Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination,as a potential natural exposure pathway

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.     

Antimony uptake by <Emphasis Type="Italic">Zea mays</Emphasis> (L.) and <Emphasis Type="Italic">Helianthus annuus</Emphasis> (L.) from nutrient solution

We investigated the extent of Sb uptake by maize (Zea mays) and sunflower (Helianthus annuus) from nutrient solutions containing concentrations from 3 to 24 mg/L of potassium antimonate, with the aim of determining the potential of Sb to enter the food chain. The maximum shoot Sb concentrations in Z. mays and H. annuus were 41 mg/kg and 77 mg/kg dry weight, respectively. There was no significant difference in Sb uptake between species. The average bioaccumulation coefficients (the plant/solution concentration quotients) were 1.02 and 1.93 for Z. mays and H. annuus, respectively. Phosphate addition did not affect plant growth or Sb uptake. Antimony uptake by both Z. mays and H. annuus is unlikely to pose a health risk to animals and humans.     

石墨烯对高等植物幼苗的毒性及机理探究

随着石墨烯产品的广泛应用和潜在的环境释放,其对生态环境的影响已引起广泛关注。为探讨石墨烯对高等植物生长的影响,探究了其对黄瓜幼苗和玉米幼苗生长的影响及其致毒机理。结果表明,水培条件下,不同浓度的石墨烯(10、50、100、500、1 000和2 000 mg·L~(-1))处理植物幼苗15 d后,对植物幼苗的生长具有抑制作用。且随着处理时间和石墨烯浓度的增加,植物幼苗生长的所有指标,包括根/地上部鲜重和干重、根长、根尖数、株高和叶面积均相应降低。另外,黄瓜幼苗比玉米幼苗对石墨烯更加的敏感。进一步研究发现,石墨烯与黄瓜幼苗根部直接接触导致的物理损伤、氧化损伤,以及营养耗竭是其致毒机理。而石墨烯对玉米幼苗的致毒机理包括物理损伤和营养耗竭。本研究为石墨烯的环境风险评价提供了基础数据。     

Aristolochic acid content of South-East Asian troidine swallowtails (Lepidoptera: Papilionidae) and of Aristolochia plant species (Aristolochiaceae)

Summary. Analysis of South-East Asian troidine swallowtails revealed high variability in the content of aristolochic acids among individuals. The presence or absence of these compounds depends on the Aristolochia species available as food plant for the larvae. Only one plant species (Aristolochia philippinensis) contained a high concentration of aristolochic acids, while other species from various localities contained none or only marginal amounts. Whether aristolochic acids have a distinct function in chemical defense of these swallowtails is still an open question. Received 11 December 2000; accepted 4 August 2001.     

Experimente zum Verbleib von Cyanid nach Aufnahme in Pflanzen

Free cyanide (prussic acid) can be degraded by many organisms. Experimental data on the kinetics of degradation by plants, however, are rare. We experimentally determined the mass balance of potassium cyanide in the system nutrient solution/plant/air In the presence of living willows (Salix alba) and light, free cyanide is almost completely eliminated from the nutrient solution, also by a willow cell suspension (> 99%) and even by a dried-out stem (> 80%). The loss is smaller when the willow is autoclaved (30%). Without a willow, the CN can be recovered to more than 89%. The loss depends on light and transpiration. Volatilization and the formation of complexed cyanide are of minor relevance for the elimination of free CN. The most plausible loss process is metabolism by plants. The transfer into the roots and stem does not change with the light/dark-rhythm. The transfer factor (mg CN/kg plant fw/mg CN/l solution) after 24 h, and related to the initial concentration in solution, is between 2 and 3 for roots, 0.1 to 0.6 for leaves and 0.2 for stems. In the roots of willows and elderberries which were growing on the former gas worksite in Holte, Denmark, high concentrations of cyanides were found. In leaves and fruits, concentrations are a factor of 10 to 1000-fold lower and up to as high as that found in non-exposed control plants. Due to these results, a cyanide-polluted site in Denmark has been remediated with poplar trees.     

