Dongmu-1 Medicago sativa seedlings were used as the test material; the variation characteristics soluble protein, soluble sugar, malondialdehyde, proline, chlorophyll, and relative water content were studied under the artificial simulated freeze-thaw (10, 5, 0, − 3, 0, 5, and 10 °C) and combined with deicing salt stress and buffer. The results showed that freeze-thaw and high-salt stress conditions will lead to the damage in the seedling including the membrane system, lipid peroxidation, and severe dehydration. Because of the self-regulating system as well as a certain degree of resistance, the plants can accumulate plenty of substances such as soluble protein, soluble sugar, and proline so as to regulate the osmotic potential. The content of soluble protein, malondialdehyde, soluble sugar, and proline in different treatment groups rose first and then decreased within a freeze-thaw cycle, among which the content of soluble protein reached the maximum value at 0 °C (t3), 20.82, 18.96, and 17.97 mg/g, respectively. The figure for malondialdehyde and proline peaked at − 3 °C (t4) while soluble sugar content peaked at 0 °C (t5). However, during this period, there were no apparent regulations for chlorophyll content and relative water content in each treatment group. Beyond that, due to the different intensity of compound stress, the seedlings showed different adaptability, and the degree of changes in physiological indexes appeared to be combined freeze-thaw and deicing salt stress > single freeze-thaw stress > combined freeze-thaw, deicing salt stress, and buffer, illustrating that buffer can alleviate the degree of the damage from freeze-thaw and deicing salt stress on M. sativa seedlings to some extent.

Aluminum/iodine pentoxide (Al/I₂O₅) composites are currently receiving much attention for their capabilities as potential anthrax combatants. Their high halogen gas release, coupled with high temperature evolution from combustion, renders them effective in bacterial deactivation. Despite extensive research on the energetic capacities of these compounds, limited information is currently available in relation to their potential environmental (non-target) effects. We evaluated the effects of Al/I₂O₅ on aquatic (Daphnia magna) and terrestrial (Eisenia fetida, Acheta domesticus) invertebrates, as well as alfalfa (Medicago sativa) seed germination. Polytetrafluoroethylene (PTFE, or DuPont Teflon® MP1150) was examined concurrently to assess whether observations were general to halogen, or element specific. Our observations were based on mortality and reproduction (hatchability) for the terrestrial and aquatic assays. In all Al/I₂O₅ assays, mortality was concentration dependent, ranging from 0% mortality in the control samples to partial and complete mortality in the contaminated cases. The PTFE assays showed no mortality at all contaminant concentrations. At a maximum Al/I₂O₅ contaminant concentration of 1000 μg/g (ppm), 100% mortality was observed in cricket assay conducted in sand within 72 h exposure and earthworm assay conducted in soil within 4 days exposure. In the aquatic assay, a water concentration of 200 μg/mL (ppm) caused 100% mortality to D. magna in less than 12 h. The effect of aluminum/iodine pentoxide on earthworm cocoon hatching success was also determined. At soil concentrations ≤ 400 μg/g, hatching success for earthworm cocoons was equivalent to control (un-contaminated) soil; above this concentration, hatching success was reduced by a factor of 2. Alfalfa germination tests were performed at a single contaminant concentration of 1000 μg/g. This soil concentration was completely inhibitory to seed germination.

The effects of phosphate processing wastewater (PPWW) on heavy metal accumulation in a Mediterranean soil (Tunisia, North Africa) were investigated. Moreover, the residual toxicities of PPWW-irrigated soils extracts were assessed. Results showed that heavy metal accumulation was significantly higher in PPWW-irrigated soil extracts than in control soil. The heavy metal accumulation increased over time in treated soil samples and their average values followed the following order: Iron (Fe 252.72 mg l⁻¹) > Zinc (Zn 152.95 mg l⁻¹) > Lead (Pb 128.35 mg l⁻¹) > Copper (Cu 116.82 mg l⁻¹) > Cadmium (Cd 58.03 mg l⁻¹). The residual microtoxicity and phytotoxicity of the various treated soil samples extracts were evaluated by monitoring the bioluminescence inhibition (BI %) of Vibrio ficheri and the measurement of the germination indexes (GI %) of Lepidium sativum and Medicago sativa seeds. The results showed an important increase of residual toxicities of PPWW-treated soil extracts over time.

