Article pubs.acs.org/est
Solanaceae Plant Malformation in Chongqing City, China, Reveals a Pollution Threat to the Yangtze River Hongbo Zhang,*,† Guanshan Liu,‡ Michael P. Timko,§ Jiana Li,† Wenjing Wang,† and Haoran Ma† †
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China § Department of Biology, University of Virginia, Charlottesville, Virginia 22904, United States ‡
S Supporting Information *
ABSTRACT: Water quality is under increasing threat from industrial and natural sources of pollutants. Here, we present our findings about a pollution incident involving the tap water of Chongqing City in China. In recent years, Solanaceae plants grown in greenhouses in this city have displayed symptoms of cupped, strappy leaves. These symptoms resembled those caused by chlorinated auxinic herbicides. We have determined that these symptoms were caused by the tap water used for irrigation. Using a bioactivity-guided fractionation method, we isolated a substance with corresponding auxinic activity from the tap water. The substance was named “solanicide” because of its strong bioactivity against Solanaceae plants. Further investigation revealed that the solanicide in the water system of Chongqing City is derived from the Jialing River, a major tributary of the Yangtze River. Therefore, it is also present in the Yangtze River downstream of Chongqing after the inflow of the Jialing River. Biological analyses indicated that solanicide is functionally similar to, but distinct from, other known chlorinated auxinic herbicides. Chemical assays further showed that solanicide structurally differs from those compounds. This study has highlighted a water pollution threat to the Yangtze River and its floodplain ecosystem.
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pounds,15,17,18 whose potential carcinogenicity has long been controversial.22−26 Over recent years, scientists at the Southwest University (SWU) campus in Chongqing, a city in the southwest of China, have encountered a problem when cultivating Solanaceae plants in greenhouses. The leaves of the affected plants showed a cupped or strappy appearance, which was similar to those caused by chlorinated auxinic herbicides.13,14,19 This phenomenon was observed in several districts of Chongqing City. In this study, we conducted studies at the SWU campus to determine the cause of this phenomenon. Tobacco (Nicotiana tabacum L.) was used as the test plant to track the active compound, because the injury symptoms were easily observed, even on young seedlings. The bioactive compound that caused this phenomenon was isolated, and its distribution and biological functions were investigated.
INTRODUCTION Water quality is of critical importance to public health. It has become an issue of global concern because of increasing threats from natural and industrial pollutants.1−3 Pesticides and industrial organic products are the major sources of persistent organic pollutants (POPs), which have attracted particular attention because of their potential significant effects on the environment and human health.4,5 Pesticides, including herbicides, insecticides, fungicides, bactericides, and so forth,6 are major pollutants derived from agriculture.7,8 Herbicides make up the largest proportion of pesticides used worldwide as they are increasingly used by farmers.9,10 Plant injury by offtarget herbicides has long been a major concern.11−14 Among commercial herbicides, the growth-regulator-type herbicides are structurally similar to the natural auxin indole-3acetic acid (IAA).14−19 This type of herbicide includes the following families: phenoxyacetic acids (e.g., 2,4-D), benzoic acids (e.g., dicamba), pyridine carboxylic acids (e.g., picloram), and quinoline carboxylic acids (e.g., quinclorac),15,17,19 which are classified based on their aromatic group type and the position of the carboxylic acid.20 These herbicides are generally foliar applied to control weed growth.13,19,21 Common symptoms of injury caused by this type of herbicide include leaf malformations such as cupped or strappy leaves.14,16,19 This type of herbicide can also result in stout roots and the formation of callus tissue.14,16,19 Most of the commercial growth-regulator-type herbicides are chlorinated com© 2014 American Chemical Society
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MATERIALS AND METHODS Plant Growth Conditions. Tobacco (N. tabacum L. cv. TN90) was used as the test plant for bioassays in this study. Plants grown in the greenhouse were irrigated once per week with tap water, except that control plants were watered with double-distilled water (ddH2O). Plants cultured in the indoor Received: Revised: Accepted: Published: 11787
March 27, 2014 September 20, 2014 September 22, 2014 September 23, 2014 dx.doi.org/10.1021/es501502y | Environ. Sci. Technol. 2014, 48, 11787−11793
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procedure could recover almost all of the solanicide in the pellet, as determined by the tobacco seedling bioassay. These steps were repeated several times to obtain a 1 × 105 times concentrated solanicide sample from 1000−2000 L of tap water. Then, the concentrated sample was separated by preparative thin layer chromatography (TLC) on F254 silica gel plates using a solvent system of n-butanol/acetic acid/H2O (8:1:1). The substance (Rf = 6.5) determined to be the active compound in the bioactivity assay was scraped from the silica gel plates and then recovered by eluting three times with nbutanol. The sample was boiled to remove n-butanol, until 0.5 mL of sample remained. This sample was again separated on F254 silica gel plates with the solvent system described above to obtain a purified sample. Then, the sample was recovered from the silica gel plates and purified by repeated recrystallization to produce pure solanicide. Image of the crystals was photographed under a stereoscopic microscope. A blank purification without sample loading was performed to verify that no toxity to tobacco was produced during sample preparation. NMR Assay. About 5 mg of purified solanicide was dissolved in deuterated H2O (D2O, Sigma−Aldrich) to a final volume of 0.5 mL in a 5 mm diameter NMR sample tube (Wilmad, Buena, NJ, USA). The NMR assay was performed on a Bruker Avance II 600 MHz spectrometer (Bruker), equipped with a 5 mm PABBO probe, operating at 600.13 MHz. 1H NMR data were acquired with 64 scans. When acquiring the 13 C NMR spectrum, sample recrystallized from methanol was dissolved in deuterated H2O to a final volume of 0.4 mL in a 5 mm diameter NMR sample tube, and another 1H NMR spectrum was acquired with 16 scans as a reference (Figure S4c, Supporting Information). The 13C NMR data were acquired with 65 536 scans. Mass Spectrum Analysis. Electrospray ionization (ESI)− high-resolution mass spectrometry was conducted using a Bruker microTOF-Q II mass spectrometer (Bruker, Rheinstetter, Germany), with direct injection of the sample. Triplicate mass spectra were acquired in the m/z 50−3000 range in negative ionization mode. Mass spectra data were analyzed using Bruker Data Analysis (V4.0) software. The background signal was subtracted from the mass spectra. Annotated peaks were detected in all replicates.
