Propylene glycol vapor contamination in controlled environment growth chambers: toxicity to corn and soybean plants

A major, often unrecognized variable regulating plant growth in semi-closed environment is air contaminant. The vapor of propylene glycol (PG), which was used as coolant in growth chambers, has been found to be toxic to corn (Zea mays L.) and soybean (Glycine max L.) plants. PG solution leaked from a valve packing system and volatilized to vapor, which was trapped in a semi-closed growth chamber. Symptoms of leaf edge chlorosis, later developing into necrosis, were observed on the third day of emergence or on the third day after moving healthy plants into the chamber. For young soybean plants, symptoms were slightly different from those observed in corn plants; the chlorosis symptoms were not uniformly distributed on all leaves. Some soybean leaves curled up and others had white spots. This problem was identified by using a portable photoionization detector to obtain instantaneous readings of total volatile organic compound concentrations inside the chambers. The presence of PG in selected chambers was verified using sample collection with solid phase microextraction (SPME) followed by analysis with multi-dimensional gas chromatography mass spectrometry (MD-GC-MS). This information is pertinent to researchers who use controlled environment to grow plants.     

Manganese toxicity as indicated by visible foliar symptoms of Japanese white birch (Betula platyphylla var. japonica)

For the purpose of a field diagnosis of Mn toxicity, we showed the possibility of using visible foliar symptoms of Japanese white birch (Betula platyphylla var. japonica Hara) as indicator. To examine the relationship between the expression of visible symptoms and leaf Mn concentrations, white birch seedlings were grown under four different Mn levels: 1 mg Mn l-1 as control, 10, 50 and 100 mg Mn l-1. Foliar symptoms of Mn toxicity for white birch were: (1) chlorosis at entire young leaves in the 50 and 100 mg Mn l-1 treatments; and (2) brown speckles at the leaf marginal and interveinal area for old leaves in the treatments greater than 1 mg Mn l-1. Mn preferably accumulated into the leaf marginal and interveinal area, where the brown speckles were observed. The mechanism determining the expression of symptoms seems to be associated with the physiological state related to leaf age as well as Mn distribution and concentration within a leaf.     

Influence of Sulfur Dioxide and Hydrogen Fluoride as a Mix or Reciprocal Exposure on Citrus Growth and Development

The influence of exposure to mixtures of SO₂ and HF on Koethen sweet orange and mixtures and alternate exposure to these gases on Satsuma mandarin were tested using a rotating fumigation greenhouse. Effects of HF-SO₂ mixtures on linear growth and leaf area of Koethen orange were additive, not synergistic. No necrosis was observed on Koethen oranges exposed to HF, SO₂, or a mixture of HF and SO₂. Effects of the mixture on chlorosis of Satsuma mandarin foliage was also not synergistic. No significant difference in linear growth of Satsuma mandarin was found among all treatments. Alternate exposure to SO₂ followed by HF produced no synergistic injury to Satsuma mandarin. Satsuma mandarin appeared more sensitive than Koethen orange to HF, SO₂, and mixtures of these two gases using degree of chlorosis and leaf abscission as the criteria of sensitivity. If iinear growth and leaf area were the principal criteria considered, Koethen orange would appear more sensitive.     

