Root Elongation Bioassays Research Articles

Soil remediation approach and bacterial community structure in a long-term contaminated soil by a mining spill (Aznalcóllar, Spain)

The Aznalcóllar accident, which occurred in 1998, spilled 36 × 105 m3 of pyritic sludge and 9 × 105 m3 of acidic water around an area of 43 km2 in the south of Spain. This spill is considered one of the most important metal-mining associated accidents worldwide. In this study, two soil remediation techniques were evaluated: the addition of marble sludge (liming treatment, LS) and the mixing of recovered soils (RC) with contaminated soils (CT) (biopile treatment, BS). Both LS and BS significantly reduce the solubility of Cu, Zn, As, and Pb mainly due to the increase in pH and organic matter content, respectively. Soil basal respiration rate and the seed germination and root elongation bioassay with Lactuca sativa were used to evaluate the toxicity of the potential pollution in the sampled soils. Both bioassays showed that the CT soils exhibited the highest toxicity with a significant reduction in the toxicity of the amended soils (LS and BS). The abundance and structure of microbial communities in the soils were determined by qPCR and Illumina 16S rRNA sequencing, respectively. The absolute abundances of total bacterial and archaeal populations, ammonium oxidising bacteria, and denitrifiers in the CT soils were statistically lower than these found in the other three soils. Similarly, the structure of the bacterial community was highly different in the CT soils. Our results underline the persistence of the detrimental effect of pollutants in CT soils compared to the recuperated (RC) and amended soils (LS and BS). We also highlight the uses of liming or biopile as remediation techniques as satisfactory tools to reduce the impact of heavy metals in the contaminated Aznalcóllar soils.

Effect of Repeated Application of Sulfonylurea Herbicides on Sulfosulfuron Dissipation Rate in Soil

Accelerated microbial degradation following previous repeated applications of the same pesticide, or another pesticide of a similar chemical structure, is a known phenomenon. Currently there is limited information regarding accelerated degradation of sulfonylurea (SU) herbicides. This study is aimed to evaluate the effect of repeated SU applications on the degradation rate of the SU herbicide sulfosulfuron in soil. The effect of repeated applications of sulfosulfuron on its degradation was assessed in two soils, using a sorghum root elongation bioassay. The effect of consecutive applications of sulfonylurea herbicides over the course of three to four seasons was further examined in controlled environment and a field study. Degradation of sulfosulfuron was determined following its application to soil samples from the field or a controlled environment, by measuring sulfosulfuron residues using liquid chromatography-tandem mass spectrometry. Following the repeated application of sulfosulfuron in the bioassay, the time to reduce sorghum root growth by 50% was shortened by up to 31.6%. However, consecutive application of SUs in the controlled environment had no effect on sulfosulfuron degradation rate constant. Yet, sulfosulfuron degradation rate was enhanced by a factor of 1.35 following consecutive application of SUs in the field, compared to untreated control soil. The data confirm that sulfosulfuron degradation could be enhanced due to repeated sulfosulfuron applications, thus potentially reducing its herbicidal efficacy.

Alteration of mixture toxicity in nonferrous metal mine tailings treated by biochar

Nonferrous metal mining activities produce enormous amounts of tailings that contain high concentrations of toxic chemicals threatening human health and the environment. This risk could be alleviated using remediation agents such as biochar, as proposed by others. However, contradictory evidence indicates that biochar can increase or sometimes decrease bioavailable concentrations depending on the selection of metal(loid)s in mine tailings. Here three biochars derived from different raw stocks were used to treat mine tailings samples. Chemical analyses indicated that all biochars favored the stabilization of Cd, Cr, Cu, Pb, and Zn, as well as the mobilization of As and Sb. The barley root elongation bioassay showed that the tailings toxicity was only partially diminished (up to 55.8%) or even elevated (up to 20.7%) by biochar treatment. Similar results were also observed from microbial enzyme assays (increased up to 28.3% or decreased up to 24.0%). Further analyses showed that these toxic effects correlated well with the relative toxicity index (R2 = 0.66 to 0.88). Toxicity testing coupled with the use of a toxicity prediction model presented here suggested that the release of As and Sb from tailings compromised the favorable effects of biochar treatment on toxic cationic metals. Such information is of paramount importance when taking countermeasures for improving bioremediation technologies.