growth room were watered twice per week with tap water or ddH2O as indicated. To culture tobacco plants under sterile (i.e., bacteria-free) conditions, tobacco seeds were surfacesterilized using 10% commercial bleach with 0.05% Tween 20. Then, the seeds were cultured for 2−3 weeks on plates containing 1/2 MS (Murashige and Skoog) medium prepared using ddH2O. The seedlings were then transferred onto plates containing 1/2 MS medium prepared using tap water or ddH2O as indicated and were cultured in the growth room for 2−4 weeks or until injury symptoms developed within 70 days. Failure to induce injury symptom within 70 days indicated the corresponding water was free from solanicide contamination. To compare the auxinic activity of the tap water with that of 1 mg/L 2,4-D, we cultivated 3 week old tobacco seedlings in pots in an indoor growth room and watered (root application) or sprayed (foliar application) the plants twice per week with ddH2O, tap water, or 1 mg/L 2,4-D (in ddH2O). Injury symptoms were assessed after 4 weeks of treatment. For the bioassay to determine the presence of solanicide in river water, tap water, and concentrated or purified samples, 2− 3 week old tobacco seedlings were cultured in bottles on four layers of filter paper soaked with the testing sample in an indoor growth room for 2−4 weeks or until injury symptoms were observed within 70 days. The 10× concentrated water sample was obtained by boiling tap water, for solanicide was shown stable during boiling in the stability test described below. We also used the tobacco bioassay to detect seasonal changes in solanicide activity in tap water. The tap water samples for this assay were collected at the SWU campus in different seasons in 2011, 2012, and 2013. We evaluated the solanicide activity in tap water by counting the number of days taken to induce typical injury symptoms (at least three strappy leaves). The results shown are the average of five replicates. To compare effects of solanicide with those of other auxinic compounds, tobacco seeds were surface-sterilized and cultured as described above to obtain seedlings. Then, the seedlings were transferred onto plates with four layers of filter paper, and 5 mL of solanicide or other auxinic compounds (indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), phenoxyacetic acid (2,4-D), dicamba (benzoic acid), picloram (pyridine carboxylic acid), or quinclorac (quinoline carboxylic acid)), all at a concentration of 1 mg/L. All of these solutions were prepared using ddH2O. The plates were sealed with Parafilm and cultured in a growth room for 2 weeks. Images of tobacco seedlings were obtained under a stereoscopic microscope. Stability Test of Solanicide. The stability of solanicide at a high temperature and at high and low pH was tested to facilitate its enrichment and purification. For these experiments, tap water was adjusted to pH 2.0 with 1 M HCl or to pH 11.0 with 1 M NaOH and then heated at 121 °C for 15 min. The pH of the treated water was adjusted to pH 6.8 with 1 M NaOH or 1 M HCl before preparing 1/2 MS medium for the tobacco seedling bioassay. Solanicide Purification. Solanicide in tap water was concentrated by evaporating off the water with a distilled water maker. The produced dH2O (distilled water) was verified no solanicide activity by the tobacco seedling bioassay. The concentrated sample was heated at 110 °C for 10 min after adding 1 M NaOH (500 μL/10 mL sample) to precipitate the undesired substances from the sample. The precipitate was removed from the concentrated sample by centrifuging at 7000g for 5 min. The residues of solanicide in the pellet were recovered by three washes with 1 volume of ddH2O. This
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RESULTS AND DISCUSSION Cause of Solanaceous Plant Malformation in Chongqing City. The greenhouse cultured solanaceous plant in Chongqing City was recently observed to exhibit injury symptoms (Figure 1 and Figure S1a, Supporting Information), which were similar to those caused by chlorinated auxinic herbicides.13,16 As illustrated for representative tobacco (N. tabacum L.) (Figure 1a), the leaves of the affected plants showed a cupped or strappy appearance. To determine the cause of this phenomenon, tobacco (N. tabacum L. cv. TN90) was used as the test plant to track the active compound. The symptoms of injury were observed in greenhouse-grown tobacco plants irrigated with tap water (Figure 1a), while plants grown in the greenhouse with ddH2O (Figure 1a) or those grown outside of the greenhouses with rainwater (Figure S2, Supporting Information) were normal. The injured plants were able to produce normal leaves after irrigation with tap water ceased (Figure 1b). Plants cultivated in pots and watered more frequently with tap water showed more severe injury symptoms and produced tumorlike callus tissue just below the shoot 11788
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concentrated tap water from the Jialing River showed typical injury symptoms, with at least three curled or strappy leaves and a protruding shoot after 2−3 weeks (Figure 1d). Using this quick bioassay, we tested the solanicide activity in water samples collected from various sampling sites. As illustrated in Figure 2a, water samples from the Jialing River induced injury symptoms in tobacco in less than 20 days, while no solanicide activity was detected in over 70 days in those from the Yangtze River upstream of Chongqing City. Moreover, solanicide activity was also present in water samples from the Yangtze River approximately 3 km downstream of Chongqing City after receiving the Jialing River (Figure 2a). This supports the hypothesis that solanicide was sourced from the Jialing River, which then carried it into the Yangtze River (Figure 2b, c), the longest river flowing across the vast land area of China. By testing solanicide activity in the tap water samples collected from different districts of Chongqing City, we found that solanicide was present in the tap water from urban land close to the Jialing River or the Yangtze River downstream of the inflow of the Jialing River, while no solanicide activity was detected in tap water from urban land close to the Yangtze River upstream of the inflow of the Jialing River (Figure 2a). The tap water in different city districts is supplied by water treatment plants on the nearest river. Therefore, the distribution pattern of solanicide in the city water system is consistent with its distribution in the rivers as a source of tap water. The Yangtze River has attracted much attention as a result of other pollution issues.28−30 Findings in this study highlighted a new type of pollution threat to this river as well as the Jialing River. Seasonal Changes in Solanicide Activity in Tap Water. Seasonal changes in solanicide activity in the tap water sourced from the Jialing River were determined using the bioassay described above and water samples collected at the SWU campus in different seasons in 2011, 2012, and 2013. As shown in Figure 3, it took around 18 days for these tap water samples to induce typical injury symptoms in tobacco. There were no significant seasonal differences in the solanicide activity of the tap water samples. This result showed that solanicide activity in the tap water sourced from the Jialing River was probably not correlated with agricultural seasons or to seasonal changes in the water level. Isolation and Chemical Characterization of Solanicide. We then isolated solanicide from tap water and analyzed its chemical properties. The bioassay described above was used to detect the presence of solanicide in concentrated and/or purified samples. We found that solanicide is stable at a high temperature (121 °C) under both acidic (pH 2.0) and alkaline (pH 11.0) conditions. Therefore, solanicide could be concentrated in tap water by evaporating off the water with a distilled water maker, and no solanicide activity was detectable in the distilled water produced during this process. The 1 × 105 times concentrated tap water sample was separated by preparative TLC on silica gel with n-butanol/acetic acid/H2O (8:1:1) as the solvent system. Only one substance (Rf = 6.5) showed solanicide activity. Approximately 15 mg of preliminarily purified solanicide was obtained from the sample concentrated from 1500 L of tap water. Thus, the recovery rate of solanicide from tap water was approximately 0.01 mg/L. Assuming that half of the solanicide was lost during the purification processes, we estimated that its content in tap water was less than or equal to 0.02 mg/L. The partially purified product was further purified by repeated recrystallization, yielding ∼7 mg of pure solanicide.
Figure 1. Injury symptoms in tobacco plants grown with tap water. (a) Morphology of tobacco plants irrigated with tap water. Insets show typical symptoms in tobacco watered with tap water (right) and a tobacco plant watered with ddH2O as control (left). The percentage of plants showing injury symptom is given. (b) Recovery of injured tobacco plant after stopping irrigation with tap water. (c) Morphology of tobacco plant grown in pot and irrigated with tap water. Inset shows tumorlike callus tissue with epidermis removed. (d) Tobacco plants cultured under sterile conditions. “ddH2O” indicates plant cultured on 1/2 MS medium prepared using ddH2O, and “tap water” indicates plant cultured on medium prepared using 10× concentrated (main image) or unconcentrated (inset) tap water. Fifteen plants were tested for each treatment, and the number of plants showing injury symptom is given.
(Figure 1c). The injured leaves could recover after tap water was replaced with ddH2O. Intriguingly, the tumorlike callus tissue continued to grow in size even after the injured leaves had recovered (Figure S3, Supporting Information). These findings suggested that some bioactive compound or biosome was present in the water system. We then conducted bioassays in the laboratory using tobacco plants grown on autoclave-sterilized 1/2 MS medium.27 The tobacco plants grown on sterilized 1/2 MS medium prepared with tap water showed the injury symptom, while those cultured on 1/2 MS medium prepared with ddH2O did not show symptoms (Figure 1d). This bioassay confirmed that a bioactive compound was present in the tap water. The compound responsible for the auxinic activity in the tap water was named “solanicide” because of its strong bioactivity against members of the Solanaceae. Source and Distribution of Solanicide. The tap water at the SWU campus is supplied by the water treatment plants on the Jialing River, the source of drinking water for more than half of the inhabitants of Chongqing City, as well as many suburban residents that live along the river. A tobacco bioassay system was used to investigate the distribution of solanicide in the water system of Chongqing City. Young tobacco seedlings (2−3 weeks old) cultured on medium prepared using 10× 11789
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Figure 2. Distribution of solanicide in Jialing River and Yangtze River. (a) Solanicide is sourced from Jialing River (red), which flows into Yangtze River (blue, before inflow of Jialing River; yellow, after inflow of Jialing River) at Chongqing City. Irregular orange patch at top-left corner shows SWU campus. Pink-ringed spots show major sampling sites, and numbers on spots indicate days taken for water samples to induce typical solanicide injury symptoms in tobacco plants. “UD” indicates undetectable solanicide activity within 70 days. Blue dots indicate other sampling sites with solanicide detected, and green ones indicate those with undetectable solanicide. Samples from sites apart from the river were collected from tap water. (b) Main streams of Yangtze River (blue line) and Jialing River (red line). Red spot indicates location of Chongqing City. (c) Location of confluence of Jialing River and Yangtze River. Arrows indicate flow direction of Jialing River (yellow) and Yangtze River (purple). Satellite images were obtained from Google Maps (a and b; https://maps.google.com.hk/) and Baidu Maps (c; http://map.baidu.com/).
carbons at δ60.0−80.0 ppm (Figure S4b, Supporting Information). These data suggested the presence of an aromatic ring, a basic component of natural and synthetic auxins,17 even though signals for quarternary carbons were not detected. From the NMR data, we could speculate that solanicide contains approximately 10 carbons (including undetected quarternary aromatic carbons) and 11 hydrogens. However, the H−C correlation was not able to be determined from the current data, especially that for alkene protons, because no alkene carbon signal was detected in the 13C NMR spectrum (Figure S4b, Supporting Information). The mass spectrum of solanicide was acquired in the m/z 50−3000 range in negative ionization mode. The largest m/z value was 213.9624 (Figure 4). In searches of available chemical databases, no ion observed in the mass spectrum hit any known compounds compatible with the NMR spectrum data. Interestingly, none of the mass peaks showed an attribute of the presence of chlorine (Figure 4; Figure S5, Supporting Information), which generates characteristic isotope patterns in the mass spectrum according to the relative abundances of its natural isotopes.31 Mass spectral data for the chlorinated herbicide 2,4-D (obtained from the National Institute of Standards and Technology, http://webbook.nist. gov) are shown in Figure S5 (Supporting Information) as a reference. This suggested that solanicide is an unchlorinated compound. Taken together, the results of the chemical analyses
Figure 3. Time to effect for tap water collected in 2011, 2012, and 2013 to induce typical injury symptoms (at least three strappy leaves) in tobacco. Error bar = ± SD.