Chronic effects of vapour phase di-n-butyl phthalate (DBP) on six plant species

A fumigation experiment was performed in which six plant species representing the European flora were exposed to a range of DBP concentrations. Controlled amounts of DBP-saturated air were injected into the ingoing air-streams of plant fumigation chambers, maintaining constant concentrations there for a period of up to 76 days. The target concentrations were a control, 0.8, 1.5, 3.5, and 10.0 microg m(-3). The variation in sensitivity between plant species to atmospheric DBP was quantified on the basis of whole plant biomass in order to derive no-observed-effect-concentrations (NOECs). Significant dose-response relationships, based on realised concentrations, were thus derived using non-linear regression, resulting in NOECs of 0.51 microg m(-3) for Trifolium repens, 0.96 microg m(-3) for Brassica campestris, 1.87 microg m(-3) for Phaseolus vulgaris and 2.21 microg m(-3) for Plantago major. A significant effect was also observed for Holcus lanatus at 12.4 microg m(-3) DBP, but due to the variation at lower levels of DBP exposure, no dose-response relationship could be derived. No significant effect on growth of current year needles in Picea abies was observed, even at the highest level of DBP, 13.7 microg m(-3). Based on statistical extrapolation according to Aldenberg and Slob [Ecotox. Environ. Safety, 25 (1993) 48], an overall predicted no-effect concentration (PNEC) for the plant-atmosphere compartment of 0.33 microg m(-3) DBP was calculated. The PNEC was calculated using the mean and standard deviation of the NOEC for four of the tested species and an extrapolation factor. In addition to changes in leaf colour, leaf crinkling and growth reduction, a number of not quantified observations are described, indicating that DBP affects the physiology as well as the morphology of these species.     

Excess copper induced physiological and proteomic changes in germinating rice seeds

Copper is an essential micronutrient for plants. Present at a high concentration in soil, copper is also regarded as a major toxicant to plant cells due to its potential inhibitory effects against many physiological and biochemical processes. The interference of germination-related proteins by heavy metals has not been well documented at the proteomic level. In the current study, physiological, biochemical and proteomic changes of germinating rice seeds were investigated under copper stress. Germination rate, shoot elongation, plant biomass, and water content were decreased, whereas accumulation of copper and TBARS content in seeds were increased significantly with increasing copper concentrations from 0.2mM to 1.5mM followed by germination. The SDS-PAGE showed the preliminary changes in the polypeptides patterns under copper stress. Protein profiles analyzed by two-dimensional electrophoresis (2-DE) revealed that 25 protein spots were differentially expressed in copper-treated samples. Among them, 18 protein spots were up-regulated and 7 protein spots were down-regulated. These differentially displayed proteins were identified by MALDI-TOF mass spectrometry. The up-regulation of some antioxidant and stress-related proteins such as glyoxalase I, peroxiredoxin, aldose reductase, and some regulatory proteins such as DnaK-type molecular chaperone, UlpI protease, and receptor-like kinase clearly indicated that excess copper generates oxidative stress that might be disruptive to other important metabolic processes. Moreover, down-regulation of key metabolic enzymes like alpha-amylase or enolase revealed that the inhibition of seed germinations after exposure to excess copper not only affects starvation in water uptake by seeds but also results in failure in the reserve mobilization processes. These results indicate a good correlation between the physiological and biochemical changes in germinating rice seeds exposed to excess copper.     

Studies of mobility of di-iso-butyl phthalate (DiBP), di-N-butyl phthalate (DBP), and di-(2-ethyl hexyl) phthalate (DEHP) by plant foliage treatment in a closed terrestrial simulation chamber

A climate chamber was constructed for model studies of mobility and effects of chemical substances within simplified terrestrial systems. The chamber functions and its performance were tested by foliar applications of phthalates on higher plants. A low elimination rate from foliage was observed for DEHP. More than 95% of DiBP and DBP were eliminated within 15 days. 1.5 μg cm⁻² of DBP caused chlorosis on the leaves of Sinapis alba L.     

California black oak response to nitrogen amendment at a high O3, nitrogen-saturated site

In a nitrogen (N) saturated forest downwind from Los Angeles, California, the cumulative response to long-term background-N and N-amendment on black oak (Quercus kelloggii) was described in a below-average and average precipitation year. Monthly measurements of leaf and branch growth, gas exchange, and canopy health attributes were conducted. The effects of both pollutant exposure and drought stress were complex due to whole tree and leaf level responses, and shade versus full sun leaf responses. N-amended trees had lower late summer carbon (C) gain and greater foliar chlorosis in the drought year. Leaf water use efficiency was lower in N-amended trees in midsummer of the average precipitation year, and there was evidence of poor stomatal control in full sun. In shade, N-amendment enhanced stomatal control. Small differences in instantaneous C uptake in full sun, lower foliar respiration, and greater C gain in low light contributed to the greater aboveground growth observed.     

Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

The interaction of SO2 and root-knot nematode on tomato

Intermittent exposure of tomato plants (cv. Pusa Ruby) to SO(2) at 286 microg m(-3) (3 h every heavy third day for 75 days) induced slight chlorosis of leaves. At 571 microg m(-3), considerable chlorosis with browning developed on the foliage. These symptoms were more pronounced and appeared earlier on SO(2)-exposed plants infected with Meloidogyne incognita race 1 (Mi), especially in post- and concomitant-inoculation exposures. Mi and/or SO(2) significantly reduced different parameters of plant growth. Synergistic (positive) interactions between SO(2) and Mi occurred in concomitant- and post-inoculation exposures at 286 and 571 microg m(-3), respectively. In other treatments, an antagonistic (negative) interaction was observed. However, in a few cases, additive effects of SO(2) and Mi were also recorded. Intensity of root-knot (galling) was enhanced at both concentrations of SO(2), while reproduction (egg mass production) of Mi was enhanced in concomitant-inoculation exposures at 286 microg m(-3) and inhibited at 571 micro m(-3). Exposure to SO(2) and/or Mi decreased the number and size of stomata but increased the number and length of trichomes on both the leaf surfaces. Stomatal aperture was significantly wider in the plants exposed to 571 microg SO(2) m(-3) alone and in pre-, post-, and concomitant-inoculation exposures at 286 or 571 microg m(-3). Stomatal aperture was directly related to foliar injury and reductions in growth, yield, and leaf pigments.     

Boron toxicity in the rare serpentine plant, Streptanthus morrisonii

The release of boron-laden mist from the cooling towers of some geothermal power stations in northern California potentially threatens nearby populations of the rare serpentine plant, Streptanthus morrisonii F. W. Hoffm. To assess the tolerance of S. morrisonii to high levels of boron, the effect of boron on leaf condition, life history, germination rate, growth rate, allocation and photosynthesis was measured on plants grown in a greenhouse. Relative to other species, S. morrisonii was tolerant of excess boron. On serpentine soil, mild to moderate toxicity symptoms (older leaves exhibiting chlorosis and necrosis, but few leaves killed) were apparent when the boron concentration in applied nutrient solutions was 240-650 microm. Severe toxicity symptoms (significant leaf loss, young leaves with toxicity symptoms) were apparent when the applied solution was over 1000 microm boron. Above 1000 microm boron, S. morrisonii appeared unable to complete its life cycle. On a tissue basis, boron toxicity was first observed when leaf boron content was 40-90 micromol g(-1) dry weight. In leaves with severe boron toxicity (> 35% injury), the boron content was generally above 130 micromol g(-1) dry weight. These levels were an order of magnitude above the tissue boron content of plants in the field. Prior to the onset of pronounced boron toxicity symptoms, growth rate, allocation patterns, and photosynthesis were unaffected by high boron. These results indicate that inhibition of growth and photosynthesis occurred because of a loss of viable tissue due to boron injury, rather than a progressive decline as leaf boron levels increased.     

Seasonal development of ozone-induced foliar injury on tall milkweed (Asclepias exaltata) in Great Smoky Mountains National Park

The goals of this study were to document the development of ozone-induced foliar injury, on a leaf-by-leaf basis, and to develop ozone exposure relationships for leaf cohorts and individual tall milkweeds (Asclepias exaltata L.) in Great Smoky Mountains National Park. Plants were classified as either ozone-sensitive or insensitive based on the amount of foliar injury. Sensitive plants developed injury earlier in the season and to a greater extent than insensitive plants. Older leaf cohorts were more likely to belong to high injury classes by the end of each of the two growing seasons. In addition, leaf loss was more likely for older cohorts (2000) and lower leaf positions (2001) than younger cohorts and upper leaves, respectively. Most leaves abscised without prior ozone-like stippling or chlorosis. Failure to take this into account can result in underestimation of the effects of ozone on these plants.     

Discharge Electrodes and Electrostatic Precipitators

Controlled fumigation experiments were conducted to determine the dose-response relationships for four species of urban trees exposed to sulfur dioxide. The species chosen were ginkgo, Norway maple, pin oak, and Chinese elm.