Influences of soil properties and long-time aging on phytotoxicity of antimony to barley root elongation

Antimony (Sb) is a toxic element of global concern. To date, the most previous researches about phytotoxicity of Sb failed to fully consider the effects of soil properties and long-time aging. To address this, the toxicity of exogenous Sb(III) and Sb(V) were studied using the standardized barley root elongation bioassay. The results indicated that in ten soils aged only for 1 d, the EC10 (concentrations causing 10% inhibition) values were 221–3164 mg kg−1 and 135–4260 mg kg−1 in Sb(III)- and Sb(V)-treated soils, respectively. The EC50 values (concentrations causing 50% inhibition) were more than the setting highest concentration of 6400 mg kg−1 in half of ten soils. The regression analysis showed that the amorphous Fe oxide and pH were the most foremost single soil factor explaining above-mentioned variance in EC10, respectively, which suggested that the dominant soil factors were related to Sb forms. The inclusion of amorphous Mn oxide in above these two simple regression model could best explain the toxicity variance. After aged for 116 and 365 d, the phytotoxicity of Sb in Sb-treated soils significantly decreased and the phytotoxicity were even not found in the majority of Sb(V)-treated soils. The extent of aging varied with soils, and correlation analysis indicated that the aging effects negatively correlated with soil pH and positively correlated with clay and amorphous Al oxide in the Sb(III)-treated test soils.

Milli-channel array for direct and quick reading of root elongation bioassays

A novel platform to perform systematic analysis and direct reading of root elongation bioassays is presented. The device was designed to include multiplexed microenvironments for the germination and growth of individual seeds, which allows observation by the naked eye or by optical systems, notably cellphone cameras. Prototypes were fabricated by laser micromachining on a highly transparent material that is fully compatible with biological systems. The effectiveness of the milli-channel array was verified against the conventional system (Petri dish). Lactuca sativa was chosen as a model species and glyphosate as a typical toxic agent. All tests were run according to standardized procedures and data analysis was carried out through different statistical indicators such as the root elongation and germination indexes. Results attained in the milli-channel array were identical to those in Petri dish, with the remarkable benefit that several steps required in the conventional system were avoided, which enormously decreases the operation time and the possibility of experimental errors. Further advantages of the milli-channel array are also reported, such as the capability to achieve live imaging of plant organs growth through a simple experiment. The developed device has been proven to be effective, versatile, easy-to-use, and integrable to cellphones, which naturally provide facilities for data recording, analysis, and networking. These improvements open the route to novel applications of bioassays in the wide field of ecotoxicology and environmental studies.

Heavy metal content and toxicity of mine and quarry soils

Soils formed in metallic mines and serpentinite quarries, among other unfavourable features, have high levels of heavy metals. They can release into the environment causing surface and subsurface water contamination, uptake by plants, their accumulation in the food chain and adverse effects on living organisms. In this work, we studied the magnitude of the soils’ toxic effects not only on spontaneous plants but also on two species with phytoremediation potential. Several soils from two different exploitations were selected: a lead and zinc mine and a serpentinite quarry. Soils were characterized, and the pseudo-total and extractable contents of Co, Cr and Ni in soils from a serpentinite quarry were determined. The Cd, Pb and Zn pseudo-total and extractable contents were determined in soils developed in the Pb/Zn abandoned mine. Using a biotest, the chronic toxicity of the soil samples on higher plants was determined. Festuca ovina L., Cytisus scoparius (L.) Link., Sinapis alba L. and Brassica juncea L. were selected, the first two because they are spontaneous plants in the study areas and the last two because they have heavy metal phytoremediation potential. Pseudo-total contents of Co, Cr and Ni in the serpentinite quarry soils and of Zn, Pb and Cd in the Zn/Pb mine soils exceed generic reference levels. CaCl2 is the reactant that extracts the highest proportion of Co, Cr and Ni in the quarry soils and EDTA the largest proportion of Pb Zn and Cd content in the mine soils. The germination index values based on seed germination and root elongation bioassays revealed increasing plant sensitivity to the mine soils in the following order: B. juncea < S. alba < F. ovina < C. scoparius. The wide range of GI values indicates that the response of test plants to soil heavy metals depended on their concentrations and soil characteristics, especially pH and organic matter content. The pollution index indicates severe Cd, Pb and Zn contamination in the mine soils, as well as high Cr and Ni and moderate Co contamination in the serpentinite quarry soils. The performed biotests were suitable for identifying toxic soils and showed that the studied soils are toxic to the spontaneous plants, more to C. scoparius than to F. ovina. They also indicate that the mine soils are more toxic than the quarry soils for both species.