The crystallization of solanicide from water gave slightly yellowish needles (Figure S4a, Supporting Information), but it did not form single crystals. The 1H NMR (600 MHz, D2O) analysis revealed the presence of four aromatic protons at δ8.01 (d, J = 7.8 Hz, 2H) ppm, δ7.58 (td, J = 8.4, 1.2 Hz, 1H) ppm, and δ7.52 (m, 1H) ppm, two alkene protons at δ6.49 (s, 1H) ppm and δ6.94 (s, 1H) ppm, and five protons at δ3.0−4.0 ppm that might be adjacent to electronegative atoms (Figure S4a, Supporting Information). The 13C NMR (600 MHz, D2O) spectrum revealed the presence of four aromatic carbons at δ122.89, δ123.80, δ127.53, and δ127.67 ppm and at least four 11790
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Figure 4. Mass spectrum of solanicide.
indicated that solanicide may be an unknown compound that differs from known chlorinated auxinic herbicides. However, further analyses are required to determine its molecular structure in detail. To achieve this goal, a full chemical analysis is necessary. At present, the main difficulty for further analysis is sample preparation, because a full chemical analysis requires at least 20 mg of purified product. More than 8000 L of water would have to be processed to obtain this quantity. Functional Characterization of Solanicide. Currently, the known compounds that induce symptoms similar to those induced by solanicide in solanaceous plants are chlorinated auxinic herbicides used for weed control.13,16,17,19 2,4-D is the most widely used chlorinated auxinic herbicide,32 which could induce typical symptoms of injury by chlorinated auxinic herbicides.16,32 To reveal the functional similarities and differences between solanicide and other chlorinated auxinic herbicides, the auxinic activity in the tap water was compared with that of 2,4-D by watering (root application) or spraying (foliar application) tobacco plants with tap water or 1 mg/L 2,4-D (in ddH2O). After 1 month of treatment, plants watered with either tap water or 2,4-D solution showed injury symptoms (Figure 5a). Tumorlike callus tissue was present only in the plants irrigated with tap water (Figure 5a). Whereas tobacco plants irrigated with 2,4-D solution formed stout roots, the roots of those irrigated with tap water were normal (Figure 5a). The leaves of plants sprayed with tap water were normal, while those of plants sprayed with 2,4-D solution showed severe symptoms of injury (Figure 5a). These findings showed that solanicide is more active than 2,4-D in causing leaf injury to tobacco via watering (root application) but inactive when applied by foliar spraying. The known chlorinated auxinic herbicides are all active by foliar application and are generally applied in this way to control weed growth.13,19,21 Also, the chlorinated auxinic herbicides cause root injury symptoms in various plants,14 including monocots (Figure S1, Supporting Information), while none of the tap water-irrigated plants showed root symptoms. Thus, solanicide and chlorinated auxinic herbicides likely may have different mechanisms of action. The fact that the tap water was able to induce more severe symptoms of shoot injury than those induced by 1 mg/L 2,4-D solution in tobacco plants (Figure 5a) suggested that
Figure 5. Functional characterization of solanicide. (a) Comparison between auxinic activity of control (ddH2O), tap water, and 1 mg/L 2,4-D (in ddH2O). The red arrow in the top panels shows tumorlike callus tissue, and arrows in middle panels show stout roots. Fifteen plants were tested for each treatment, and the number of plants showing injury symptom in the displayed tissue is given. (b) Comparison of effects of solanicide (1 mg/L) with those of other chlorinated auxinic herbicides (1 mg/L) on tobacco. Insets show enlargements of cotyledon nodes. Red arrows show callus tissue; white arrows show induced lateral root primordial (or lateral roots); red open brackets show the protruded shoot (epicotyl). Thirty plants were tested for each treatment, and the representative image is displayed. 11791
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auxinic herbicide,32 showed that 2,4-D content in the tap water was ∼0.01 mg/L (Figure S6, Supporting Information). This value is below the World Health Organization guidelines for 2,4-D in drinking water (0.03 mg/L).24 Furthermore, no significant seasonal change in solanicide activity in the tap water sourced from the Jialing River was observed (Figure 3). These findings imply that the contaminant is not an agriculturalderived herbicide. We are currently unable to fully solve the chemical structure of solanicide because of its special characteristics and the difficulty in preparing samples. However, our findings show that solanicide is an unchlorinated chemical with a strong auxinic effect. The unique chemical characteristics and bioactivity of solanicide suggest that it differs from the known compounds. Our results demonstrated that solanicide pollution is the cause of malformation of Solanaceae plants in Chongqing City and further revealed that solanicide is sourced from the Jialing River and carried by this river into the Yangtze River, the longest river flowing across the vast land area of China. Thus, a pollution threat to the Yangtze River and massive scale ecosystem in its floodplain is highlighted. The presence of solanicide in tap water in Chongqing City also implies that the current water treatment process (a combination of coagulation, sedimentation, filtration, and disinfection) by water treatment plants is not reliable for removing organic pollutants.
solanicide has very strong bioactivity, as its content in tap water is less than or equal to 0.02 mg/L. To further reveal the functional characteristics of solanicide, we compared the effects of 1 mg/L solanicide (prepared using the purified product) on tobacco seedlings with those of other auxinic compounds including IAA, the unchlorinated synthetic auxin NAA, and the chlorinated auxinic herbicides from all families of growth-regulator herbicides13,15,17,19 (all at a concentration of 1 mg/L). The symptoms caused by 2,4-D and picloram in tobacco were taken as the representative symptoms of injury by chlorinated auxinic herbicides, for comparison with those caused by solanicide. The results showed that the symptoms in tobacco seedlings induced by 1 mg/L solanicide differed from those induced by the other auxinic compounds (Figure 5b). After 2 weeks of treatment, plants treated with solanicide showed curled leaves, protruded shoots, and formation of callus tissue from the cotyledonary node. The unchlorinated auxin IAA and NAA as well as the chlorinated auxin 2,4-D did not induce leaf curling (Figure 5b). Picloram, a fast-acting chlorinated herbicide,13,16 induced leaf curling as rapidly as did solanicide, but also caused the seedlings to wilt (Figure 5b). Also, picloram did not induce extensive callus formation but only several tiny callus initiation spots around the cotyledonary node (Figure 5b). Another obvious difference between solanicide and the other auxinic compounds was that the other auxinic compounds induced formation of stout lateral roots (or callus tissue) in the maturation zone, while solanicide induced callus tissue in taproot apex (Figure 5b). Interestingly, formation of a normal adventitious root from hypocotyl is occasionally observed in solanicide treated tobacco plants as in control (Figure 5b). In terms of symptoms, the most noticeable difference between solanicide and the other auxinic compounds was that solanicide caused the shoot of tobacco seedlings to protrude, whereas the shoots of tobacco seedlings treated with the other auxinic compounds were enclosed in the rosettes (Figure 5b). Solanicide-induced shoot protrusion was also obvious in older seedlings irrigated with tap water (Figure 1d). These findings suggested that solanicide is functionally different from other natural or synthetic auxinic compounds. The roles of solanicide in promoting shoot protrusion and inducing callus tissue in taproot apex imply that it predominantly functions in the apical meristem. Yet, it was less active in the root, as tap water induced injury symptoms in shoots but not in roots (Figure 5a). These findings showed that the function of solanicide is similar to but distinct from other chlorinated auxinic compounds. In this study, we determined that the injury symptoms in greenhouse-grown solanaceous plants in Chongqing City were caused by the tap water used for irrigation. Plant injury by auxinic compounds is generally observed in areas where there has been excessive application of chlorinated auxinic herbicides.13,14 This finding is unusual in that the presence of high auxinic activity in the water system, especially drinking water, is a rare occurrence, even though there are detectable levels of herbicides in some rivers.33 Therefore, it ever raised considerable concerns about water contamination by commercial chlorinated auxinic herbicides that were applied seasonally by farmers, for the auxinic activity of the polluted water was somewhat similar to this kind of compounds. The presence of chlorinated auxinic herbicides in drinking water is strictly monitored because of their potential toxity.22−26 Analyses of tap water using an enzyme-linked immunosorbent assay (ELISA) kit to detect 2,4-D, the most widely used chlorinated
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ASSOCIATED CONTENT
* Supporting Information S
Growth condition for other plants, method for ELISA assay, supporting data for plant injury symptoms from solanicide or other auxinic herbicides, phenotype of tobacco plants grown with rainwater, tap water induced tumorlike callus tissue in tobacco, crystals and NMR spectra of solanicide, mass spectrum of 2,4-D, and 2,4-D content in tap water. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-23-6825-0744; fax: +86-23-6825-1264; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to B. Lei at Guizhou Tobacco Research Institute, and Q. Guo and Q. Luo at the School of Chemistry and Chemical Engineering at SWU for the technical assistant. We also thank the SWU scientists whose work on Solanaceae plants provided help to this study. The authors were supported by the Fundamental Research Funds for the Central Universities of China (XDJK2012C084; 2362014xk09), the “111” Project (B12006), China National Tobacco Corporation (110201301005(JY-05)), State Education Ministry (SRF for ROCS), and the Doctoral Fund of Southwest University (SWU110053).