Acid deposition and temperature variation could lead to changes of physiological characteristics of plants in response to stress. In this paper, Medicago sativa CV. Dongmu–1 was investigated to test the effects of freeze-thaw circle and acid deposition upon the changes of osmotic adjustment substances, biological membrane permeability, and antioxidant enzymes. The experiment was conducted under laboratory conditions, and the seedlings were divided into four groups (group I: no treatment, group II: acid stressed only, group III: freeze-thaw stressed only, group IV: both freeze-thaw and acid stressed). Results indicated that under freeze-thaw circle and acid deposition, the contents of malondialdehyde (MDA) and proline increased respectively by 0.6~203.4 and 19.3~68.8% when compared with group I, while protein content declined by 4.1~31.7%, and the effects were even significant than freeze-thaw-only stressed groups. In the freeze-thaw process, superoxide dismutase (SOD) activity dropped at first and then increased with the increase of temperature, peaking at − 3 °C by 1118.45 U g⁻¹; peroxidase (POD) activity showed a brief rise and declined rapidly below 0 °C. By increasing the potentials of antioxidant enzymes and MDA, the membrane lipid peroxidation inside alfalfa was prevented; meanwhile, several indexes changed adaptively in resisting hurts. Variation of SOD and POD was induced by the defense mechanism, which showed alfalfa’s satisfactory cold resistance and acid tolerance. Further research on acid deposition and freeze-thaw circle would be beneficial for the global cultivation of forage grass.

In this study, 1-year greenhouse pot experiments were conducted to investigate the effect of Phyllobacterium myrsinacearum strain RC6b on the growth and phytoextraction efficiency of heavy metals by a Zn/Cd hyperaccumulator (Sedum alfredii) and alfalfa (Medicago sativa L.) in a co-cropping system. The treated soil sample was collected from a land reclamation site of Pb/Zn mine tailings in Hanzhong City, Shaanxi Province, China. Results showed that, with the inoculation of RC6b, shoot biomass yields of plants were significantly increased by 15.9–20.2 % and 17.2–19.9 % for alfalfa and S. alfredii, respectively, compared to the non-inoculated plants. Biomass yield of alfalfa was higher than that of S. alfredii. RC6b inoculation increased metal concentrations by 18.6–31.2 % (Pb), 23.8–37.5 % (Cd), and 26.4–38.3 % (Zn) in S. alfredii shoots, and by 13.8–24.7 % (Pb), 15.8–26.6 % (Cd), and 24.8–35.6 % (Zn) in alfalfa shoots, respectively. After six consecutive harvests of shoots, RC6b inoculation increased the phytoextraction efficiencies of Pb, Cd, and Zn by shoots of the co-planting system by 16.9, 46.3, and 60.9 %, respectively. Nevertheless, phytoextraction of Cu was not improved by RC6b inoculation. In the co-planting/inoculation system, the percentage removals of metals from soil by the plant shoots were 6.09, 30.97, 11.10, and 1.68 % for Pb, Cd, Zn, and Cu, respectively, after six harvests of shoots. Inoculation with RC6b significantly increased the soil microbial activity and the carbon utilization ability of the soil microbial community.

Zinc toxicity thresholds for reclamation plants are largely unknown. As a result, ecological risk assessments often rely on toxicity thresholds for agronomic species, which may differ from those of restoration species. Our objective was to provide Zn toxicity thresholds for forb species that are commonly used in reclamation activities. We used a greenhouse screening study where seedlings of yarrow (Achillea millefolium L.), Bigelow's tansyaster (Machaeranthera bigelovii (Gray) Greene var. bigelovii), blue flax (Linum perenne L. var. Appar), alfalfa (Medicago sativa L. var. Ladak), Palmer's penstemon (Penstemon palmeri Gray), and Rocky Mountain penstemon (Penstemon strictus Benth. var. Bandera) were grown in sand culture and exposed to increasing concentrations of Zn. Lethal concentrations (LC50 - substrate Zn concentration resulting in 50% mortality), effective concentrations (EC50 - substrate Zn concentration resulting in 50% biomass reduction), and phytotoxicity thresholds (PT50 - tissue Zn concentration resulting in 50% biomass reduction) were then determined. Phytotoxicity thresholds and effective concentrations for these reclamation species were relatively consistent between species. Our estimates of PT50-shoot for these species range from 1258 to 3214 mg Zn kg-¹ . Measures of EC50-plant for these restoration forbs ranged from 82 to 214 mg Zn L-¹ . These thresholds might be more useful for risk assessors working on reclamation sites than those based on non-reclamation species that are widely used.

Grazing animals act as a bioindicator to study the heavy metal status in the pasture lands because excessive amount of toxic metals in the animal diet either disturb their normal activity or deposit the contaminants into their tissues. The aim of this study was to appraise the chromium status in soil and pasture crops with respect to the nutritional requirement of grazing animals. Three different sites were selected to collect soil, forages, and animal samples from District Jhang. All the samples were processed through atomic absorption spectrophotometer to analyze the chromium concentration in them. Chromium concentration was varied as 0.703–4.20 mg/kg in soil, 0.45–2.85 mg/kg in forages, and 0.588–2.37 mg/kg in all collected animal samples. Both the soil and forage samples displayed the maximum chromium concentration in the Capparis decidua, whereas animal samples revealed maximum concentration in animal blood. Results of pollution load index (0.078 to 0.463 mg/kg) exhibited that all the sample values are less than unity while enrichment factor (1.57–8.25mg/kg) showed that significant level of chromium is enriched in these sites. The maximum value of daily intake (0.0007–0.0055mg/kg/day) and health risk index (0.0004–0.00370055mg/kg/day) was observed in the buffalo that feed on the Capparis decidua. Bio-concentration factor (0.398–2.09mg/kg) value was the maximum in the Medicago sativa. It is concluded that all the animal samples showed chromium concentration beyond their standards. Thus, proper measures should be taken to reduce the metal contamination in these areas that ultimately lessen the availability of toxic metals to grazing animals.