The sensitivity of an hydroponic lettuce root elongation bioassay to metals, phenol and wastewaters

The root elongation bioassay is one of the most straightforward test methods used for environmental monitoring in terms of simplicity, rapidity and economy since it merely requires filter paper, distilled water and Petri dishes. However, filter paper as a support material is known to be problematic as it can reduce the sensitivity of the test.The newly developed hydroponic method reported here differs from the conventional root elongation method (US EPA filter paper method) in that no support material is used and the exposure time is shorter (48h in this test versus 120h in the US EPA test). For metals, the hydroponic test method was 3.3 (for Hg) to 57 (for Cu) times more sensitive than the US EPA method with the rank orders of sensitivity, estimated from EC50 values, being Cu≥Cd>Ni≥Zn≥Hg for the former and Hg≥Cu≥Ni≥Cd≥Zn for the latter methods. For phenol, the results did not differ significantly; EC50 values were 124mgL−1 and 108–180mgL−1 for the hydroponic and filter paper methods, respectively. Lettuce was less sensitive than daphnids to wastewaters, but the root elongation response appears to be wastewater-specific and is especially sensitive for detecting the presence of fluorine.The new hydroponic test thus provides many practical advantages, especially in terms of cost and time-effectiveness requiring only a well plate, a small volume of distilled water and short exposure period; furthermore, no specialist expertise is required. The method is simpler than the conventional EPA technique in not using filter paper which can influence the sensitivity of the test. Additionally, plant seeds have a long shelf-life and require little or no maintenance.

Field assessment of the effectiveness of organic amendments for aided phytostabilization of a Pb–Zn contaminated mine soil

A field trial was carried out to assess the effectiveness of organic amendments (COW: cow slurry; POULTRY: poultry manure; PAPER: paper mill sludge mixed with poultry manure) for the aided phytostabilization of vegetated and non-vegetated sites of an abandoned Pb/Zn contaminated mine. A native ecotype of Festuca rubra L. was used for colonizing the initially non-vegetated sites. Two and six months after amendment application, soil Pb and Zn availability (CaCl2-extractability) decreased. COW and POULTRY treatments resulted in highest reduction in Pb extractability in non-vegetated sites. In vegetated sites, COW-treated plots showed the highest decrease in metal extractability at 6months. All amendments, especially POULTRY, led to higher F. rubra biomass and vegetation cover in vegetated and initially non-vegetated sites, respectively. Two months after soil treatment, soil basal and substrate-induced respiration peaked in vegetated sites. Based on a root elongation bioassay with Lactuca sativa L., beneficial effects of soil amendments were also observed in terms of soil phytotoxicity. The problem of high variability (spatial, temporal and environmental) of vegetated and non-vegetated mine soils for an effective aided phytostabilization is discussed.

Germination and Root Elongation Bioassays in Six Different Plant Species for Testing Ni Contamination in Soil

In vitro short-term chronic phytotoxicity germination and root elongation test were applied to test the effects of nickel (Ni) in seed germination and root elongation in six plants species: Cucumis sativus (Cucurbitaceae), Lepidium sativum and Brassica nigra (Brassicaceae), Trifolium alexandrinum and Medicago sativa (Fabaceae), Phacelia tanacetifolia (Boraginaceae). A naturally Ni rich soil was used to compare the results obtained. Unlike root elongation, germination was not affected by Ni in any of the six species tested. EC50 values, calculated on the root elongation, showed that Ni toxicity decreases in the following order: P. tanacetifolia > B. nigra > C. sativus > L. sativum > M. sativa > T. alexandrinum. The test conducted using soil elutriate revealed a significantly lower effect in both seed germination and root elongation when compared to the results obtained using untreated soil. Conversely, the test performed on soil confirmed the high sensitivity of C. sativus, P. tanacetifolia and L. sativum to Ni.