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REFERENCES
(1) Qiu, J. China faces up to groundwater crisis. Nature 2010, 466 (7304), 308. (2) Rodriguez-Lado, L.; Sun, G.; Berg, M.; Zhang, Q.; Xue, H.; Zheng, Q.; Johnson, C. A. Groundwater arsenic contamination throughout China. Science 2013, 341 (6148), 866−868.
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(3) Qiu, J. China to spend billions cleaning up groundwater. Science 2011, 334 (6057), 745. (4) Sharma, B. M.; Bharat, G. K.; Tayal, S.; Nizzetto, L.; Cupr, P.; Larssen, T. Environment and human exposure to persistent organic pollutants (POPs) in India: A systematic review of recent and historical data. Environ. Int. 2014, 66C, 48−64. (5) Wong, M. H.; Leung, A. O.; Chan, J. K.; Choi, M. P. A review on the usage of POP pesticides in China, with emphasis on DDT loadings in human milk. Chemosphere 2005, 60 (6), 740−752. (6) Randall, C.; Hock, W.; Crow, E.; Hudak-Wise, C.; Kasai, J. National Pesticide Applicator Certification - Core Manual; National Association of State Departments of Agriculture Research Foundation: Washington, DC, 2006. (7) Ritter, L.; Solomon, K.; Sibley, P.; Hall, K.; Keen, P.; Mattu, G.; Linton, B. Sources, pathways, and relative risks of contaminants in surface water and groundwater: A perspective prepared for the Walkerton inquiry. J. Toxicol. Environ. Health, Part A 2002, 65 (1), 1− 142. (8) Shukla, G.; Kumar, A.; Bhanti, M.; Joseph, P. E.; Taneja, A. Organochlorine pesticide contamination of ground water in the city of Hyderabad. Environ. Int. 2006, 32 (2), 244. (9) Kiely, T.; Donaldson, D.; Grube, A. Pesticides Industry Sales and Usage: 2000 and 2001 Market Estimates; United States EPA: Washington, DC, 2004. (10) Grube, A.; Donaldson, D.; Kiely, T.; Wu, L. Pesticides Industry Sales and Usage: 2006 and 2007 Market Estimates; United States EPA: Washington, DC, 2011. (11) Wall, D. A. Potato (Solanum-Tuberosum) response to simulated drift of dicamba, clopyralid and tribenuron. Weed Sci. 1994, 42 (1), 110−114. (12) Al-Khatib, K.; Peterson, D. Soybean (Glycine max) response to simulated drift from selected sulfonylurea herbicides, dicamba, glyphosate, and glufosinate. Weed Technol. 1999, 13 (2), 264−270. (13) Israel, T. D.; Rhodes, G. N.; Denton, P. Diagnosing Suspected Off-Target Herbicide Damage to Tobacco, W 290-B; University of Tennessee Extension: Knoxville, TN, 2012. (14) Bradley, K. W.; Johnson, B.; Smeda, R.; Boerboom, C. Practical Weed Science for the Field Scout - Corn and Soybean; University of Missouri Extension: Columbia, MO, 2009; Vol. IPM1007, pp 12−14. (15) Mallory-Smith, C. A.; Retzinger, E. J. Revised Classification of Herbicides by Site of Action for Weed Resistance Management Strategies. Weed Technology 2003, 17 (3), 605. (16) Gunsolus, J. L.; Curran, W. S. Herbicide Mode of Action and Injury Symptoms; BU-03832; University of Minnesota Extension Service: St. Paul, MN, 1999. (17) Grossmann, K. Mode of action of auxin herbicides: A new ending to a long, drawn out story. Trends Plant Sci. 2000, 5 (12), 506− 508. (18) Mithila, J.; Hall, J. C.; Johnson, W. G.; Kelley, K. B.; Riechers, D. E. Evolution of resistance to auxinic herbicides: Historical perspectives, mechanisms of resistance, and implications for broadleaf weed management in agronomic crops. Weed Sci. 2011, 59 (4), 445. (19) Sterling, T. M.; Hall, J. C. Mechanism of action of natural auxins and the auxinic herbicides. In Herbicide Activity: Toxicology, Biochemistry and Molecular Biology; Roe, R. M., Burton, J. D., Kuhr, R. J., Eds. IOS Press: Amsterdam, The Netherlands, 1997; pp 111− 141. (20) Ashton, F. M.; Crafts, A. S. Phenoxys. In Mode of Action of Herbicides; Ashton, F. M., Crafts, A. S., Eds.; John Wiley and Sons: Toronto, Canada, 1981; pp 272−302. (21) Kay, S. H. Response of two alligatorweed biotypes to quinclorac. J. Aquat. Plant Manage. 1992, 30, 35−40. (22) Kogevinas, M.; Becher, H.; Benn, T.; Bertazzi, P. A.; Boffetta, P.; Bueno-de-Mesquita, H. B.; Coggon, D.; Colin, D.; Flesch-Janys, D.; Fingerhut, M.; Green, L.; Kauppinen, T.; Littorin, M.; Lynge, E.; Mathews, J. D.; Neuberger, M.; Pearce, N.; Saracci, R. Cancer mortality in workers exposed to phenoxy herbicides, chlorophenols, and dioxins. An expanded and updated international cohort study. Am. J. Epidemiol. 1997, 145 (12), 1061−1075.
(23) World Health Organization. Dioxins and Their Effects on Human Health; fact sheet no. 225; WHO: Geneva, Switzerland, 2010. (24) World Health Organization. Guidelines for Drinking-Water Quality, 4th ed.; WHO: Geneva, Switzerland, 2011; p 347. (25) Dad, N. K.; Tripathi, P. S. Acute toxicity of herbicides to freshwater fish and midge larvae, Chironomus tentans. Environ. Int. 1980, 4 (5−6), 435−437. (26) Alpöz, A. R.; Tosun, N.; Eronat, C.; Delen, N.; Şen, B. H. Effects of 2,4-dichlorophenoxy acetic acid dimethyl amine salt on dental hard tissue formation in rats. Environ. Int. 2001, 26 (3), 137. (27) Murashige, T.; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962, 15 (3), 473. (28) Yang, H.; Xie, P.; Ni, L.; Flower, R. J. Pollution in the Yangtze. Science 2012, 337 (6093), 410. (29) Wong, C. M.; Williams, C. E.; Pittock, J.; Collier, U.; Schelle, P. World’s Top 10 Rivers at Risk; WWF International: Gland, Switzerland, 2007. (30) Gfrerer, M.; Martens, D.; Gawlik, B. M.; Wenzl, T.; Zhang, A.; Quan, X.; Sun, C.; Chen, J.; Platzer, B.; Lankmayr, E.; Kettrup, A. Triazines in the aquatic systems of the Eastern Chinese Rivers Liao-He and Yangtse. Chemosphere 2002, 47 (4), 455−466. (31) Crews, P.; Rodriguez, J.; Jaspars, M. Organic Structure Analysis; Oxford University Press: New York, 1998; pp 257−257. (32) Environmental Protection Agency. U.S. Pesticides: Reregistration 2,4-D RED Facts, EPA-738-F-05-002; EPA: Washington, DC, 2005. (33) Villeneuve, A.; Larroudé, S.; Humbert, J. F. Herbicide contamination of freshwater ecosystems: Impact on microbial communities. In Pesticides - Formulations, Effects, Fate; Stoytcheva, M., Ed.; InTech: Rijeka, Croatia, 2011; pp 285−312.
