Chromium bioaccumulation: comparison of the capacity of two floating aquatic macrophytes

The capacity of Salvinia herzogii and Pistia stratiotes to remove Cr (III) from water and their behaviour at different Cr (III) concentrations were studied in outdoor experiments. Cr distribution in aerial parts and roots with time and the possible mechanisms of Cr uptake were analyzed. Both macrophytes efficiently removed Cr from water at concentrations of 1, 2, 4 and 6 mgCrL(-1). S. herzogii was the best adapted species. At a greater initial concentration, greater bioaccumulation rates were observed. Root Cr uptake was a rapid process that was completed within the first 24h. Cr uptake through direct contact between the leaves and the solution is the main cause of the increase of Cr in the aerial parts, Cr being poorly translocated from the roots to the aerial parts. Both mechanisms were fast processes. The Cr uptake mechanism involves two components: a fast component and a slow one. The former occurs mainly due to the roots and leaves adsorption and is similar for both species. The slow component is different for each species probably because in P. stratiotes a Cr precipitation occurs induced by the roots.     

Influence of iron plaque on uptake and accumulation of Cd by rice (Oryza sativa L.) seedlings grown in soil

Iron plaque is ubiquitously formed on the root surfaces of rice. However, little is known about the role of iron plaque in Cd movement from soil to the plant aboveground parts. A pot experiment was conducted to investigate the influence of iron plaque in Cd uptake and accumulation by rice seedlings in soil. Rice seedlings were pre-cultivated in solution culture for 16 days. Two seedlings were transplanted in a nylon bag containing no substrate but surrounded by soil amended with Fe and Cd combined at rates of 0, 1, or 2 g Fe kg(-1) and 0, 2.0, or 10 mg Cd kg(-1) soil. Fe was added to induce different amounts of iron plaque, and Cd to simulate Cd-polluted soils. Plants were grown for a further 43 days and then harvested. The length of the longest leaf and SPAD values of the newly mature leaves were measured during plant growth. Fe and Cd concentrations were determined in dithionite-citrate-bicarbonate (DCB) soil extracts and in plant roots and shoots. Shoot and root dry weights were significantly affected by Fe supply level but not by added Cd. Root dry weight declined with increasing Fe supply but shoot dry weight decreased at 2 g Fe kg(-1) and increased at 1 g Fe kg(-1) (except at 2 mg Cd kg(-1)). The length of the longest leaf and SPAD values of the newly mature leaves were significantly affected by plant growth stage and added Fe and Cd. Fe tended to diminish the negative effect of Cd on these two parameters. Cd concentrations in DCB extracts increased with increasing Cd and Fe supply. In contrast, external Fe supply markedly reduced shoot and root Cd concentrations and there was generally no significant difference between the two Fe supply levels. Shoot and root Cd concentrations increased with increasing Cd addition. Root Cd concentrations were negatively correlated with root Fe concentrations. The proportion of Cd in DCB extracts was significantly lower than in roots or shoots. The results indicate that enhanced Fe uptake by plants can diminish the negative effects of Cd to some extent and that iron plaque on root surfaces is of little significance in affecting uptake and accumulation of Cd by rice plants.     

Arsenic uptake potential of water lettuce (Pistia Stratiotes L.)

Arsenate uptake by aquatic plant water lettuce (Pistia stratiotes L.) was studied in the laboratory condition to investigate a low cost natural aquatic treatment system for pollutant removal. The plants were harvested from a local pollution‐free pond in young condition and hydroponically cultured in the laboratory. Bio‐accumulation was noticed to be both concentration and duration dependent. The results show that the plant could effectively absorb arsenic between a range of 0.25 to 5.0 mg/l to the extent of 82.0 to 22.8% for a biomass of 20 g/l at pH 7.0 after 144 h. The results were also plotted in Langmuir and Freundlich isotherms and the data were well fitted. The sorption capacity was evaluated as 1.43 mg/g for the Langmuir isotherm and 1.01 mg/g for the Freundlich isotherm. The removal efficiency was, however, noted to be maximum (87.5%) at pH 6.5. The effect of biomass quantity has also been investigated along with some metabolic parameters.     