Heavy metal (HM) pollution and the need to preserve the environment have gathered increasing scientific attention. The immobilization of HMs into less-soluble, less mobile, and less toxic forms in addition to the improvement of Medicago sativa L. growth and HMs accumulation were evaluated after the application of marble waste (MW) and/or beneficial PGP rhizobacteria and mycorrhizae to the mining soil compost. A greenhouse assay was conducted to elucidate the influence of both amendment and beneficial microorganisms. The application of marble waste to the soil-compost resulted in decreasing the bioavailability of metals (Cu, Zn, Pb, and Cd), thus ameliorating the installation of the vegetal cover for 6 months of culture. Cultivation of M. sativa under 5% MW-amended soil for 6 months increased the shoot dry weight by almost twofold, while the inoculation with rhizobacteria-mycorrhizae combined with the application of 15% MW resulted in an improvement of 3.5-fold in case of shoot dry weight. In addition, the application of marble waste amendment or their combination with metallo-resistant bacteria resulted in decreasing HM accumulation leading to HM content below the threshold recommended for animal grazing. Thus, the application of amendments and beneficial microorganisms appeared to guarantee the safe cultivation of alfalfa for 6 months of culture. The dual combination amendments and beneficial microorganisms showed the good potential to restore HM polluted soils and could stand as a novel approach for restoration of HM-contaminated soils.

Zinc oxide nanoparticles (ZnO-NPs) are among the most commonly used nano-fertilizers (NF). However, elevated levels of ZnO-NPs in soil may affect plant growth and development due to its potential toxicity when accumulated in large amounts in plant tissues. This research was conducted using an in situ rhizobox system with the aims of evaluating zinc uptake from nano-zinc oxide amended rhizosphere soil by alfalfa plant and the effect of plant growth-promoting microorganisms on alleviating the phytotoxicity of ZnO-NPs. Treatments included microbial inoculations (Sinorhizobium meliloti, Serendipita indica) and different ZnO-NP concentrations (0, 400, and 800 mg kg⁻¹) with three replications. The results indicated that S. indica minimized the phytotoxicity of ZnO-NPs to alfalfa by enhancing growth rate and decreasing zinc (Zn) translocation from root to shoot. Compared with plants inoculated with S. meliloti, co-inoculation with S. indica increased the shoot dry weight by 18.33% and 8.05% at 400 and 800 mg kg⁻¹ ZnO-NPs, respectively. However, at the highest level of ZnO-NPs (800 mg kg⁻¹), root inoculation of S. indica and S. indica + S. meliloti decreased Zn translocation factor by 60.2% and 44.3% compared to S. meliloti, respectively. Furthermore, a distinct relation between tolerance of S. indica–colonized plant to ZnO-NPs and the ability of S. indica in inhibiting or retarding degradation of polyunsaturated lipids through prevention of excess reactive oxygen species formation was observed. Malondialdehyde content of inoculated plants with S. indica either alone or in combination with S. meliloti was significantly lower than non-inoculated plants (p< 0.01). Zn-induced oxidative stress was mitigated by S. indica through enhanced activities of catalase and peroxidase enzymes. The findings of the present study indicate the potential use of endophytes fungus S. indica for ensuring food safety and security, and human health in heavy metal-polluted soil by reducing the phytoavailability of heavy metals in the aerial parts of the host plants.

The response of soil microbiota to hydrocarbon contamination has been studied normally several months after the event. However, as those studies represent a “long-term” context for microbial processes, since protozoa succession can be achieved after 28 days, we wonder how fast does trophic structure of ciliates and flagellates recover from a strong pulse of petroleum contamination under the influence of Medicago sativa. We hypothesized that the root effect of M. sativa would promote faster recovery of the protozoan trophic structure, in comparison with the level reached in unplanted microcosms. The abundances of individuals, species, and trophic groups of ciliates and flagellates were determined at 1, 7, 14, and 30 days after a single pulse of 50,000 ppm of light petroleum on soil microcosms unplanted and planted with M. sativa, and their respective controls. Protozoan diversity and trophic groups were strongly reduced immediately after the pulse of contamination. Ciliates and flagellate trophic diversity increased steadily in all microcosms after 7 days. However, unplanted contaminated and planted contaminated microcosms remained the poorest communities and reached full recovery of trophic groups after 30 days. Also, the protozoan communities were segregated into 2 groups: the first from petroleum and the second formed by non-polluted microcosms. These results suggest that petroleum is a strong selection factor leading to an alternative protozoan community composition and the root effect of M. sativa promotes faster recovery of ciliate and flagellate communities after a devastation produced by a strong pulse of petroleum contamination.