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Solanaceae Plant Malformation in Chongqing City, China, Reveals a Pollution Threat to the Yangtze River Hongbo Zhang,*,† Guanshan Liu,‡ Michael P. Timko,§ Jiana Li,† Wenjing Wang,† and Haoran Ma† †
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China § Department of Biology, University of Virginia, Charlottesville, Virginia 22904, United States ‡
S Supporting Information *
ABSTRACT: Water quality is under increasing threat from industrial and natural sources of pollutants. Here, we present our findings about a pollution incident involving the tap water of Chongqing City in China. In recent years, Solanaceae plants grown in greenhouses in this city have displayed symptoms of cupped, strappy leaves. These symptoms resembled those caused by chlorinated auxinic herbicides. We have determined that these symptoms were caused by the tap water used for irrigation. Using a bioactivity-guided fractionation method, we isolated a substance with corresponding auxinic activity from the tap water. The substance was named “solanicide” because of its strong bioactivity against Solanaceae plants. Further investigation revealed that the solanicide in the water system of Chongqing City is derived from the Jialing River, a major tributary of the Yangtze River. Therefore, it is also present in the Yangtze River downstream of Chongqing after the inflow of the Jialing River. Biological analyses indicated that solanicide is functionally similar to, but distinct from, other known chlorinated auxinic herbicides. Chemical assays further showed that solanicide structurally differs from those compounds. This study has highlighted a water pollution threat to the Yangtze River and its floodplain ecosystem.
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pounds,15,17,18 whose potential carcinogenicity has long been controversial.22−26 Over recent years, scientists at the Southwest University (SWU) campus in Chongqing, a city in the southwest of China, have encountered a problem when cultivating Solanaceae plants in greenhouses. The leaves of the affected plants showed a cupped or strappy appearance, which was similar to those caused by chlorinated auxinic herbicides.13,14,19 This phenomenon was observed in several districts of Chongqing City. In this study, we conducted studies at the SWU campus to determine the cause of this phenomenon. Tobacco (Nicotiana tabacum L.) was used as the test plant to track the active compound, because the injury symptoms were easily observed, even on young seedlings. The bioactive compound that caused this phenomenon was isolated, and its distribution and biological functions were investigated.
INTRODUCTION Water quality is of critical importance to public health. It has become an issue of global concern because of increasing threats from natural and industrial pollutants.1−3 Pesticides and industrial organic products are the major sources of persistent organic pollutants (POPs), which have attracted particular attention because of their potential significant effects on the environment and human health.4,5 Pesticides, including herbicides, insecticides, fungicides, bactericides, and so forth,6 are major pollutants derived from agriculture.7,8 Herbicides make up the largest proportion of pesticides used worldwide as they are increasingly used by farmers.9,10 Plant injury by offtarget herbicides has long been a major concern.11−14 Among commercial herbicides, the growth-regulator-type herbicides are structurally similar to the natural auxin indole-3acetic acid (IAA).14−19 This type of herbicide includes the following families: phenoxyacetic acids (e.g., 2,4-D), benzoic acids (e.g., dicamba), pyridine carboxylic acids (e.g., picloram), and quinoline carboxylic acids (e.g., quinclorac),15,17,19 which are classified based on their aromatic group type and the position of the carboxylic acid.20 These herbicides are generally foliar applied to control weed growth.13,19,21 Common symptoms of injury caused by this type of herbicide include leaf malformations such as cupped or strappy leaves.14,16,19 This type of herbicide can also result in stout roots and the formation of callus tissue.14,16,19 Most of the commercial growth-regulator-type herbicides are chlorinated com© 2014 American Chemical Society
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MATERIALS AND METHODS Plant Growth Conditions. Tobacco (N. tabacum L. cv. TN90) was used as the test plant for bioassays in this study. Plants grown in the greenhouse were irrigated once per week with tap water, except that control plants were watered with double-distilled water (ddH2O). Plants cultured in the indoor Received: Revised: Accepted: Published: 11787
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procedure could recover almost all of the solanicide in the pellet, as determined by the tobacco seedling bioassay. These steps were repeated several times to obtain a 1 × 105 times concentrated solanicide sample from 1000−2000 L of tap water. Then, the concentrated sample was separated by preparative thin layer chromatography (TLC) on F254 silica gel plates using a solvent system of n-butanol/acetic acid/H2O (8:1:1). The substance (Rf = 6.5) determined to be the active compound in the bioactivity assay was scraped from the silica gel plates and then recovered by eluting three times with nbutanol. The sample was boiled to remove n-butanol, until 0.5 mL of sample remained. This sample was again separated on F254 silica gel plates with the solvent system described above to obtain a purified sample. Then, the sample was recovered from the silica gel plates and purified by repeated recrystallization to produce pure solanicide. Image of the crystals was photographed under a stereoscopic microscope. A blank purification without sample loading was performed to verify that no toxity to tobacco was produced during sample preparation. NMR Assay. About 5 mg of purified solanicide was dissolved in deuterated H2O (D2O, Sigma−Aldrich) to a final volume of 0.5 mL in a 5 mm diameter NMR sample tube (Wilmad, Buena, NJ, USA). The NMR assay was performed on a Bruker Avance II 600 MHz spectrometer (Bruker), equipped with a 5 mm PABBO probe, operating at 600.13 MHz. 1H NMR data were acquired with 64 scans. When acquiring the 13 C NMR spectrum, sample recrystallized from methanol was dissolved in deuterated H2O to a final volume of 0.4 mL in a 5 mm diameter NMR sample tube, and another 1H NMR spectrum was acquired with 16 scans as a reference (Figure S4c, Supporting Information). The 13C NMR data were acquired with 65 536 scans. Mass Spectrum Analysis. Electrospray ionization (ESI)− high-resolution mass spectrometry was conducted using a Bruker microTOF-Q II mass spectrometer (Bruker, Rheinstetter, Germany), with direct injection of the sample. Triplicate mass spectra were acquired in the m/z 50−3000 range in negative ionization mode. Mass spectra data were analyzed using Bruker Data Analysis (V4.0) software. The background signal was subtracted from the mass spectra. Annotated peaks were detected in all replicates.