Results indicated that resistance to SO₂ increased among the species in the following order: Chinese elm, Norway maple, ginkgo, pin oak. Elm showed almost 100% leaf necrosis at exposures over 2 ppm for 6 hr, and severe chlorosis and necrosis at 0.25 ppm for 30 days. Fifty per cent leaf necrosis occurred on Norway maple at 3 ppm for 6 hr, and on ginkgo at 4 ppm for 6 hr, and both species developed moderate marginal chlorosis at 0.50 ppm for 30 days. Injury on pin oak was minor, even at 8 ppm for 8 hr, but at 0.50 ppm for 30 days, a slight overall chlorosis developed on the leaves.

The relative susceptibilities of the four species were the same in the long-term as in the short-term exposures. The shapes of the dose-response surfaces indicated that duration of exposure and concentration of the pollutant were of equal importance in producing injury on Chinese elm and probably on pin oak, but on Norway maple and ginkgo, concentration of SO₂ was of greater importance than the duration of exposure.     

Response to ambient ozone of two white clover (Trifolium repens L.cv. "Regal") clones, one resistant and one sensitive, grown in a Mediterranean environment

Two clones of white clover (Trifolium repens L.) differing in ozone tolerance were grown in southern Italy during 1997 and 1998 to study the effects of ambient ozone exposure on yield, leaf morphology and water use. Ambient ozone levels were high in both years with values exceeding the threshold for leaf injury reported in the literature. In both years ozone injury was observed on the sensitive clone (NC-S) but not on the resistant one (NC-R), and leaf and stolon dry matter production was significantly lower in NC-S than in NC-R. However, it cannot be excluded that other factors, such as high temperature, interacted with the effect of ozone on biomass production. The clones differed in morphological characteristics. Lower total leaf area in NC-S plants was due to a smaller number of leaves per plant, but the average area per leaf was higher in NC-S. Specific leaf weight and net assimilation rate were higher in the more productive clone (NC-R). Cumulative plant water use was higher in NC-R in each growth period because of the larger leaf area; by contrast, water use per unit leaf area was higher in NC-S, indicating higher leaf conductance to water vapour. The results suggest that ozone significantly reduces the yield of sensitive white clover plants under well-watered conditions, and that the difference in ozone tolerance between clover clones is related to differences in leaf morphology and water use.     

Bioremediation of environmental endocrine disruptor di-n-butyl phthalate ester by Rhodococcus ruber

In this study DBP-degradation strain CQ0301 was isolated from rubbish landfill soil. According to the biophysical, biochemical characteristics and analysis of 16S rRNA, the strain was identified as Rhodococcus ruber. Three new protein bands could be fractioned after DBP-inducing, which were suspected to participate the process of DBP-degrading. Catechol was suspected to be an intermediate product of DBP and cleaving the benzene ring was catalyzed by catechol 1,2-dioxygenase, because a highly activity of catechol 1,2-dioxygenase could be detected after DBP-inducing. The results of this study also showed the optimal pH value, optimal temperature which influenced the degradation rate in soil: pH 7.0-8.0, 30-35 degrees C. Kinetics of degradation reaction had been performed at different initial concentration and different time. Analyzed with SPSS10.0 software, the DBP degradation can be described as the same exponential model when the initial DBP concentration was lower than 50 mg/kg. The kinetics equation was lnC=-0.1332t + A, with the degradation half-life of DBP in soil (5.20 d). Inoculating CQ0301 could relieve DBP content in plant. We also found that adding nutrient materials into soil was useful for decreasing the DBP content in plant. In summary, we isolated a bacterium capable of degrading DBP and decreasing DBP content in plant. We also explored the mechanism of biodegradation and characterized the environmental factors influencing the degradation process in contaminated soil. Based on this work, we hope that these findings can provide some information for applying of bioremediation of DBP contaminated soil.     

Proteomic analysis of chromate-induced modifications in Pseudokirchneriella subcapitata

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1 ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis.The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.     

