The downside of copper pesticides: An earthworm's perspective.

The widespread use of copper-based pesticides, while effective in controlling plant diseases, has been identified as a major source of copper contamination in soils. This raises concerns about potential adverse effects on earthworms, key players in soil health and ecosystem function. To inform sustainable pesticide practices, this study aimed to establish copper toxicity thresholds for earthworm avoidance in agricultural soils impacted by copper-based pesticides. We collected 40 topsoil samples (0-5 cm) from orchards and vineyards in the O'Higgins Region of central Chile, and 10 additional soils under native vegetation as background references. A standardized avoidance bioassay using Eisenia fetida assessed the impact of copper-based pesticides on the soils. Total copper concentrations ranged between 23 and 566 mg kg-1, with observed toxic effects on earthworms in certain soils. The effective concentration at 50% (EC50) for total soil copper, determined by Eisenia fetida's avoidance response, was 240 mg kg-1, with a 95% confidence interval of 193-341 mg kg-1. We further compared our EC50 values with existing data from agricultural soils impacted by mining activities. Interestingly, the results revealed a remarkable similarity between the thresholds for earthworm avoidance, regardless of the source of copper contamination. This observation underscores the universality of copper toxicity in agricultural ecosystems and its potential impact on soil biota. This study provides novel insights into copper toxicity thresholds for earthworms in real-world, pesticide-contaminated soils.

Gypsum soil amendment in metal-polluted soils-an added environmental hazard.

Scientists around the world have long been searching for effective strategies to reduce the bioavailability of metals in contaminated soils. In case of metal-spiked soils, some studies have proposed gypsum as a soil amendment to alleviate metal phytotoxicity. However, for real field-collected soils, evidence on the efficacy of gypsum as a metal phytotoxicity amendment is limited. Therefore, the present study was designed to examine the effect of gypsum on plant growth in soils polluted by a copper smelter. We grew perennial ryegrass on untreated and gypsum-treated soils (at a dose of 3% by weight) under laboratory conditions. We found that gypsum had no effect on alleviating metal phytotoxicity in our soils. We also demonstrated - for the first time - that gypsum increased the concentrations of soluble metals in the soil, enhancing metal uptake by plants. The calcium ions from gypsum displace metals in the soil exchangeable complex; however, the metals do not get immobilized in soils because gypsum is a neutral salt. While our results contrast with the Terrestrial Biotic Ligand Model, that Model has never been tested on real industrially polluted soils but only on metal-spiked soils. Our main conclusion is that gypsum is ineffective in alleviating metal phytotoxicity in real industrially polluted soils and, moreover, its use is inappropriate as a soil remediation method, because it increases the environmental hazard rather than reducing it. Our study is the very first attempt to recognize that gypsum is a hazardous material when used to ameliorate soils polluted by metals.

The role of leaf litter as a protective barrier for copper-containing pesticides in orchard soils.

This study assessed the capacity of leaf litters to adsorb copper ions applied as a copper-based pesticide. Leaf litters of two fruit tree species with different lignin/N ratios were examined to determine their protective role against the incorporation of Cu into soil. A leaf litter Cu-adsorption capacity assay and a degradation assay were performed using table grape (lignin/N = 2.35) and kiwi (lignin/N = 10.85) leaf litters. Table grape leaf litter had a significantly (p = 0.001) higher Cu-adsorption capacity (15,800 mg kg-1) than kiwi leaf litter (14,283 mg kg-1). Following leaf litter degradation, significant differences (p = 0.011) were observed in the release of Cu from Cu-enriched leaf litter into soil, showing that kiwi litter has a greater protective effect against the incorporation of Cu into soil, regardless of the amount of Cu applied. This protective role is reflected in a significantly higher (p = 0.015) Cu concentration in table grape soil (41.71 ± 2.14 mg kg-1) than in kiwi soil (35.87 ± 0.69 mg kg-1). Therefore, leaf litter with higher lignin/N ratio has greater protective role against copper incorporation into soil.

The microbial community from the early-plant colonizer (Baccharis linearis) is required for plant establishment on copper mine tailings.

Plants must deal with harsh environmental conditions when colonizing abandoned copper mine tailings. We hypothesized that the presence of a native microbial community can improve the colonization of the pioneer plant, Baccharis linearis, in soils from copper mining tailings. Plant growth and microbial community compositions and dynamics were determined in cultivation pots containing material from two abandoned copper mining tailings (Huana and Tambillos) and compared with pots containing fresh tailings or surrounding agricultural soil. Controls without plants or using irradiated microbe-free substrates, were also performed. Results indicated that bacteria (Actinobacteria, Gammaproteobacteria, and Firmicutes groups) and fungi (Glomus genus) are associated with B. linearis and may support plant acclimation, since growth parameters decreased in both irradiated (transiently without microbial community) and fresh tailing substrates (with a significantly different microbial community). Consistently, the composition of the bacterial community from abandoned copper mining tailings was more impacted by plant establishment than by differences in the physicochemical properties of the substrates. Bacteria located at B. linearis rhizoplane were clearly the most distinct bacterial community compared with those of fresh tailings, surrounding soil and non-rhizosphere abandoned tailings substrates. Beta diversity analyses showed that the rhizoplane bacterial community changed mainly through species replacement (turnover) than species loss (nestedness). In contrast, location/geographical conditions were more relevant than interaction with the plants, to explain fungal community differences.

Mayten Tree Seed Oil: Nutritional Value Evaluation According to Antioxidant Capacity and Bioactive Properties.