growth room were watered twice per week with tap water or ddH2O as indicated. To culture tobacco plants under sterile (i.e., bacteria-free) conditions, tobacco seeds were surfacesterilized using 10% commercial bleach with 0.05% Tween 20. Then, the seeds were cultured for 2−3 weeks on plates containing 1/2 MS (Murashige and Skoog) medium prepared using ddH2O. The seedlings were then transferred onto plates containing 1/2 MS medium prepared using tap water or ddH2O as indicated and were cultured in the growth room for 2−4 weeks or until injury symptoms developed within 70 days. Failure to induce injury symptom within 70 days indicated the corresponding water was free from solanicide contamination. To compare the auxinic activity of the tap water with that of 1 mg/L 2,4-D, we cultivated 3 week old tobacco seedlings in pots in an indoor growth room and watered (root application) or sprayed (foliar application) the plants twice per week with ddH2O, tap water, or 1 mg/L 2,4-D (in ddH2O). Injury symptoms were assessed after 4 weeks of treatment. For the bioassay to determine the presence of solanicide in river water, tap water, and concentrated or purified samples, 2− 3 week old tobacco seedlings were cultured in bottles on four layers of filter paper soaked with the testing sample in an indoor growth room for 2−4 weeks or until injury symptoms were observed within 70 days. The 10× concentrated water sample was obtained by boiling tap water, for solanicide was shown stable during boiling in the stability test described below. We also used the tobacco bioassay to detect seasonal changes in solanicide activity in tap water. The tap water samples for this assay were collected at the SWU campus in different seasons in 2011, 2012, and 2013. We evaluated the solanicide activity in tap water by counting the number of days taken to induce typical injury symptoms (at least three strappy leaves). The results shown are the average of five replicates. To compare effects of solanicide with those of other auxinic compounds, tobacco seeds were surface-sterilized and cultured as described above to obtain seedlings. Then, the seedlings were transferred onto plates with four layers of filter paper, and 5 mL of solanicide or other auxinic compounds (indole-3-acetic acid (IAA), naphthalene acetic acid (NAA), phenoxyacetic acid (2,4-D), dicamba (benzoic acid), picloram (pyridine carboxylic acid), or quinclorac (quinoline carboxylic acid)), all at a concentration of 1 mg/L. All of these solutions were prepared using ddH2O. The plates were sealed with Parafilm and cultured in a growth room for 2 weeks. Images of tobacco seedlings were obtained under a stereoscopic microscope. Stability Test of Solanicide. The stability of solanicide at a high temperature and at high and low pH was tested to facilitate its enrichment and purification. For these experiments, tap water was adjusted to pH 2.0 with 1 M HCl or to pH 11.0 with 1 M NaOH and then heated at 121 °C for 15 min. The pH of the treated water was adjusted to pH 6.8 with 1 M NaOH or 1 M HCl before preparing 1/2 MS medium for the tobacco seedling bioassay. Solanicide Purification. Solanicide in tap water was concentrated by evaporating off the water with a distilled water maker. The produced dH2O (distilled water) was verified no solanicide activity by the tobacco seedling bioassay. The concentrated sample was heated at 110 °C for 10 min after adding 1 M NaOH (500 μL/10 mL sample) to precipitate the undesired substances from the sample. The precipitate was removed from the concentrated sample by centrifuging at 7000g for 5 min. The residues of solanicide in the pellet were recovered by three washes with 1 volume of ddH2O. This
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RESULTS AND DISCUSSION Cause of Solanaceous Plant Malformation in Chongqing City. The greenhouse cultured solanaceous plant in Chongqing City was recently observed to exhibit injury symptoms (Figure 1 and Figure S1a, Supporting Information), which were similar to those caused by chlorinated auxinic herbicides.13,16 As illustrated for representative tobacco (N. tabacum L.) (Figure 1a), the leaves of the affected plants showed a cupped or strappy appearance. To determine the cause of this phenomenon, tobacco (N. tabacum L. cv. TN90) was used as the test plant to track the active compound. The symptoms of injury were observed in greenhouse-grown tobacco plants irrigated with tap water (Figure 1a), while plants grown in the greenhouse with ddH2O (Figure 1a) or those grown outside of the greenhouses with rainwater (Figure S2, Supporting Information) were normal. The injured plants were able to produce normal leaves after irrigation with tap water ceased (Figure 1b). Plants cultivated in pots and watered more frequently with tap water showed more severe injury symptoms and produced tumorlike callus tissue just below the shoot 11788
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concentrated tap water from the Jialing River showed typical injury symptoms, with at least three curled or strappy leaves and a protruding shoot after 2−3 weeks (Figure 1d). Using this quick bioassay, we tested the solanicide activity in water samples collected from various sampling sites. As illustrated in Figure 2a, water samples from the Jialing River induced injury symptoms in tobacco in less than 20 days, while no solanicide activity was detected in over 70 days in those from the Yangtze River upstream of Chongqing City. Moreover, solanicide activity was also present in water samples from the Yangtze River approximately 3 km downstream of Chongqing City after receiving the Jialing River (Figure 2a). This supports the hypothesis that solanicide was sourced from the Jialing River, which then carried it into the Yangtze River (Figure 2b, c), the longest river flowing across the vast land area of China. By testing solanicide activity in the tap water samples collected from different districts of Chongqing City, we found that solanicide was present in the tap water from urban land close to the Jialing River or the Yangtze River downstream of the inflow of the Jialing River, while no solanicide activity was detected in tap water from urban land close to the Yangtze River upstream of the inflow of the Jialing River (Figure 2a). The tap water in different city districts is supplied by water treatment plants on the nearest river. Therefore, the distribution pattern of solanicide in the city water system is consistent with its distribution in the rivers as a source of tap water. The Yangtze River has attracted much attention as a result of other pollution issues.28−30 Findings in this study highlighted a new type of pollution threat to this river as well as the Jialing River. Seasonal Changes in Solanicide Activity in Tap Water. Seasonal changes in solanicide activity in the tap water sourced from the Jialing River were determined using the bioassay described above and water samples collected at the SWU campus in different seasons in 2011, 2012, and 2013. As shown in Figure 3, it took around 18 days for these tap water samples to induce typical injury symptoms in tobacco. There were no significant seasonal differences in the solanicide activity of the tap water samples. This result showed that solanicide activity in the tap water sourced from the Jialing River was probably not correlated with agricultural seasons or to seasonal changes in the water level. Isolation and Chemical Characterization of Solanicide. We then isolated solanicide from tap water and analyzed its chemical properties. The bioassay described above was used to detect the presence of solanicide in concentrated and/or purified samples. We found that solanicide is stable at a high temperature (121 °C) under both acidic (pH 2.0) and alkaline (pH 11.0) conditions. Therefore, solanicide could be concentrated in tap water by evaporating off the water with a distilled water maker, and no solanicide activity was detectable in the distilled water produced during this process. The 1 × 105 times concentrated tap water sample was separated by preparative TLC on silica gel with n-butanol/acetic acid/H2O (8:1:1) as the solvent system. Only one substance (Rf = 6.5) showed solanicide activity. Approximately 15 mg of preliminarily purified solanicide was obtained from the sample concentrated from 1500 L of tap water. Thus, the recovery rate of solanicide from tap water was approximately 0.01 mg/L. Assuming that half of the solanicide was lost during the purification processes, we estimated that its content in tap water was less than or equal to 0.02 mg/L. The partially purified product was further purified by repeated recrystallization, yielding ∼7 mg of pure solanicide.
Figure 1. Injury symptoms in tobacco plants grown with tap water. (a) Morphology of tobacco plants irrigated with tap water. Insets show typical symptoms in tobacco watered with tap water (right) and a tobacco plant watered with ddH2O as control (left). The percentage of plants showing injury symptom is given. (b) Recovery of injured tobacco plant after stopping irrigation with tap water. (c) Morphology of tobacco plant grown in pot and irrigated with tap water. Inset shows tumorlike callus tissue with epidermis removed. (d) Tobacco plants cultured under sterile conditions. “ddH2O” indicates plant cultured on 1/2 MS medium prepared using ddH2O, and “tap water” indicates plant cultured on medium prepared using 10× concentrated (main image) or unconcentrated (inset) tap water. Fifteen plants were tested for each treatment, and the number of plants showing injury symptom is given.
(Figure 1c). The injured leaves could recover after tap water was replaced with ddH2O. Intriguingly, the tumorlike callus tissue continued to grow in size even after the injured leaves had recovered (Figure S3, Supporting Information). These findings suggested that some bioactive compound or biosome was present in the water system. We then conducted bioassays in the laboratory using tobacco plants grown on autoclave-sterilized 1/2 MS medium.27 The tobacco plants grown on sterilized 1/2 MS medium prepared with tap water showed the injury symptom, while those cultured on 1/2 MS medium prepared with ddH2O did not show symptoms (Figure 1d). This bioassay confirmed that a bioactive compound was present in the tap water. The compound responsible for the auxinic activity in the tap water was named “solanicide” because of its strong bioactivity against members of the Solanaceae. Source and Distribution of Solanicide. The tap water at the SWU campus is supplied by the water treatment plants on the Jialing River, the source of drinking water for more than half of the inhabitants of Chongqing City, as well as many suburban residents that live along the river. A tobacco bioassay system was used to investigate the distribution of solanicide in the water system of Chongqing City. Young tobacco seedlings (2−3 weeks old) cultured on medium prepared using 10× 11789
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Figure 2. Distribution of solanicide in Jialing River and Yangtze River. (a) Solanicide is sourced from Jialing River (red), which flows into Yangtze River (blue, before inflow of Jialing River; yellow, after inflow of Jialing River) at Chongqing City. Irregular orange patch at top-left corner shows SWU campus. Pink-ringed spots show major sampling sites, and numbers on spots indicate days taken for water samples to induce typical solanicide injury symptoms in tobacco plants. “UD” indicates undetectable solanicide activity within 70 days. Blue dots indicate other sampling sites with solanicide detected, and green ones indicate those with undetectable solanicide. Samples from sites apart from the river were collected from tap water. (b) Main streams of Yangtze River (blue line) and Jialing River (red line). Red spot indicates location of Chongqing City. (c) Location of confluence of Jialing River and Yangtze River. Arrows indicate flow direction of Jialing River (yellow) and Yangtze River (purple). Satellite images were obtained from Google Maps (a and b; https://maps.google.com.hk/) and Baidu Maps (c; http://map.baidu.com/).
carbons at δ60.0−80.0 ppm (Figure S4b, Supporting Information). These data suggested the presence of an aromatic ring, a basic component of natural and synthetic auxins,17 even though signals for quarternary carbons were not detected. From the NMR data, we could speculate that solanicide contains approximately 10 carbons (including undetected quarternary aromatic carbons) and 11 hydrogens. However, the H−C correlation was not able to be determined from the current data, especially that for alkene protons, because no alkene carbon signal was detected in the 13C NMR spectrum (Figure S4b, Supporting Information). The mass spectrum of solanicide was acquired in the m/z 50−3000 range in negative ionization mode. The largest m/z value was 213.9624 (Figure 4). In searches of available chemical databases, no ion observed in the mass spectrum hit any known compounds compatible with the NMR spectrum data. Interestingly, none of the mass peaks showed an attribute of the presence of chlorine (Figure 4; Figure S5, Supporting Information), which generates characteristic isotope patterns in the mass spectrum according to the relative abundances of its natural isotopes.31 Mass spectral data for the chlorinated herbicide 2,4-D (obtained from the National Institute of Standards and Technology, http://webbook.nist. gov) are shown in Figure S5 (Supporting Information) as a reference. This suggested that solanicide is an unchlorinated compound. Taken together, the results of the chemical analyses
Figure 3. Time to effect for tap water collected in 2011, 2012, and 2013 to induce typical injury symptoms (at least three strappy leaves) in tobacco. Error bar = ± SD.