Phytoremediation potential of three aquatic macrophytes in manganese‐contaminated water

Manganese (Mn) uptake capacities of water hyacinth (Eichhornia crassipes (Mart.) Solms, water lettuce (Pistia stratiotes L.) and slligator alternenthera (Alternanthera philoxeroides) were investigated with four Mn levels: 0(control), 50, 200, 400 mg/L. With increased Mn exposure, the tested plants showed similar responses with a decrease in relative growth rate and an increase in plant Mn concentrations. Comparatively, water lettuce had the significantly higher total Mn concentrations, shoots enrichment factor and translocation factor at all Mn‐treated treatments and the much greater total Mn removal at concentrations of 200 and 400 mg/L Mn. In most cases, Mn pollution significantly decreased Mg concentrations for all three plants. Concomitantly, increment trends of Fe, Cu and Zn concentrations were observed for water lettuce with the increasing external Mn concentrations, which could partly contribute to its higher Mn concentrations. The results indicated that water lettuce have standout potentials as a phytoremediation plant for Mn contaminated waters.     

Influence of organic matter on the uptake of cadmium, zinc, copper and iron by sorghum plants

This article describes an experiment, carried out under controlled environment conditions, to investigate the effects of a fulvic acid fraction of soil organic matter on growth, cadmium (Cd) uptake and redistribution by sorghum. In addition the uptake of copper (Cu), zinc (Zn) and iron (Fe) was also determined. Sorghum was grown in nutrient solutions with 0, 0.1, 1 and 10 mg Cd dm(-3), in the absence and presence of organic matter (32 mg C dm(-3)), for various periods up to 20 days. A decrease in sorghum biomass due to Cd toxicity was observed at 10 mg Cd dm(-3), but for concentrations of 0.1 and 1 mg Cd dm(-3) the biomass was increased compared with control, without visual toxicity symptoms. The presence of organic matter (OM) further increased biomass production. Cadmium was mainly retained in sorghum roots, as usually found in tolerant plants, but Cd accumulation in sorghum was greater than in other Gramineae, or even more tolerant plants such as lettuce. The presence of OM decreased the bioavailability of Cd that was partially retained in solution by the OM ligands. However, OM promoted the translocation of Cd to shoots, an effect that may pose a risk to public health because plant-animal transfer of Cd could be enhanced. The presence of OM decreased the uptake of Cu, Zn and Fe. The presence (vs. absence) of 0.1 mg Cd dm(-3) enhanced the uptake of Fe, both in the absence and presence of OM.     

Enhanced uptake of phosphorus by activated sludge-effect of substrate addition

Studies were undertaken to examine the effect of substrate addition upon the release and subsequent uptake of phosphorous by a biological wastewater treatment laboratory scale system.A train of six reactors were fed at a rate of 16 ml min⁻¹ raw wastewater using a sludge recycle ratio of 0.75 (12 ml min⁻¹). The first two reactors were simply stirred (anoxic) without the addition of air and the remaining four were aerated with excess air.Various substrates were added to the first reactor (anoxic) at different concentrations. It was determined that all the short chain substrates tested enhanced the release of phosphorus in the anoxic stages and subsequently led to increased biological uptake (removal) of phosphorus. The substrates tested included sodium acetate, acetic acid, butyric acid, ethanol and methanol.It was determined that 30 mg l⁻¹ sodium acetate was the optimum dose for biological release and uptake of phosphorus and the addition of any greater concentration had marginal effect on the ultimate removal of phosphorus.The effect of these substrates showed some specificity regarding phosphorus release, with butyric acid causing the greatest release and sodium acetate the least. However as far as phosphorus uptake (removal) was concerned, this phenomenon of substrate-specificity was less significant. For all substrates, effluent phosphorus concentration was within ± 1 mg l⁻¹ with an approximate mean concentration of 1 mg l⁻¹ residual.Phosphorus released (approx. 14 mg l⁻¹) at higher temperature (29°C) was 75% greater than at the lower temperature (24°C). Similarly the final residual phosphorus at 29°C was 33% lower than at 24°C.     