Pb in medicinal plants from Jordan

This is the first report on Pb in medicinal herbs from Jordan. Medicinal herbs may present a health risk due to the presence of toxic metals. Seventy-nine dry medicinal plant samples were collected from herbalist shops in Jordan. The plants were digested with acids and analyzed for total Pb concentration using atomic absorption spectrometry. Mean Pb concentration was 15.9 μg/g on a dry weight basis. Our results show that Pb concentrations in Jordan medicinal plants are higher than published data in other countries. The highest level of 33.4 μg Pb/g was determined in Inula viscosa, and the lowest level of 3.0 μg Pb/g was found in Nigella sativa. Calculated daily intakes of Pb of most analyzed herbs were high; most of them are higher than recommended values by the world health organization (WHO). Fortunately, the herbs that contain the highest Pb levels are the less commonly used medicinal herbs in Jordan. The mean Pb levels in the most commonly, commonly and less commonly used herbs in Jordan are 13.9, 13.1 and 16.9 μg Pb/g, respectively. The average dietary intake of Pb through a mixture of these medicinal herbs consumption, assuming 5.0 g herbs is consumed daily, is 79.5 μg Pb/day, which is higher than the maximum daily limit allowed by WHO. We conclude that most of the medicinal plants consumed in Jordan contain significant amount of Pb, and therefore, people of Jordan should not consume large amounts of these herbs.     

Short-term influence of nutrient availability on the uptake and translocation of phenanthrene in mangrove seedlings

Abstract

The uptake and distribution of phenanthrene, a typical polycyclic aromatic hydrocarbon, in plant tissues of Aegiceras corniculatum and Avicennia marina and the relationship with nutrient (nitrate, ammonium, and soluble reactive phosphorus) availability were investigated. After 12?h of exposure, enhancements in the concentration of nitrate and soluble reactive phosphorus markedly decreased the residual level of phenanthrene in roots, while the addition of ammonium significantly increased the residual concentration. Due to the similar enzymatic degradation potential between treatment groups, the variation of phenanthrene concentration in mangrove roots may result from the H⁺/phenanthrene cotransport at the root surface that was influenced by nutrient uptake. Moreover, both nitrate and soluble reactive phosphorus amendments significantly increased translocation of phenanthrene from roots to leaves, which likely resulted from the change of hydraulic conductivity in mangrove plants triggered by different nutrient availability.     

Stabilization of the As-contaminated soil from the metal mining areas in Korea

Phytoextraction is a remediation technology with a promising application for removing arsenic (As) from soils and waters. Several plant species were evaluated for their As accumulation capacity in hydroponic culture amended with As. Cucumis sativus (cucumber) displayed the highest tolerance against As among 4 plants tested in this study (corn, wheat, sorghum and cucumber). The germination ratio of Cucumis sativus was more than 50% at the high concentration of 5,000 mg-As/l. In Cucumis sativus grown in a solution contaminated with 25 mg-As/l, the accumulated As concentrations in the shoot and root were 675.5 ± 11.5 and 312.0 ± 163.4 mg/kg, respectively, and the corresponding values of the translocation and bioaccumulation factors for As were 1.9 ± 0.9 and 21.1 ± 8.4, respectively. These results indicate Cucumis sativus is to be a candidate plant for phytoextraction of As from soils and water.     

Occupational exposure to airborne aromatic amines in rubber manufacturing: Determination by gas chromatography‐mass spectrometry

Aromatic amines (AAs) are used in a variety of chemical industries and consequently they are the object of great attention in occupational hygiene owing to the carcinogenic effects that many of them have shown. This work outlines a procedure for the determination of occupational exposure to airborne AAs in the rubber industry and the application of this method in a tyre manufacturing plant using p‐phenylenediamines as antiozonants. Samples were collected on a glass fibre filter followed by a silica gel tube and analyzed by GC‐MS/SIM using a capillary column coated with methyl silicone. p‐Phenylendiamines and other AAs probably formed as thermodegradation products were found in concentrations of up to 10 μg/m³ during rubber vulcanization.     