Phytotoxicity and potential genotoxicity of Radiowo municipal landfill leachate

Phytotoxicity and potential genotoxicity of Radiowo municipal landfill leachate Phytotoxicity and potential genotoxicity of Radiowo municipal landfill leachate. The most common method of municipal solid waste disposal is storing at landfills. Many hazardous substances are present in garbage and some more are formed during degradation. They may be assimilated by numerous organisms, pass through the food chain and bioaccumulate by long-term exposure in animals and humans. Therefore it seems an important tusk to monitor the toxic and genotoxic potential of municipal landfill leachates, which may contaminate waters and soils. Higher plants provide a very useful, uncomplicated and inexpensive tool for first-step screening of environmental pollutants. The objective of our study was to evaluate the phytotoxicity and potential genotoxicity of Radiowo municipal landfill leachate by the means of plant bioassays and to preliminary valuate the efficiency of zeolite as a leachate purifier. Phytotoxicity was assessed by the Allium cepa Root Elongation Bioassay and genotoxicity by the means of Allium and Vicia Root Tip Assays (RTA). Results were subjected to the F-test for the two way analysis of variance (ANOVA 2) and subsequently the Tukey's test, with statistical significance set at p = 0.05. Applied plant bioassays indicated significant phytotoxicity and potential genotoxicity of Radiowo landfill leachate The process of zeolite filtration did not eliminate, but evidently decreased phytotoxicity. It did not cause significant change in genotoxic potential of 100% leachate, but resulted in its more rapid decrease in a concentration dependent manner.

A Terrestrial Biotic Ligand Model. 1. Development and Application to Cu and Ni Toxicities to Barley Root Elongation in Soils

A Terrestrial Biotic Ligand Model (TBLM) was developed using noncalcareous soils from Europe based on Cu and Ni speciation and barley (Hordeum vulgare cv. Regina) root elongation bioassays. Free metal ion (M2+) activity was computed by the WHAM VI model using inputs of soil metal, soil organic matter, and alkali and alkaline earth metals concentrations, and pH in soil solution. The TBLM assumes that metal in soil and in the solution are in equilibrium. Metal ions react with the biotic ligand, the receptor site, and inhibit root elongation. Other ions, principally H+, Ca2+ and Mg2+, compete with M2+ and, therefore, affect its toxicity. Toxicity is correlated only to the fraction of the total biotic ligand sites occupied by M2+. Compared to other models using either the soil metal concentration or M2+ activity as the toxic dose, the TBLM provides a more consistent method to normalize and compare Cu and Ni toxicities to root elongation among different soils. The TBLM was able to predictthe EC50 soil Cu and Ni concentrations generally within a factor of 2 of the observed values, a level of precision similar to that for the aquatic Biotic Ligand Model, indicating its potential utility in metals risk assessment in soils.

Nutrient status and plant growth effects of forest soils in the Basin of Mexico

The nutrient status of forest soils in the Mexico City Air Basin was evaluated by observing plant growth responses to fertilization with N, P or both nutrients combined. P deficiency was the most frequent condition for soil from two high pollution sites and N deficiency was greatest at a low N deposition site. Concentrations of Pb and Ni, and to a lesser extent Zn and Co, were higher at the high pollution sites. However, positive plant growth responses to P and sometimes to N, and results of wheat root elongation bioassays, suggest that heavy metal concentrations were not directly phytotoxic. Further studies are needed to determine if heavy metal toxicity to mycorrhizal symbionts of eucalyptus ( Eucalyptus camaldulensis Dehnh.) from high pollution sites may explain the P deficiency and stunted growth. P deficiency is expected to limit the capacity for biotic N retention in N saturated forested watersheds in the Basin of Mexico dominated by Andisols.