Effect of EDTA and Fe-EDTA complex concentration on TCF Kraft mill effluent degradability. Batch and continuous treatments

The effect of ethylenediaminetetracetic acid (EDTA) and Fe-EDTA complex on synthetic totally chlorine-free (TCF) effluent degradability in batch and continuously operating reactors was evaluated. Under batch treatment, the addition of EDTA and Fe-EDTA complex was studied in the range of 80 to 320 mg l(-1). Under continuously operated reactors, the Fe-EDTA complex concentration varied from 20 to 80 mg l(-1), and the hydraulic retention time (HRT) varied from 48 to 24 h. Sludge oxygen uptake rate (OUR) and chemical oxygen demand (COD) removal decreased when EDTA concentration increased in the influent under batch treatment; however, this inhibitory effect was reduced by the addition of Fe-EDTA complex. Without the addition of EDTA, COD removal decreased from 71% to 8%. The most efficient EDTA removal treatment (almost 10%) was the treatment of 80 mg l(-1) Fe-EDTA. Under continuously operated reactors, COD removal was greater than 57% in the synthetic TCF effluent with a Fe-EDTA concentration that varied from 20 to 80 mg l(-1); however, EDTA removal was lower than 25% in all cases. Synthetic TCF effluent with a Fe -EDTA concentration higher than 80 mg l(-1) could not be treated by the activated sludge treatment due to EDTA's inhibitory effect on the sludge.     

Removal of lead ions from industrial waste water by different types of natural materials

The adsorption capacity of some natural materials for lead such as animal bone powder, active carbon, Nile rose plant powder, commercial carbon and ceramics was studied. The V/m ratio has been chosen to be 500 ml/g. The adsorption process was affected by various parameters such as contact time, pH and concentration of lead solution. The lead uptake percent reaches equilibrium state after 15, 30, 45 and 120 min for bone powder, active carbon, plant powder and commercial carbon, respectively. The uptake percent of lead increased by increasing pH value. The sequence of lead uptake percent (% adsorption) at constant pH from certain concentration of lead nitrate solution by the different natural materials is in the order: bone powder > active carbon > plant powder > commercial carbon. The uptake percent of lead is increased by decreasing the concentration of lead at constant pH. The capacity of lead adsorbed from nitrate solution by the different natural materials increased by increasing pH value. The synthetic and industrial waste-water samples were treated by using the different natural materials (contact time 3h, pH = 4). The percent removal of lead was 100% by bone powder, 90% by active carbon, 80% by plant powder and 50% by commercial carbon. There was no removal of lead by ceramics. This may be due to the presence of high percent of lead in the constituent of ceramics (372 mg/g).     

Response of graywater recycling systems based on hydroponic plant growth to three classes of surfactants

Anionic (sodium laureth sulfate, SLES), amphoteric (cocamidopropyl betaine, CAPB) and nonionic (alcohol polyethoxylate, AE) surfactants were added to separate nutrient film technique (NFT) hydroponic systems containing dwarf wheat (Triticum aestivum cv. USU Apogee) in a series of 21 day trials. Surfactant was added either in a (1). temporally dynamic mode (1-3 g surfactant m(-2) growing area d(-1)) as effected by automatic addition of a 300 ppm surfactant solution to meet plant water demand, or (2). continuous mode (2 g surfactant m(-2) growing area d(-1)) as effected by slow addition (10 mLh(-1)) of a 2000 ppm surfactant solution beginning at 4d after planting. SLES showed rapid primary degradation in both experiments, with no accumulation 24 h after initial addition. CAPB and AE were degraded less rapidly, with 30-50% remaining 24 h after initial addition, but CAPB and AE levels were below detection limit for the remainder of the study. No reductions in vegetative growth of wheat were observed in response to SLES, but biomass was reduced 20-25% with CAPB and AE. Microbial communities associated with both the plant roots and wetted hardware surfaces actively degraded the surfactants, as determined by monitoring surfactant levels following pulse additions at day 20 (with plants) and day 21 (after plant removal). In order to test whether the biofilm communities could ameliorate phytotoxicity by providing a microbial community acclimated for CAPB and AE decay, the continuous exposure systems were planted with wheat seeds after crop removal at day 21. Acclimation resulted in faster primary degradation (>90% within 24h) and reduced phytotoxicity. Overall, the studies indicate that relatively small areas (3-5m(2)) of hydroponic plant systems can process per capita production of mixed surfactants (5-10 g x person(-1)d(-1)) with minimal effects on plant growth.     