Mercury contamination and health risk to crops around the zinc smelting plant in Huludao City,northeastern China

The Huludao zinc plant in Liaoning province, northeast China was the largest in Asia, and its smelting activities had seriously contaminated soil, water and atmosphere in the surrounding area. For the first time, we investigated the total mercury (THg) content in maize, soybean, broomcorn, 22 vegetables, and the soil around their roots from eight sampling plots near the Huludao zinc plant. THg contents of the seeds of maize, soybean, and broomcorn are 0.008, 0.006, and 0.057 mg kg⁻¹, respectively, with the broomcorn being the highest, exceeding the maximum level of contaminant in food (GB2762-2005) by 4.7 times. The edible parts of vegetables are also contaminated with a range of mercury contents of 0.001–0.147 mg kg⁻¹ (dry weight). THg contents in plant tissue decrease in the order of leaves > root > stalk > grain. Using correlation analysis, we show that mercury in the roots of these plants is mainly derived from soil, and the uptake of gaseous mercury is the predominant path by which the mercury accumulated in the foliage. The average and maximum mercury daily intake (DI) of adult around the Huludao zinc plant via consuming vegetables are 0.015 and 0.051 μg/kg/d, respectively, and those of children are 0.02 and 0.07 μg/kg/d, respectively. The average and maximum weekly intakes of total mercury for adult are 2.1 and 7.1%, respectively, of the provisional tolerable weekly intake (PTWI), and 2.8 and 9.7%, respectively, of the PTWI for children.     

Nutrient fluxes at the landscape level and the R* rule

Nutrient cycling in terrestrial ecosystems involves not only the vertical recycling of nutrients at specific locations in space, but also biologically driven horizontal fluxes between different areas of the landscape. This latter process can result in net accumulation of nutrients in some places and net losses in others. We examined the effects of such nutrient-concentrating fluxes on the R* rule, which predicts that the species that can survive in steady state at the lowest level of limiting resource, R*, can exclude all competing species. To study the R* rule in this context, we used a literature model of plant growth and nutrient cycling in which both nutrients and light may limit growth, with plants allocating carbon and nutrients between foliage and roots according to different strategies. We incorporated the assumption that biological processes may concentrate nutrients in some parts of the landscape. We assumed further that these processes draw nutrients from outside the zone of local recycling at a rate proportional to the local biomass density. Analysis showed that at sites where there is a sufficient biomass-dependent accumulation of nutrients, the plant species with the highest biomass production rates (roughly corresponding to the best competitors) do not reduce locally available nutrients to a minimum concentration level (that is, minimum R*), as expected from the R* rule, but instead maximize local nutrient concentration. These new results require broadening of our understanding of the relationships between nutrients and vegetation competition on the landscape level. The R* rule is replaced by a more complex criterion that varies across a landscape and reduces to the R* rule only under certain limiting conditions.     

Effect of arsenic species on the growth and arsenic accumulation in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

The effects of arsenic (As) species, such as As(III), As(V) and dimethylarsinic acid (DMA), on the accumulation of As in cucumber (Cucumis sativus), as well as on its growth in a soil mesocosm were evaluated. When Cucumis sativus was cultivated in soils contaminated with 20 and 50 mg/kg of As(III), As(V) or DMA for 40 days, the growth was markedly inhibited by the inorganic As (As(III) and As(V)) rather than the organic As (DMA). Irrespective of the As species, the As concentrations accumulated in Cucumis sativus increased with increasing As concentration in the soil. The As bioaccumulation factors from soil into the tissue of Cucumis sativus were 17.5–35.4, 29.3–42.7 and 17.6–25.7 for As(III), As(V) and DMA, respectively. In addition, the As translocation factors from the roots to shoots were 0.025–0.031, 0.018–0.032 and 0.014–0.026 for As(III), As(V) and DMA, respectively. In conclusion, Cucumis sativus mainly accumulated As in its roots rather than its shoots and easily accumulated inorganic rather than organic As from the soil into its tissue.     