The crystallization of solanicide from water gave slightly yellowish needles (Figure S4a, Supporting Information), but it did not form single crystals. The 1H NMR (600 MHz, D2O) analysis revealed the presence of four aromatic protons at δ8.01 (d, J = 7.8 Hz, 2H) ppm, δ7.58 (td, J = 8.4, 1.2 Hz, 1H) ppm, and δ7.52 (m, 1H) ppm, two alkene protons at δ6.49 (s, 1H) ppm and δ6.94 (s, 1H) ppm, and five protons at δ3.0−4.0 ppm that might be adjacent to electronegative atoms (Figure S4a, Supporting Information). The 13C NMR (600 MHz, D2O) spectrum revealed the presence of four aromatic carbons at δ122.89, δ123.80, δ127.53, and δ127.67 ppm and at least four 11790
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Figure 4. Mass spectrum of solanicide.
indicated that solanicide may be an unknown compound that differs from known chlorinated auxinic herbicides. However, further analyses are required to determine its molecular structure in detail. To achieve this goal, a full chemical analysis is necessary. At present, the main difficulty for further analysis is sample preparation, because a full chemical analysis requires at least 20 mg of purified product. More than 8000 L of water would have to be processed to obtain this quantity. Functional Characterization of Solanicide. Currently, the known compounds that induce symptoms similar to those induced by solanicide in solanaceous plants are chlorinated auxinic herbicides used for weed control.13,16,17,19 2,4-D is the most widely used chlorinated auxinic herbicide,32 which could induce typical symptoms of injury by chlorinated auxinic herbicides.16,32 To reveal the functional similarities and differences between solanicide and other chlorinated auxinic herbicides, the auxinic activity in the tap water was compared with that of 2,4-D by watering (root application) or spraying (foliar application) tobacco plants with tap water or 1 mg/L 2,4-D (in ddH2O). After 1 month of treatment, plants watered with either tap water or 2,4-D solution showed injury symptoms (Figure 5a). Tumorlike callus tissue was present only in the plants irrigated with tap water (Figure 5a). Whereas tobacco plants irrigated with 2,4-D solution formed stout roots, the roots of those irrigated with tap water were normal (Figure 5a). The leaves of plants sprayed with tap water were normal, while those of plants sprayed with 2,4-D solution showed severe symptoms of injury (Figure 5a). These findings showed that solanicide is more active than 2,4-D in causing leaf injury to tobacco via watering (root application) but inactive when applied by foliar spraying. The known chlorinated auxinic herbicides are all active by foliar application and are generally applied in this way to control weed growth.13,19,21 Also, the chlorinated auxinic herbicides cause root injury symptoms in various plants,14 including monocots (Figure S1, Supporting Information), while none of the tap water-irrigated plants showed root symptoms. Thus, solanicide and chlorinated auxinic herbicides likely may have different mechanisms of action. The fact that the tap water was able to induce more severe symptoms of shoot injury than those induced by 1 mg/L 2,4-D solution in tobacco plants (Figure 5a) suggested that
Figure 5. Functional characterization of solanicide. (a) Comparison between auxinic activity of control (ddH2O), tap water, and 1 mg/L 2,4-D (in ddH2O). The red arrow in the top panels shows tumorlike callus tissue, and arrows in middle panels show stout roots. Fifteen plants were tested for each treatment, and the number of plants showing injury symptom in the displayed tissue is given. (b) Comparison of effects of solanicide (1 mg/L) with those of other chlorinated auxinic herbicides (1 mg/L) on tobacco. Insets show enlargements of cotyledon nodes. Red arrows show callus tissue; white arrows show induced lateral root primordial (or lateral roots); red open brackets show the protruded shoot (epicotyl). Thirty plants were tested for each treatment, and the representative image is displayed. 11791
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auxinic herbicide,32 showed that 2,4-D content in the tap water was ∼0.01 mg/L (Figure S6, Supporting Information). This value is below the World Health Organization guidelines for 2,4-D in drinking water (0.03 mg/L).24 Furthermore, no significant seasonal change in solanicide activity in the tap water sourced from the Jialing River was observed (Figure 3). These findings imply that the contaminant is not an agriculturalderived herbicide. We are currently unable to fully solve the chemical structure of solanicide because of its special characteristics and the difficulty in preparing samples. However, our findings show that solanicide is an unchlorinated chemical with a strong auxinic effect. The unique chemical characteristics and bioactivity of solanicide suggest that it differs from the known compounds. Our results demonstrated that solanicide pollution is the cause of malformation of Solanaceae plants in Chongqing City and further revealed that solanicide is sourced from the Jialing River and carried by this river into the Yangtze River, the longest river flowing across the vast land area of China. Thus, a pollution threat to the Yangtze River and massive scale ecosystem in its floodplain is highlighted. The presence of solanicide in tap water in Chongqing City also implies that the current water treatment process (a combination of coagulation, sedimentation, filtration, and disinfection) by water treatment plants is not reliable for removing organic pollutants.
solanicide has very strong bioactivity, as its content in tap water is less than or equal to 0.02 mg/L. To further reveal the functional characteristics of solanicide, we compared the effects of 1 mg/L solanicide (prepared using the purified product) on tobacco seedlings with those of other auxinic compounds including IAA, the unchlorinated synthetic auxin NAA, and the chlorinated auxinic herbicides from all families of growth-regulator herbicides13,15,17,19 (all at a concentration of 1 mg/L). The symptoms caused by 2,4-D and picloram in tobacco were taken as the representative symptoms of injury by chlorinated auxinic herbicides, for comparison with those caused by solanicide. The results showed that the symptoms in tobacco seedlings induced by 1 mg/L solanicide differed from those induced by the other auxinic compounds (Figure 5b). After 2 weeks of treatment, plants treated with solanicide showed curled leaves, protruded shoots, and formation of callus tissue from the cotyledonary node. The unchlorinated auxin IAA and NAA as well as the chlorinated auxin 2,4-D did not induce leaf curling (Figure 5b). Picloram, a fast-acting chlorinated herbicide,13,16 induced leaf curling as rapidly as did solanicide, but also caused the seedlings to wilt (Figure 5b). Also, picloram did not induce extensive callus formation but only several tiny callus initiation spots around the cotyledonary node (Figure 5b). Another obvious difference between solanicide and the other auxinic compounds was that the other auxinic compounds induced formation of stout lateral roots (or callus tissue) in the maturation zone, while solanicide induced callus tissue in taproot apex (Figure 5b). Interestingly, formation of a normal adventitious root from hypocotyl is occasionally observed in solanicide treated tobacco plants as in control (Figure 5b). In terms of symptoms, the most noticeable difference between solanicide and the other auxinic compounds was that solanicide caused the shoot of tobacco seedlings to protrude, whereas the shoots of tobacco seedlings treated with the other auxinic compounds were enclosed in the rosettes (Figure 5b). Solanicide-induced shoot protrusion was also obvious in older seedlings irrigated with tap water (Figure 1d). These findings suggested that solanicide is functionally different from other natural or synthetic auxinic compounds. The roles of solanicide in promoting shoot protrusion and inducing callus tissue in taproot apex imply that it predominantly functions in the apical meristem. Yet, it was less active in the root, as tap water induced injury symptoms in shoots but not in roots (Figure 5a). These findings showed that the function of solanicide is similar to but distinct from other chlorinated auxinic compounds. In this study, we determined that the injury symptoms in greenhouse-grown solanaceous plants in Chongqing City were caused by the tap water used for irrigation. Plant injury by auxinic compounds is generally observed in areas where there has been excessive application of chlorinated auxinic herbicides.13,14 This finding is unusual in that the presence of high auxinic activity in the water system, especially drinking water, is a rare occurrence, even though there are detectable levels of herbicides in some rivers.33 Therefore, it ever raised considerable concerns about water contamination by commercial chlorinated auxinic herbicides that were applied seasonally by farmers, for the auxinic activity of the polluted water was somewhat similar to this kind of compounds. The presence of chlorinated auxinic herbicides in drinking water is strictly monitored because of their potential toxity.22−26 Analyses of tap water using an enzyme-linked immunosorbent assay (ELISA) kit to detect 2,4-D, the most widely used chlorinated
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ASSOCIATED CONTENT
* Supporting Information S
Growth condition for other plants, method for ELISA assay, supporting data for plant injury symptoms from solanicide or other auxinic herbicides, phenotype of tobacco plants grown with rainwater, tap water induced tumorlike callus tissue in tobacco, crystals and NMR spectra of solanicide, mass spectrum of 2,4-D, and 2,4-D content in tap water. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-23-6825-0744; fax: +86-23-6825-1264; e-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to B. Lei at Guizhou Tobacco Research Institute, and Q. Guo and Q. Luo at the School of Chemistry and Chemical Engineering at SWU for the technical assistant. We also thank the SWU scientists whose work on Solanaceae plants provided help to this study. The authors were supported by the Fundamental Research Funds for the Central Universities of China (XDJK2012C084; 2362014xk09), the “111” Project (B12006), China National Tobacco Corporation (110201301005(JY-05)), State Education Ministry (SRF for ROCS), and the Doctoral Fund of Southwest University (SWU110053).