Heavy metal and micronutrient uptake in soybeans as influenced by sewage sludge amendment

Soybean (Glycine max (L.) Merr.) uptake of the elements, Cd, Ni, Pb, Cu, Zn and Mn, from a sewage sludge-amended Mecklenburg soil was conducted in the greenhouse. “Bragg” soybeans were grown in pots for five weeks at which time the tops and roots were sampled separately for elemental analysis. Soil samples from each pot were extracted with DTPA (diethylenetriaminepentaacetic acid) and the concentration of extractable elements correlated with the elemental content in the soybean plant. There was a significant increase in dry matter production with sludge treatment. Concentrations of Cd, Ni and Pb in the soybean shoots and roots increased from sludge-amended soil as compared to the control. The metal concentration in the soybean tissue increased with increasing levels of sludge amendment. Uptake of the heavy metals was greater by the roots than by the shoots indicating some barrier to movement of the metals from roots to shoots. The DTPA extractable Cd in sludge-amended soil increased significantly, and showed correlation to the soybean tissue metal concentrations. As for the micronutrients, Cu increased in the soybean shoot as the extractable Cu increased. There was no significant relationship between soybean tissue Zn and Mn and extractable Zn and Mn.     

Adsorption kinetics of fluoride on low cost materials

Adsorption is one important technique in fluoride removal from aqueous solutions. The viability of adsorption techniques is greatly dependent on the development of adsorptive materials. A large number of materials have been tested at a fluoride concentration greater than 2 mg/l, and the lowest limit for fluoride reduction by them is about 2 mg/l. Decreasing the fluoride concentration to less than 2 mg/l, most of the tested materials displayed a very low capacity of fluoride removal.

This paper has concentrated on investigating the adsorption kinetics and adsorption capacity of low cost materials at a low initial fluoride concentration. The experiments were carried out at a natural pH, and radioisotope ¹⁸F rather than ¹⁹F was used since ¹⁸F can be rapidly measured by measuring the radioactivity with a resolution of 1×10⁻¹³ mg or 0.01 μCi. The tested materials are hydroxyapatite, fluorspar, calcite, quartz and quartz activated by ferric ions. Their adsorption capacities follow the order:

Hydroxyapatite>Fluorspar>Quartz activated using ferric ions>Calcite>Quartz

The uptake of fluoride on hydroxyapatite is an ion-exchange procedure and follows the pseudo-first- and second-order equations, while the uptake of fluoride on the others is a surface adsorption and follows the pseudo-second-order equation. Calcite has been seen as a good adsorbent in fluoride removal and has been patented. However, our data suggested that its adsorption capacity is only better than quartz.

The external mass transfer is a very slow and rate-determining step during fluoride removal from the aqueous solution. Under static conditions, there was no relative movement between adsorbents and solutions, the fluoride uptake was at a very slow rate and the adsorbent properties did not significantly affect the fluoride uptake. Under shaken conditions, the adsorption of fluoride was controlled by the adsorbent structure and chemical properties.     

The assessment of air and soil as contributors of some trace metals to vegetable plants. III. Experiments with field-grown plants

Plants have been grown on common soils at a number of field sites with differing air metal concentrations. Moss bags were co-located with the plants to evaluate atmospheric deposition. Values of the concentration factor (CF) have been estimated (soil-derived metal in plant divided by soil metal concentration) and are generally found to be in the order Zn greater than Cd greater than Ni greater than Cr greater than Pb (sometimes Cd greater than Zn), with values for Pb comparable with earlier studies in which the atmospheric contribution was subtracted. Values of the air accumulation factor (AAF) (air-derived metal in plant divided by air metal concentration) showed values of 15-30 m3g-1 for Cd and Pb for the exposed parts of plants, and lower values for non-exposed parts. Values of both CF and AAF are comparable with those determined in Part I, using a laboratory growth chamber.     

