and proposed with support from literature data.     

Effect of earthworms (Eisenia fetida) on the fractionation and bioavailability of rare earth elements in nine Chinese soils

The effect of earthworm (Eisenia fetida) activity on soil pH, dissolved organic carbon (DOC), fraction distribution pattern and bioavailability of rare earth elements (REEs) Y, La, Ce, Pr and Nd in nine Chinese soils were investigated using pot experiments. A three-step extraction procedure recommended by the European Community (Standards, Measurements and Testing Programme) was used to fractionate REEs in soils into water soluble, exchangeable and carbonate bound (B1), Fe- and Mn-oxides bound (B2) and organic matter and sulfide bound (B3). Inoculated with earthworms, the soil pH, DOC and water-soluble rare earth elements fraction increased. A significant correlation was obtained between the increased DOC and the increased water-soluble REEs. REEs in fraction B1 increased after earthworm inoculation, while those in fraction B3 decreased. No significant differences were observed for REEs in fraction B2. The biomass and the concentrations of REEs in wheat shoots and roots increased after the treatment with earthworms. The results demonstrated that earthworm activity increased the mobility and bioavailability of REEs in soils.     

Lead accumulation and elimination in tissues of Prussian carp, Carassius gibelio (Bloch, 1782), after long-term dietary exposure, and depuration periods

We studied the bioaccumulation of lead in selected tissues of Prussian carp Carassius gibelio (Bloch, 1782) during 12 and 24 months exposure to different doses of this metal in feed and the elimination of lead from tissues during the following 12-month depuration period. Lead concentration was determined using atomic absorption spectrometry method. The highest lead concentrations were observed at 2.0?±?0.54 to 7.4?±?1.1 mg?kg^?1 in the kidney, 3.0?±?0.13 to 5.2?±?0.17 mg?kg^?1 in the bone, and 4.5 (±0.4)?mg?kg^?1 in the hepatopancreas of fish from groups exposed to lead dietary concentration from 8 to 49 mg?kg^?1 for 24 months. The rate of accumulation were generally the highest at the beginning of exposure as evidenced by the highest monthly increments of bioaccumulation observed after 3 months of contamination for muscles, hepatopancreatic gland, intestine, and gills. Also analysis of the monthly increments of lead bioaccumulation in bone tissue and the highly significant coefficients of correlation indicate that the dynamics of accumulation are clearly dependent on dose of exposure. Depuration of accumulated lead from the organs depended mainly on tissue and duration of elimination period. Very rapid depuration was observed in soft tissues such as the intestine or muscles. Very low elimination was observed for scales and bones where until the end of the experiment highly significant lead concentration differences were observed in all groups in relation to the control group. Chronic dietary exposure in the range of 8–49 mg Pb?kg^?1 resulted in no significant effects on the growth and survival of Prussian carp females.     

Effect of La3+ on the activities of antioxidant enzymes in wheat seedlings under lead stress in solution culture

In order to improve the plant ability to resist lead stress, effect of 0.05 mg/l La(NO₃)₃ on the activities of catalase (CAT), superoxide dismutase (SOD), the level of malondialdehyde (MDA) in wheat seedlings under lead stress was studied. The effect of La³⁺ on plant growth, chlorophyll content in wheat seedlings after adding 0, 50, 100 mg/l Pb(NO₃)₃ to the nutrient solution for 12 days was observed. The plants were grown in nutrient solution in a strictly controlled climate growth room. Effects of La³⁺ (with La treatment) compared with check groups was evidently observed. The activities of SOD and CAT in root were enhanced 0.45–1.69 times and 33.20–77.77% respectively and MDA content was reduced 11.05–27.49% in root after treatments from the second day till the end of the experiment. The activities of SOD and CAT was found to be increased slightly (P<0.05) and MDA content decreased in shoot and root of wheat seedlings by La³⁺ under lead stress within five days after treatments compared with Pb1 and Pb2 groups. It was assumed that antioxidant enzymes was found to be increased by La(NO₃)₃, the antioxidant potential of the wheat seedlings to resist lead stress enhanced. It is suggested that La³⁺ could be used to resist lead stress at the beginning under stress while the stress was not so serious.     

Bioaccumulation and elimination of tetrachlorobenzyltoluene (TCBT) by the rat and by fish

A comparative study has been made of the bioaccumulation and elimination by fish and the rat of (i) tetrachlorobenzyltoluene (TCBT), a substance recently proposed as a substitute for polychlorobiphenyl (PCB), and (ii) DP5, a pentachlorobiphenyl mixture (PCB-5 C1).

The study relating to fish (Brachydanio Rerio) comprised a 30-day accumulation period followed by a 30-day elimination period. Nominal concentrations of TCBT and PCB-5 C1, respectively, in the water, were 1mg/litre in both cases.