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REFERENCES
(1) Qiu, J. China faces up to groundwater crisis. Nature 2010, 466 (7304), 308. (2) Rodriguez-Lado, L.; Sun, G.; Berg, M.; Zhang, Q.; Xue, H.; Zheng, Q.; Johnson, C. A. Groundwater arsenic contamination throughout China. Science 2013, 341 (6148), 866−868.
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(3) Qiu, J. China to spend billions cleaning up groundwater. Science 2011, 334 (6057), 745. (4) Sharma, B. M.; Bharat, G. K.; Tayal, S.; Nizzetto, L.; Cupr, P.; Larssen, T. Environment and human exposure to persistent organic pollutants (POPs) in India: A systematic review of recent and historical data. Environ. Int. 2014, 66C, 48−64. (5) Wong, M. H.; Leung, A. O.; Chan, J. K.; Choi, M. P. A review on the usage of POP pesticides in China, with emphasis on DDT loadings in human milk. Chemosphere 2005, 60 (6), 740−752. (6) Randall, C.; Hock, W.; Crow, E.; Hudak-Wise, C.; Kasai, J. National Pesticide Applicator Certification - Core Manual; National Association of State Departments of Agriculture Research Foundation: Washington, DC, 2006. (7) Ritter, L.; Solomon, K.; Sibley, P.; Hall, K.; Keen, P.; Mattu, G.; Linton, B. Sources, pathways, and relative risks of contaminants in surface water and groundwater: A perspective prepared for the Walkerton inquiry. J. Toxicol. Environ. Health, Part A 2002, 65 (1), 1− 142. (8) Shukla, G.; Kumar, A.; Bhanti, M.; Joseph, P. E.; Taneja, A. Organochlorine pesticide contamination of ground water in the city of Hyderabad. Environ. Int. 2006, 32 (2), 244. (9) Kiely, T.; Donaldson, D.; Grube, A. Pesticides Industry Sales and Usage: 2000 and 2001 Market Estimates; United States EPA: Washington, DC, 2004. (10) Grube, A.; Donaldson, D.; Kiely, T.; Wu, L. Pesticides Industry Sales and Usage: 2006 and 2007 Market Estimates; United States EPA: Washington, DC, 2011. (11) Wall, D. A. Potato (Solanum-Tuberosum) response to simulated drift of dicamba, clopyralid and tribenuron. Weed Sci. 1994, 42 (1), 110−114. (12) Al-Khatib, K.; Peterson, D. Soybean (Glycine max) response to simulated drift from selected sulfonylurea herbicides, dicamba, glyphosate, and glufosinate. Weed Technol. 1999, 13 (2), 264−270. (13) Israel, T. D.; Rhodes, G. N.; Denton, P. Diagnosing Suspected Off-Target Herbicide Damage to Tobacco, W 290-B; University of Tennessee Extension: Knoxville, TN, 2012. (14) Bradley, K. W.; Johnson, B.; Smeda, R.; Boerboom, C. Practical Weed Science for the Field Scout - Corn and Soybean; University of Missouri Extension: Columbia, MO, 2009; Vol. IPM1007, pp 12−14. (15) Mallory-Smith, C. A.; Retzinger, E. J. Revised Classification of Herbicides by Site of Action for Weed Resistance Management Strategies. Weed Technology 2003, 17 (3), 605. (16) Gunsolus, J. L.; Curran, W. S. Herbicide Mode of Action and Injury Symptoms; BU-03832; University of Minnesota Extension Service: St. Paul, MN, 1999. (17) Grossmann, K. Mode of action of auxin herbicides: A new ending to a long, drawn out story. Trends Plant Sci. 2000, 5 (12), 506− 508. (18) Mithila, J.; Hall, J. C.; Johnson, W. G.; Kelley, K. B.; Riechers, D. E. Evolution of resistance to auxinic herbicides: Historical perspectives, mechanisms of resistance, and implications for broadleaf weed management in agronomic crops. Weed Sci. 2011, 59 (4), 445. (19) Sterling, T. M.; Hall, J. C. Mechanism of action of natural auxins and the auxinic herbicides. In Herbicide Activity: Toxicology, Biochemistry and Molecular Biology; Roe, R. M., Burton, J. D., Kuhr, R. J., Eds. IOS Press: Amsterdam, The Netherlands, 1997; pp 111− 141. (20) Ashton, F. M.; Crafts, A. S. Phenoxys. In Mode of Action of Herbicides; Ashton, F. M., Crafts, A. S., Eds.; John Wiley and Sons: Toronto, Canada, 1981; pp 272−302. (21) Kay, S. H. Response of two alligatorweed biotypes to quinclorac. J. Aquat. Plant Manage. 1992, 30, 35−40. (22) Kogevinas, M.; Becher, H.; Benn, T.; Bertazzi, P. A.; Boffetta, P.; Bueno-de-Mesquita, H. B.; Coggon, D.; Colin, D.; Flesch-Janys, D.; Fingerhut, M.; Green, L.; Kauppinen, T.; Littorin, M.; Lynge, E.; Mathews, J. D.; Neuberger, M.; Pearce, N.; Saracci, R. Cancer mortality in workers exposed to phenoxy herbicides, chlorophenols, and dioxins. An expanded and updated international cohort study. Am. J. Epidemiol. 1997, 145 (12), 1061−1075.
(23) World Health Organization. Dioxins and Their Effects on Human Health; fact sheet no. 225; WHO: Geneva, Switzerland, 2010. (24) World Health Organization. Guidelines for Drinking-Water Quality, 4th ed.; WHO: Geneva, Switzerland, 2011; p 347. (25) Dad, N. K.; Tripathi, P. S. Acute toxicity of herbicides to freshwater fish and midge larvae, Chironomus tentans. Environ. Int. 1980, 4 (5−6), 435−437. (26) Alpöz, A. R.; Tosun, N.; Eronat, C.; Delen, N.; Şen, B. H. Effects of 2,4-dichlorophenoxy acetic acid dimethyl amine salt on dental hard tissue formation in rats. Environ. Int. 2001, 26 (3), 137. (27) Murashige, T.; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962, 15 (3), 473. (28) Yang, H.; Xie, P.; Ni, L.; Flower, R. J. Pollution in the Yangtze. Science 2012, 337 (6093), 410. (29) Wong, C. M.; Williams, C. E.; Pittock, J.; Collier, U.; Schelle, P. World’s Top 10 Rivers at Risk; WWF International: Gland, Switzerland, 2007. (30) Gfrerer, M.; Martens, D.; Gawlik, B. M.; Wenzl, T.; Zhang, A.; Quan, X.; Sun, C.; Chen, J.; Platzer, B.; Lankmayr, E.; Kettrup, A. Triazines in the aquatic systems of the Eastern Chinese Rivers Liao-He and Yangtse. Chemosphere 2002, 47 (4), 455−466. (31) Crews, P.; Rodriguez, J.; Jaspars, M. Organic Structure Analysis; Oxford University Press: New York, 1998; pp 257−257. (32) Environmental Protection Agency. U.S. Pesticides: Reregistration 2,4-D RED Facts, EPA-738-F-05-002; EPA: Washington, DC, 2005. (33) Villeneuve, A.; Larroudé, S.; Humbert, J. F. Herbicide contamination of freshwater ecosystems: Impact on microbial communities. In Pesticides - Formulations, Effects, Fate; Stoytcheva, M., Ed.; InTech: Rijeka, Croatia, 2011; pp 285−312.
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dx.doi.org/10.1021/es501502y | Environ. Sci. Technol. 2014, 48, 11787−11793