Using phosphate rock to immobilize metals in soil and increase arsenic uptake by hyperaccumulator Pteris vittata

This greenhouse experiment evaluated the effects of phosphate rock (PR) on arsenic and metal uptake by the arsenic hyperaccumulator Pteris vittata in a soil spiked with arsenic and heavy metals Cd, Pb and Zn. Five soil treatments were used, 1) control with no arsenic, 2) spiked with 50 mg kg(-1) As (As) as Na2H AsO4, 3) spiked with 50 mg kg(-1) As and P as PR (AsP), 4) spiked with 50 mg kg(-1) As, Pb, Cd, and Zn (AsM), and 5) spiked with 50 mg kg(-1) As, Pb, Cd, Zn and P (AsMP). The plants were harvested after growing in the soil for five weeks. Compared to the As treatment, the presence of heavy metals (AsM) reduced arsenic concentrations in the fronds from 1631 to 608 mg kg(-1). However, this effect was mitigated by PR (AsMP), with arsenic concentrations in the fronds increased from 608 to 1046 mg kg(-1). Phosphate rock also significantly reduced Pb (13.5 to 4.10 mg kg(-1)) and Cd (13.0 to 3.45 mg kg(-1)) concentrations in the fronds. Most of the arsenic in P. vittata was accumulated in the fronds (89-93%). Compared to the control, P was more concentrated in the roots along with less P being translocated to the fronds in the treatments with arsenic. While in those same treatments higher Ca concentrations in both the fronds and roots were observed. This research shows that PR was effective in increasing arsenic uptake and decreasing metal uptake by P. vittata and thus can be used as a cost-effective amendment for phytoremediation of arsenic and metal polluted soils.     

水生植物在水污染控制中的应用与发展

利用水生植物净化污水具有投资、维护和运行费用低，管理简便，污水处理效果好，可改善和恢复生态环境、回收资源和能源以及收获经济植物等优点，是一种低成本高效益的生态工程技术，在污水处理和富营养化水体净化等方面均表现出良好的效果，引起国内外专家关注．介绍了风眼莲、芦苇、水花生、香蒲、水葱和大薄等几种应用较广泛的水生植物对不同类型污水的净化效果，指出了目前水生植物处理系统中存在的问题，提出了今后的发展方向．     

Effect of scale of Cd heterogeneity and timing of exposure on the Cd uptake and shoot biomass, of plants with a contrasting root morphology

A pot experiment was conducted to investigate the influence of spatial heterogeneity of Cd distribution in soil on shoot biomass, shoot metal concentration and total shoot Cd uptake by lettuce (Lactuca sativa, variety Tom Thumb) and Indian mustard (Brassica juncea). Five different soil treatments had similar overall concentration of Cd per pot, but different scales of heterogeneity and also timing of plant exposure during the growth cycle. The presence and scale of heterogeneity and timing of exposure were found to have significant effects on shoot biomass for both plants (with one exception). The mean values of Cd mass taken up were significantly affected by the presence of heterogeneity and timing only for lettuce. Only the scale of heterogeneity affected the uptake of Cd by Indian mustard, presumably because of its larger root system (approximately 18 cm, compared with approximately 5 cm for lettuce). These findings have important implications for phytoremediation, and for human health risk assessment where leafy vegetables are grown in situations with highly elevated Cd concentrations.     

Examination of arsenic(III) and (V) uptake by the desert plant species mesquite (Prosopis spp.) using X-ray absorption spectroscopy

This study describes the effects of Arsenic(III) and (V) on the growth and their uptake by the desert plant mesquite (Prosopis spp.). Seedlings were sown in agar-based medium containing a modified Hoagland's nutrient solution. After 1 week, the seedlings were transplanted to arsenic (As) treated agar media that contained 5 mgL(-1) of As either As(III) (As(2)O(3)) or As(V) (As(2)O(5)). The plants were harvested after 14 days of growth and sectioned into roots, stems, and leaves. After digestion, As concentrations in the roots, stems, and leaves were determined using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Our results showed that the As concentrations from As(V) were significantly higher than the As concentrations from As(III) in all portions of the plant. Plants exposed to As(V) concentrated (mg As kg(-1) d wt) about 770+/-191, 326+/-94, and 119+/-18 in roots, stems, and leaves, respectively. X-ray absorption spectroscopy (XAS) showed that As(V) was reduced to As(III) inside the mesquite plant. In addition, greater than 90% of the As(III) found in the mesquite plants was bound to sulfur ligands in the roots, stems and leaves.     