The study relating to the rat comprised a 90-day treatment period followed by a 30-day elimination period. The animals were exposed by oral (gavage) to doses of 0, 5, 30, and 200 mg/kg/day in the case of TCBT and to the single dose of 5 mg/kg/day in the case of PCB-5 Cl.

Concentrations of TCBT and PCB-5 Cl in rat liver and fats, in whole body tissues of fish and in water were measured by gas chromatography, using an electron capture detector.

Results showed a clear difference between TCBT and PCB-5 Cl: in fish, bioaccumulation of TCBT was relatively slight and the 50% depuration time was approximately 26 days; in rat liver and fats, TCBT was also only weakly accumulated and rapidly eliminated. This contrasted sharply with the findings for PCB-5 Cl, viz: high accumulation potential and slow elimination.

Observed rates of bioaccumulation and elimination of TCBT and PCB-5 Cl by the rat were in good agreement with the toxicological findings for the same species.     

A new method for determining concentrations of Endod-S (Phytolacca dodecandra) in water during mollusciciding

A hemolytic method was developed for the quantitative determination of oleanane saponin (Endod-S) in Phytolacca dodecandra (Phytolaccaceae), a plant with potent molluscicidal properties. A chemical method based on thin-layer chromatography (TLC) was used as a control for comparison with a hemolysis test. Standard curves for calibration of both tests of Endod-S gave high correlation coefficients. The hemolysis method gave a correlation coefficient of 0.98 for saponin (Endod-S) levels ranging from 1.6 to 4.2 mg/l. The TLC method gave a correlation coefficient of 0.99 for saponin levels above 1.5 micrograms. Due to the action of saponins on erythrocytes, the hemolysis method could not be used at levels below 1.6 and above 4.2 mg/l. Similarly, sample size limited TLC quantitation of these saponins at levels below 1 microgram. Both the TLC and the hemolysis methods on identical Endod-S water samples gave comparable results at saponin levels between 2 and 6 mg/l. A snail bioassay showed that the LC50 and the LC90 concentrations of Endod-S to the snail Biomphalaria glabrata (albino) were 2.57 and 2.92 mg/l respectively. These values fell in the standard curve range for the hemolysis method thus indicating that the hemolysis method is of practical value for testing Endod-S concentrations that would actually be used in field applications for snail control. The hemolysis method is less expensive than TLC and can be easily adapted to field conditions.     

Spectroscopic studies of the progress of humification processes in humic substances extracted from refuse in a landfill

Humic acids (HA) and fulvic acids (FA) extracted from refuse in a landfill at different landfill ages were characterized by elemental composition, GPC, FTIR, and (13)C CP/MAS NMR. The elemental composition analysis revealed high O/C and low H/C ratios in the FA, indicating a high proportion of O-alkyl and carboxylic acids in the FA. The M(w) and M(n) values and M(w)/M(n) ratios indicated that the HA had a higher molecular weight and were more homogeneous with respect to molecular weight. FTIR spectra showed that the FA had relatively more intense bands assigned to aliphatic structures and were abundant in oxygen-containing function groups. The analytical results of (13)C CP/MAS NMR suggested that there were more oxygenated aliphatic carbons and fewer aromatic carbons in the FA than in the HA. All the results demonstrated that the degree of humification increased with landfill age.     

镉对鲫鱼组织转氨酶和过氧化氢酶活性的影响

当水中镉离子浓度为３２ｍｇ／Ｌ，６４ｍｇ／Ｌ时，肝胰脏，肾脏和鳃的谷丙转氨酶和谷草转氨酶活性显著降低，而过氧化氢酶活性则显著升高，对鲫鱼心脏３种酶活性的影响不明显，体外试验表明，镉能直接抑制ＧＰＴ、ＧＯＴ的活性，而对Ｃａｔａｌａｓｅ活性不产生直接影响。     

Rare earth elements in naturally grown fern Dicranopteris linearis in relation to their variation in soils in south-Jiangxi region (southern China)

Ferns (Dicranopteris linearis) and soils, sampled from four rare earth mining areas and one non-mining area locating in South-Jiangxi region, were chosen for analysis of 15 rare earth elements (REEs) by inductively coupled plasma-mass spectrometry (ICP-MS). The fern samples were divided into 4 parts: root, stem, petiole, and lamina. The soils of the same sites were also sampled, and 4 soil layers were observed at the profile: A (0-20 cm depth), B (20-150 cm depth), C (150-400 cm depth), and D (rock). The distribution patterns and transportation characteristics of REEs of different soil layers and of different parts of D. linearis were studied. The results showed that in the soil layers, the sigma REE (the sum of the concentrations of 15 REEs) of A layer, as well as B layer were lower than that of C layer, but Ce was relatively concentrated in A layer. The sigma REE of different parts of D. linearis were lamina, root > stem > petiole. The REE distribution patterns of D. linearis root were similar to the total REE distribution patterns of the A layer of their host soils, especially to the soluble REE distribution patterns of the A layer of their host soils. The REEs were fractionated in their transportation in D. linearis. The abundances of heavy rare earth elements (HREEs, Gd to Lu and Y) in the stem, petiole, lamina are lower than those in the root.     