Phytotoxicity in seven higher plant species exposed to di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate or bis (2-ethylhexyl) phthalate

We investigated phytotoxicity in seven plant species exposed to a range of concentrations (0–500 mg·kg^?1 soil) of di-n-butyl phthalate (DnBP) or bis (2-ethylhexyl) phthalate (DEHP), two representative phthalate esters (PAEs) nominated by USEPA as priority pollutants and known environmental estrogens. We studied seed germination, root elongation, seedling growth, biomass (fresh weight, FW) and malondialdehyde (MDA) content of shoots and roots of wheat (Triticum aestivum L.), alfalfa (Medicago sativa L.), perennial ryegrass (Lolium perenne), radish (Raphanus sativus L.), cucumber (Cucumis sativus L.), oat (Avena sativa) and onion (Allium cepa L.), together with monitoring of plant pigment content (chlorophyll a, b and carotinoids) in alfalfa, radish and onion shoots. Root elongation, seedling growth and biomass of the test species were generally inhibited by DnBP but not by DEHP, indicating a lower level of phytotoxicity of DEHP than of DnBP. MDA contents of four species were promoted by PAE exposure, but not in alfalfa, ryegrass or onion shoots, indicating lower sensitivity of these three species to PAE pollutants. Plant pigment contents were clearly affected under the stress of both pollutants, implying the potential damage to the photosynthetic system of test plants, mainly by decreasing the content of chlorophyll a and b. Results of DnBP and DEHP phytotoxicity to the primary growth of test plants has provided information for the assessment of their environmental risk in the soil and also forms a basis for the further analysis of their toxic effects over the whole growth period of different plant species.     

A two-dimensional simulation model of phosphorus uptake including crop growth and P-response

Modelling nutrient uptake by crops implies considering and integrating the processes controlling the soil nutrient supply, the uptake by the root system and relationships between the crop growth response and the amount of nutrient absorbed. We developed a model that integrates both dynamics of maize growth and phosphorus (P) uptake. The crop part of the model was derived from Monteith's model. A complete regulation of P-uptake by the roots according to crop P-demand and soil P-supply was assumed. The soil P-supply to the roots was calculated using a diffusion equation and assuming that roots behave as zero-sinks. The actual P-uptake and crop growth were calculated at each time step by comparing phosphate and carbohydrate supply–demand ratios. Model calculations for P-uptake and crop growth were compared to field measurements on a long term P-fertilization trial. Three P-fertilization regimes (no P-fertilization, 42.8 kg P ha⁻¹ year⁻¹ and 94.3 kg P ha⁻¹ year⁻¹) have led to a range of P-supply. Our model correctly simulated both the crop development and growth for all P-treatments. P-uptake was correctly predicted for the two non-limiting P-treatments. Nevertheless, for the limiting P-treatment, P-uptake was correctly predicted during the early period of growth but it was underestimated at the last sampling date (61 day after sowing). Several arguments for under-prediction were considered. However, most of them cannot explain the observed magnitude in discrepancy. The most likely reason might be the fact that biomass allocation between shoot and root must be modelled more precisely. Despite this mismatch, the model appears to provide realistic simulations of the soil–plant dynamic of P in field conditions.     

Sensitive crop species and appropriate bioassays for potential use in phytotoxicity assessment of Pb-contaminated soils

The present study aims to determine the phytotoxic effects of lead (Pb) on corn (Zea mays), wheat (Triticum aestivum), cucumber (Cucumis sativus), cabbage (Brassica oleracea) and lettuce (Lactuca sativa) and to identify the sensitive crop species and appropriate bioassays for potential use in phytotoxicity assessment of Pb-contaminated soil. In a laboratory experiment, Pb(NO₃)₂ was added to the background soil to obtain eight Pb treatments. The results indicate that the seed germination rate of lettuce decreases by 14.44%, 30.00% and 40.00% at 2000, 3000 and 4000 mg Pb kg^?1 soil, respectively. However, the germination of corn, wheat, cucumber and cabbage is not significantly influenced by the Pb-contaminated soil treated with all the tested concentrations. Furthermore, the root elongation is more sensitive to Pb than is seed germination. The minimum concentrations of adverse effect of maize, wheat, cucumber, cabbage and lettuce are 2000, 3000, 1300, 800 and 300 mg Pb kg^?1 soil, respectively. Moreover, dicotyledon species are more sensitive than monocotyledon species. In the genotoxicity study, the mitotic index (MI) fluctuates with an increasing Pb concentration. The micronuclei (MN) frequencies of cucumber, cabbage and lettuce exhibit a dose-dependent effect at concentrations ranging from 1300 to 4000 mg Pb kg^?1 soil. It can be concluded that lettuce is a good candidate for indicating the toxicity of Pb in soil. Root elongation and the micronucleus frequency of dicotyledon are appropriate bioassays for potential use in phytotoxicity assessment of Pb-contaminated soil.     