Sulfometuron‐resistant Amaranthus retroflexus: cross‐resistance and molecular basis for resistance to acetolactate synthase‐inhibiting herbicides

A biotype of Amaranthus retroflexus L. is the first weed in Israel to develop resistance to acetolactate synthase (ALS)‐inhibiting herbicides. The resistant biotype (Su‐R) was collected from Ganot, a site that had been treated for more than 3 consecutive years with sulfometuron‐methyl + simazine. On the whole‐plant basis, the resistance ratio (ED50 Su‐R)/(ED50 Su‐S) was 6–127 for sulfonylureas, 4–63 for imidazolinones, 20–35 for triazolopyrimidines and 11 for pyrithiobac‐sodium. Similar levels of resistance were found also when the herbicides were applied before emergence. Based on a root elongation bioassay, Su‐R was 3240‐fold more resistant to sulfometuron‐methyl than Su‐S. In vitro studies have shown that the Su‐R biotype was resistant at the enzyme level to all ALS inhibitors tested. The nucleotide sequences of two amplified regions between the Su‐S and the Su‐R differed in only one nucleotide. One substitution has occurred in domain A, cytosine by thymine (CCC to CTC) at position 248, that confers an exchange of the amino acid proline in the susceptible to leucine in the Su‐R. The proline to leucine change in domain A is the only difference in the amino acid primary structure of the regions sequenced, indicating that it is responsible for the ALS‐inhibitor resistance observed.

Calibration of the single‐ and double‐buffer SMP lime requirement methods by root elongation bioassay

Chemical and biological lime requirement (LR) reference values of 154 soils were obtained by six months of incubation of each soil with five levels of calcium carbonate (CaCO3). Levels of CaCO3 addition differed among soils according to their characteristics. Chemical LR values were based on the individual neutralization curves to achieve a desired pH (pHd) value of 5.5, 6.0, and 6.5 in water or 5.0, 5.5, and 6.0 in 0.01M calcium chloride (CaCl2). Biological LR values were estimated to achieve 90% relative root elongation on each soil after a growth period of 48 h using wheat cv Abe. Chemical values of LR suggest that SMP method is valid for a wide range of mineral soils from different geographic regions. However, the proportion of soil acidity reacting with the buffer is not constant. Results indicate that values obtained with the routine methods need to be calibrated with equations different from the originals. The use of curvilinear models to adjust one single pH of the soil‐buffer system improved substantially its accuracy, allowing the single‐buffer (SB) results to be comparable to the more time consuming and labourious double‐buffer (DB) technique. No advantage was noticed with the use of curvilinear equations for DB technique. The adoption of pHd in 0.01M CaCl2 leads to an increase of precision of the predicted LR. Regression equations are provided for calculating LR rates to different pHd values. Accuracy is high (r2=0.887) even for pH values (5.0 in 0.01M CaCl2) lower than normally considered in methods based on buffer solutions. An overestimation of biological LR values was observed with both SMP methods. Notwithstanding, a calibration can be also made with the root bioassay, adjusting the chemical values to lime rates based on biological constraints related with aluminum (Al) toxicity. Regression equations are provided. Once more, the use of quadratic model for SB method allows an accuracy (r2=0.836) comparable with the DB technique (r2=0.850).

Allelopathy of small everlasting (Antennaria microphylla)

Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

Effect of DBCP on Fluchloralin Persistence

Three field studies were conducted over a 2-yr period to evaluate the persistence of fluchloralin [N-(2-chloroethyl)-2,6-dinitro-N-propyl-4-(trifluoromethyl) aniline], and to determine whether DBCP (1,2-dibromo-3-chloropropane) affected persistence. Fluchloralin was applied to field plots at 1.1 kg/ha with and without DBCP at 20.5 kg/ha. In the first study, soil samples were taken periodically over a 1-yr period and assayed for fluchloralin by both gas chromatography (GC) and a sorghum (Sorghum bicolorL. Moench ‘AKS-516’) root-elongation bioassay. Both methods of analysis indicated that fluchloralin persistence was unaffected by DBCP. An oat (Avena sativaL. ‘Ora’) bioassay of soil from the field plots 41 weeks after treatment showed no residual herbicide activity. In the next two field studies, soil samples were taken periodically over a 32-week period and assayed by GC for fluchloralin. A greenhouse sorghum bioassay of soil samples taken from both tests 32 weeks after application showed residual activity of fluchloralin in one test, but differences were not attributable to DBCP. A two-phase process of fluchloralin dissipation in field soil was indicated from analysis of the data using a complex first-order regression, as opposed to a simple first-order regression. Half-life values describing fluchloralin persistence, using the complex first-order regression, ranged from 2.3 to 3.7 weeks for the first phase and 9.5 to 26.7 weeks for the second phase.