The effects of pH on fluoxetine in Japanese medaka (Oryzias latipes): acute toxicity in fish larvae and bioaccumulation in juvenile fish

Recent detection of fluoxetine in the aquatic environment and fish suggests a possibly high accumulation of fluoxetine; however, no report is available on the bioaccumulation of fluoxetine in aquatic organisms. Since bioaccumulation of fluoxetine was probably dependent on pH near the pK(a) value of 10.1, experiments were conducted approximately at pH 7, 8, and 9. Distribution coefficients between 1-octanol and water (D(ow)), and those between synthetic membrane vesicles (liposomes) and water (D(lip-wat)) were determined at pH 7, 8, and 9. The D(ow) and D(lip-wat) values increased significantly with increasing pH. Acute toxicity tests were performed using Japanese medaka (Oryzias latipes) prior to the bioaccumulation test, and 96-h LC(50) values were 5.5, 1.3, and 0.20mgl(-1) at pH 7, 8, and 9, respectively. In the bioaccumulation test, concentrations of fluoxetine and its major metabolite, norfluoxetine, in the fish body and liver were measured. The bioconcentration factors (BCF) of fluoxetine for Japanese medaka were 8.8, 3.0x10, and 2.6x10(2) in the body and 3.3x10(2), 5.8x10(2), and 3.1x10(3) in the liver at pH 7, 8, and 9, respectively. The BCF values were lower at pH 7 and higher at pH 9 mainly because of the increase in nonionized species with significantly higher hydrophobicity than the ionized species at pH values closer to pK(a). A similar trend was obtained for the concentration of norfluoxetine in the fish but the pseudo-BCF values (the ratio of the norfluoxetine concentration in the fish and the fluoxetine concentration in test water) were higher than the BCF value of fluoxetine at all pH conditions.     

柠檬酸对小麦吸收铜的影响

通过土培和水培试验,研究了柠檬酸对小麦吸收铜的影响.土培结果表明：柠檬酸对土壤中的铜有一定的活化能力,使土壤中生物有效性的铜含量提高,从而提高了小麦中铜的含量,并且表明茎叶中的元素铜含量一般比根中的铜含量要低.水培结果表明：柠檬酸对小麦积累铜存在＂高抑低促＂的生物效应.     

Soil ecotoxicity assessment using cadmium sensitive plants

Four crop plant species (sweet corn, Zea may; wheat, Triticum aestivum; cucumber, Cucumis sativus; and sorghum, Sorghum bicolor) were tested to assess an ecotoxicity in cadmium-amended soils. The measurement endpoints used were seed germination and seedling growth (shoot and root). The presence of cadmium decreased the seedling growth. The medium effective concentration values (EC50) for shoot or root growth were calculated by the Trimmed Spearman-Karber method. Due to the greater accumulation of Cd to the roots, root growth was a more sensitive endpoint than shoot growth. Bioavailability and transport of Cd within plant were related to concentration and species. The ratio of bioaccumulation factor (BAF) in the shoots to the roots indicated high immobilization of Cd in the roots. Seed germination was insensitive to Cd toxicity, and is not recommended for a suitable assay. Among the test plants and test endpoints, root growth of sorghum and cucumber appears to be a good protocol to assess ecotoxicity of soils contaminated by Cd.     

Mercury mobility in a salt marsh colonised by Halimione portulacoides

The present study intends to increase the knowledge on the mobility of mercury in a salt marsh colonised by Halimione portulacoides. Mercury distribution in the sediment layers and its incorporation into the plant biomass were assessed, as well as the potential export of mercury from the contaminated area to the adjacent environment. Mercury pools in the sediments ranged from 560 to 943 mg m(-2) and are largely associated with the solid fraction, with just a small amount being associated with the pore waters. Estimated diffusive fluxes of reactive mercury ranged from 1.3 to 103 ng m(-2) d(-1). Despite the above ground biomass values being comparatively higher than below ground biomass values, the mercury pools were much higher in the root system (0.06-0.16 mg m(-2) and 29-102 mg m(-2), respectively). The annual bioaccumulation of mercury in above ground tissues was estimated in 0.11 mg m(-2) y(-1), while in below ground biomass the values were higher (7 2mg m(-2) y(-1)). The turnover rates of H. portulacoides biomass suggest higher mercury mobility within the plant rhizosphere. Taking into account the pools of mercury in above ground biomass, the export of mercury by macro-detritus following the "outwelling hypothesis" is not significant for the mercury balance in the studied ecosystem. The mercury accumulated in the below ground part of the plant is quite mobile, being able to return to the sediment pool throughout the mineralisation process.