Phytotoxicity and uptake of chlorpyrifos in cabbage

Chinese cabbage, Brassica chinensis L., and cabbage, Brassica oleracea L. var. capitata, are the main daily foliar vegetables of the vast majority of the population of eastern and southern China. Cabbages are also planted and consumed widely in other countries. The insecticide and acaricide chlorpyrifos is registered in many countries. Chlorpyrifos controls a variety of insects in plants and soils, and chlorpyrifos is extensively used in the Chinese market. Food poisoning due to the presence of organophosphorus pesticide residues in vegetables has been reported in China provinces. Plant uptake of pesticide residues in air, water, and soil is a source of pesticide residues in vegetables. Here, phytotoxicity and uptake of chlorpyrifos by Chinese cabbage and cabbage were studied in the laboratory using the batch technique. From 0 to 16 days after chlorpyrifos treatment, vegetables roots, stems, leaves, and culture water samples were collected, and the residues of chlorpyrifos in culture water, plant tissues were analyzed using GC-FPD. The results demonstrate that culture solutions with chlorpyrifos had no significant inhibitory effects on vegetable plant height. However, at 1.0 mg/l, it had significant inhibitory effects on the root length and fresh weight of Chinese cabbage. Then, at 10.0 mg/l, it had only significant inhibitory effects on the root length and fresh weight of cabbage compared to the control treatment. The disappearance rates of chlorpyrifos in solutions were in sequence as: nutrition solution with Chinese cabbage, nutrition solution with cabbage, pond water, nutrition solution. The results showed also that chlorpyrifos can be taken up by roots of Chinese cabbage and cabbage from water and subsequently translocated as a function of time. Uptake dynamics of chlorpyrifos from culture solutions by the two cabbage plants were similar.     

Effects of Cadmium on Nutrient Uptake and Translocation by Indian Mustard

Plants that hyperaccumulate metals are ideal subjects for studying the mechanisms of metal and mineral nutrient uptake in the plant kingdom. Indian Mustard (Brassica juncea) has been shown to accumulate moderate levels of Cd, Pb, Cr, Ni, Zn, and Cu. In this experiment, 10 levels of Cd concentration treatments were imposed by adding 10–190 mg Cd kg^–1 to the soils as cadmium nitrate [Cd(NO₃)₂]. The effect of Cd on phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and the micronutrients iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) in B. juncea was studied. Plant growth was affected negatively by Cd, root biomass decreased significantly at 170 mg Cd kg^–1 dry weight soils treatment. Cadmium accumulation both in shoots and roots increased with increasing soil Cd treatments. The highest concentration of Cd was up to 300 mg kg^–1 d.w. in the roots and 160 mg kg^–1 d.w. in the shoots. The nutrients mainly affected by Cd were P, K, Ca, Fe, and Zn in the roots, and P, K, Ca, and Cu in the shoots. K and P concentrations in roots increased significantly when Cd was added at 170 mg kg^–1, and this was almost the same level at which root growth was inhibited. Zn concentrations in roots decreased significantly when added Cd concentration was increased from 50 to 110 mg kg^–1, then remained constant with Cd treatments from 110 to 190 mg kg^–1. However, Zn concentrations in the shoots seemed less affected by Cd. It is possible that Zn uptake was affected by the Cd but not the translocation of Zn within the plant. Ca and Mg accumulation in roots and shoots showed similar trends. This result indicates that Ca and Mg uptake is a non-specific process.     