Gonad differential proteins revealed with proteomics in oyster (Saccostrea cucullata) using alga as food contaminated with cadmium

As mercury and lead, cadmium (Cd) is one of the highly toxic metals in both the ocean and land environments, but its toxicological mechanism in organisms including human is still unclear because of the complex toxicological pathways in vivo. Here, the alga Chlorella vulgaris were cultivated at room temperature under the stress of cadmium (1 mg L⁻¹) to obtain a toxic food, and then the contaminated food were directly supplied to oyster (Saccostrea cucullata) in seawater. After feeding with C. vulgaris contaminated with Cd (C. vulgaris-Cd), the differential proteins in the oyster gonad (OG) were effectively separated and identified with proteomic approaches. Eleven protein spots were observed to be significantly changed in the OG feeding with C. vulgaris-Cd, which seven spots of these differential proteins were down-regulated while four spots were up-regulated. These altered spots were further excised in gels and identified by a combined technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and database searching. A portion of these differential proteins were further proofed by real-time PCR and Western blotting. The results indicate that the major functions of these differential proteins were described as follows: binding, protein translocation, catalysis, regulation of energy metabolism, reproductive function and skeleton structure. These differential proteins in part may effectively provide a few novel biomarkers for the evaluation of Cd pollution level via a food pathway for harming halobios, mammal and human health, and for understanding the complex mechanisms of Cd toxicity in vivo.     

Degradation of di-butyl-phthalate by soil bacteria

Twelve Gram-positive phthalate ester degraders were isolated from soil. Using Biolog GP2 plates, eight of them were identified as belonging to the Corynebacterium-Mycobacterium-Nocardia group, while the remaining four were unidentifiable. When cultured in the presence of di-butyl-phthalate (DBP) in basal salts solution, five of these isolates accomplished more than 90% of DBP degradation within 48 h (fast group), three were placed in the medium group, and the remaining four were placed in the slow group which caused less than 30% of DBP degradation within the same period of time. A 420 bp DNA fragment was amplified from six isolates and none of them fell within the slow group. When compared with the large subunit of phthalate dioxygenase gene (phtA) of Arthrobacter keyseri, 83% and 91% similarities were evident in the nucleotide and amino acid sequences, respectively. However, no correlation between cell surface hydrophobicity and phthalate degradation ability was evident. Six surfactants (Brij 30, Brij 35, Tergitoltype NP-10, Triton N-101, Triton X-100 and SDS) were tested for their abilities to increase degradation rate. When added at the critical micellar concentration (CMC), they all displayed strong growth inhibition against the three bacteria tested, with Brij 30 been the least toxic to isolates G2 and G11, and Brij 35 had the least inhibitory effect for G1. When half the CMC of Brij 30 was incorporated into the basal salts, the inhibitory effect on DBP degradation remained. Soil helped to minimize surfactant toxicity of surfactant and increase the degradation potential of some of the test bacteria. When DBP-amended soil had been aged for three months, decreases in bioavailability were observed but the effect varied tremendously between different organisms. For isolates G1, G2, G5, G7 and G17 the aging effects were almost non-exist. The present study indicates that selection of a suitable degrader may minimize the undesired effect of aging on bioremediation process.     

Schedule of Technical Sessions

Peroxyacyl nitrates are a family of compounds which result from photochemical reactions between contaminants released to the atmosphere by combustion of organic fuels. Peroxyacetyl nitrate (PAN) is the most abundant member of this family and is responsible for serious plant injury in some polluted areas. Plant injury has been recognized in at least 19 states and several foreign countries but economic loss has caused greatest concern in California. Characteristically, injury from PAN appears as glazing or bronzing of the lower leaf surface but the symptom syndrome may also include tissue collapse, chlorosis, and leaf drop. PAN inhibits activity of several enzyme systems by attacking the sulfhydryl group when enzyme extracts are fumigated in vitro, PAN may produce visible symptoms when sensitive plants are exposed for 4 hours to about 14 ppb. Other chemical members of the family are more toxic than PAN. Concentrations of 20 to 30 ppb PAN occur frequently at Riverside, Calif, and a maximum peak of 58 ppb was measured for one 2-hour period. Peaks as high as 54 ppb were reported at Salt Lake City, and 210 ppb at Los Angeles, Calif.