制革废水处理厂及下游综合污水厂升级改造探究

对某制革废水处理厂和下游综合污水处理厂的进出水水质和沿程工艺段进行采样分析,得出制革废水处理厂出水NH3-N和TN平均浓度分别为77. 32、160. 93 mg/L,综合污水处理厂出水COD平均浓度为106. 8 mg/L,其中大部分是难降解COD,出水TN平均浓度为89. 93 mg/L,出水COD和TN是影响污水处理厂出水达标排放的主要指标。在小试中投加500 mg/L葡萄糖(以COD浓度计)时脱氮效果明显增强,综合污水处理厂出水TN浓度可稳定在15 mg/L以下。利用臭氧、活性焦和四相催化氧化深度处理综合污水处理厂二级出水,发现臭氧对COD基本没有去除效果,活性焦和四相催化氧化都能使COD浓度降至50 mg/L以下,但四相催化氧化去除单位COD的成本约是活性焦的29%、再生活性焦的49%。     

Alkaline biosolids and EDTA for phytoremediation of an acidic loamy soil spiked with cadmium

A greenhouse experiment was conducted to investigate the growth of Brassica juncea and Cd phytoextraction in a mimicked Cd contaminated acidic loamy soil amended with alkaline biosolids, prepared from sewage sludge and coal fly ash, in the presence and absence of EDTA at 2 mmol kg(-1). The acidic loamy soil was spiked with 0, 5, 20, 50 and 100 mg Cd kg(-1) in the form of CdCO(3) and then amended with 4% alkaline biosolids (w/w). Alkaline biosolids and 0.12% CaCO(3) amendments resulted in a higher biomass than unamended soil spiked with 20 mg kg(-1) Cd where plants did not survive and of the two amendments, alkaline biosolids amendment had higher plant dry weight yield and phytoextraction of Cd. Adding 2 mmol kg(-1) EDTA to alkaline biosolids amended soil significantly increased the solubility of Cd ions by 9- to 29-fold, but plant Cd accumulation decreased by a factor of 24-48%. The results indicate that alkaline biosolids amendment is an effective approach for assisting growth of B. juncea and phytoextraction of Cd from the contaminated acidic loamy soil, but further application of chelating agents did not enhance the phytoextraction efficiency of Cd.     

The effect of water hardness on nitrilotriacetate removal and microbial acclimation in activated sludge

The effect of water hardness on microbial acclimation to the removal of nitrilotriacetate (NTA) in the wastewater treatment process was examined in 4 laboratory-scale activated sludge reactors. Consistent high removal (i.e. >90%) was observed in all 4 reactors after acclimation occurred. Removals of greater than 60% of added NTA after 8 days and greater than 90% after 15 days were observed in a continuously fed, hard water (250 mg l⁻¹ as CaCO₃) reactor. In soft water (50 mg l⁻¹ as CaCO₃) comparable removals were observed after 20 and 28 days respectively. In 24 h, semi-continuous activated sludge reactors (24 h, fill-and-draw type) removal was greater in hard water than in soft water initially, but was greater than 95% in both hard and soft water after 4 weeks.The proportion of bacteria in the activated sludge with the ability to utilize NTA increased approx. 100-fold during the acclimation period. This proportion increased faster in hard water than in soft water and faster in semi-continuous activated sludge reactors than continuous activated sludge reactors.Ultimate biodegradation of ¹⁴C[U]NTA was greater than 85% in both hard and soft water acclimated units. Accumulation of ¹⁴C in the sludge solids was negligible (<1%).     

The distribution of cadmium,copper, lead,and zinc in eelgrass (Zostera marina L.)

The distribution of Cd, Cu, Pb, and Zn in eelgrass (Zostera marina L.) was studied at three locations with different heavy metal loads in the Limfjord, Denmark.The eelgrass was fractionated into roots, rhizome, stem, and leaves according to age, and the heavy metal concentrations in each fraction were determined. The distribution patterns of the four heavy metals in eelgrass were independent of the heavy metal loads at the sampling stations. The concentrations of all metals were greater in the roots than in the rhizomes. In the aerial¹ parts two different age-dependent distribution patterns were observed. The concentrations of Cd, Pb, and Zn increased with age while the opposite was true for Cu. The distribution of lead correlated with the distribution of ash content. These age-dependent distribution patterns were maintained throughout the observation period and were most pronounced for Cu and Zn in winter.The heavy metal distribution in eelgrass is discussed in relation to gross morphology, especially age-structure. It is suggested that the accumulation of Cd, Pb, and Zn is due to a slow irreversible uptake or to the existence of more binding sites in old leaves. The distribution of Cu can be explained by translocation within the plant, dilution due to growth or leakage from the older leaves.