Kelp (<Emphasis Type="Italic">Laminaria digitata</Emphasis>) increases germination and affects rooting and plant vigour in crops and native plants from an arable grassland in the Outer Hebrides,Scotland

Kelp and other seaweeds are traditionally used in many parts of the world as a soil amendment on arable fields. Seaweeds contain biochemical compounds that can act as plant growth regulators in terrestrial plants. In a low-intensity arable grassland in northwest Scotland an organic fertilizer, kelp (Laminaria digitata) has been used for hundreds of years, due to its anticipated positive effect as a soil conditioner and provider of plant nutrients. In this study the effects of kelp on germination and rooting of crops and native plants from this area were investigated in soil-free media. Germination was studied by incubation in the presence of kelp solutions. Rooting of plant cuttings was assessed after a pulse treatment with kelp solutions, and indole-3 acetic acid (IAA) as a reference plant growth regulator. Germination percentage of Plantago lanceolata, Trifolium repens and Avena strigosa seeds increased significantly when incubated with 0.05% kelp solutions. Total root weight and the individual weight of roots produced in cuttings of Vigna radiata and P. lanceolata were significantly increased when exposed to a 0.5% solution of kelp. Plant vigour, assessed visually, decreased significantly for P. lanceolata exposed to kelp at concentrations of 0.5 and 5.0% indicating the presence of a threshold level for an inhibitory effect of kelp at these concentrations, which may be due to high salinity. The results confirmed the presence of plant growth regulators in kelp, and indicates that amendment with kelp may potentially affect plant community composition. The threshold levels where some plants responded negatively to kelp amendment were close to or lower than the theoretical concentrations of kelp in soil water at field conditions with the current doses used on the machair, indicating that care should be taken in either administering kelp at the appropriate dose or leaching out salt before application.     

Assessment of the impacts of municipal solid waste dumps on soils and plants

The study aimed at evaluating the impacts of open municipal solid wastes dumps on soil and vegetation near the main roads linking major cities in Nigeria. We hypothesised that the metals from the wastes exerted substantial impacts at the dump sites which affect the soil and plants. Data were analysed from five dump sites and five control sites. The result revealed that the effects of the heavy metals (HM) were significant and higher at the dump sites where their concentrations were far above the EU, and Canadian environmental quality permissible limits for agricultural soils and vegetation. In contrast with dump sites, a significant relationship (R²?=?0.70; p??Cr?>?Pb at both dump sites and control sites. Further study on the effects of more HM on soil and plant is recommended in the area. Recycling and bio-phytoremediation processes should also be introduced.     

Efficient arsenic depollution in water using modified maize powder

This article reports the synthesis of an efficient, low-cost material from maize powder to depollute arsenic-contaminated water. Arsenic is toxic for humans and other organisms even at low concentrations. The most well-known and severe case of arsenic poisoning through drinking water has been found in India and Bangladesh. Numerous inorganic materials have been tested for the removal of arsenic from water bodies over the last two decades. However, all such materials have several disadvantages such as unpredictable arsenic ion removal, high cost and the generation of toxic sludge that is often more difficult to manage. Alternatively, organic material from agricultural waste may be modified to enrich functional groups responsible for As sorption and, in turn, used to depollute contaminated waters. Here, Zea mays cob powder has been modified to remove arsenic species from water. Two modified materials were produced: an aminated maize powder and a thiolated maize powder. Amination was done using epichlorohydrin and dimethylamine. Thiolation was done using thioglycolic acids. Amination increased As (III) sorption from 70 to 75.8 % and As (V) sorption from 85 to 94.42 %, compared with unmodified maize powder. Thiolation increased As (III) sorption from 70 to 81.7 % and As (V) sorption from 85 to 90 %. Amination increased usability cycles from 3 to 5. Thiolation increased usability cycles from 3 to 6. The novel modified maize biosorbent has enough potential for the development of a low-cost technological pre-treatment step, prior to high-tech chemical treatments.