The Mayten tree (Maytenus boaria Mol.), a native plant of Chile that grows under environmentally limiting conditions, was historically harvested to extract an edible oil, and may represent an opportunity to expand current vegetable oil production. Seeds were collected from Mayten trees in north-central Chile, and seed oil was extracted by solvent extraction. The seed oil showed a reddish coloration, with quality parameters similar to those of other vegetable oils. The fatty acid composition revealed high levels of monounsaturated and polyunsaturated fatty acids. Oleic and linoleic acids, which are relevant to the human diet, were well represented in the extracted Mayten tree seed oil. The oil displayed an antioxidant capacity due to the high contents of antioxidant compounds (polyphenols and carotenoids) and may have potential health benefits for diseases associated with oxidative stress.

A novel fast-vegetative propagation technique of the pioneer shrub Baccharis linearis on mine tailings by adding compost.

Interest in Baccharis linearis has increased as an alternative for assisted phytostabilization due to its spontaneous colonization of tailings dumps. The search for a novel fast-vegetative propagation technique to accelerate its coverage on mine tailings is a promising research area for sustainable mine closure plans. In this study, we determined the optimal proportion of compost and tailings as growing media to promote fast B. linearis propagation through a compound layering technique. The assessed growing substrates were: 100% tailings, 70% tailings + 30% compost, and 50% tailings + 50% compost. After 84 days of growth, the change in number and height of layering branches, root and shoot dry mass, percentage of ground coverage, and substrate chemical properties were assessed. The main results showed that compound layering of B. linearis is possible with compost addition. The growth of new roots and layering branches was significantly improved by either 30% or 50% compost addition into tailings, due to chemical improvements of substrate (higher nutrients and pH and decreased copper bioavailability). The study confirms that the compound layering of B. linearis may be an effective and novel technique for speeding the reclamation of post-operative mine tailings, which is improved by the incorporation of compost.

Increase in Phytoextraction Potential by Genome Editing and Transformation: A Review.

Soil metal contamination associated with productive activities is a global issue. Metals are not biodegradable and tend to accumulate in soils, posing potential risks to surrounding ecosystems and human health. Plant-based techniques (phytotechnologies) for the in situ remediation of metal-polluted soils have been developed, but these have some limitations. Phytotechnologies are a group of technologies that take advantage of the ability of certain plants to remediate soil, water, and air resources to rehabilitate ecosystem services in managed landscapes. Regarding soil metal pollution, the main objectives are in situ stabilization (phytostabilization) and the removal of contaminants (phytoextraction). Genetic engineering strategies such as gene editing, stacking genes, and transformation, among others, may improve the phytoextraction potential of plants by enhancing their ability to accumulate and tolerate metals and metalloids. This review discusses proven strategies to enhance phytoextraction efficiency and future perspectives on phytotechnologies.

Use of Zinc Carbonate Spiking to Obtain Phytotoxicity Thresholds Comparable to Those in Field-Collected Soils.

Several studies have reported the presence of smithsonite (ZnCO3 ) in soils polluted by zinc mining. The present study aimed to determine upper critical threshold values of Zn phytotoxicity in a substrate spiked with ZnCO3 and to compare them with those obtained in field-collected soils. We studied Zn toxicity to perennial ryegrass (Lolium perenne L.) grown in pots with unpolluted peat treated with increasing concentrations of ZnCO3 that produced nominal total Zn concentrations of 0, 0.7, 1.3, 2.0, 2.6, and 3.3%. To keep constant near-neutral pH value in all the treatments, we used decreasing concentrations of dolomitic lime. In the treatment with total soil Zn of 3.3% (pH 6.8), the foliar Zn concentration of L. perenne was 1914 ± 211 mg kg-1 , falling into the range of 2400 ± 300 mg kg-1 reported for Lolium species grown under similar laboratory conditions in a polluted soil (total soil Zn 5.4%, pH 7.3) collected near a Zn smelter. The value of 92 ± 98 mg kg-1 was obtained for the median effective concentration (EC50) values of 0.01 M KNO3 -extractable Zn using the responses of shoot dry biomass, shoot length, and total pigments. This value falls within the range of 95 ± 46 mg kg-1 reported in other studies for the EC50 values of salt-extractable Zn using field-collected soils. The application of ZnCO3 for spiking was able to mimic foliar Zn concentrations of Lolium species observed in field-collected soils. The effective concentrations of soil Zn obtained in the present study are comparable to those obtained in field-collected soils. Future research should determine effective concentrations of metals using soils spiked with metal-containing compounds that mimic a real source of contamination. Environ Toxicol Chem 2020;39:1790-1796. © 2020 SETAC.

Evaluation of phytotoxic effects of cationic polyacrylamide polymers: implication for the use of sludges as organic soil amendments in assisted phytostabilization.

Sludges, as biosolids, are organic soil amendments commonly used in assisted phytostabilitation. Extensive studies on their environmental impacts exists, particularly for improper land application and contents of trace elements, organic compounds and pathogens, but not for their content of cationic polyacrylamide polymers (C-PAMs). Direct toxicity of C-PAMs on aquatic organisms has been demonstrated but scarce information about plant toxicity is available. In this study, the effect of C-PAMs on early plant growth was evaluated by means of standard toxicity assays. Firstly, increasing doses of C-PAMs were applied as solutions to seeds of Avena sativa, Lactuca sativa and Solanum lycopersycum to evaluate germination and root elongation. Secondly, the effect of increasing doses of C-PAMs spiked in pig manure and mixed with sand (75 t ha-1 dry base) was evaluated on the emergence, radicle elongation and biomass of A. sativa. Results showed high phytotoxicity of C-PAMs in solutions above 1,000 mg L-1, but no effect was detected when spiked into experimental substrate; a significant effect was only observed above 5,000 mg L-1 for radicle elongation, aerial biomass, and radicle biomass. Results demonstrate direct phytotoxic effects of C-PAMs, which can be mitigated when spiked into an organic (pig manure) and mineral (sand) matrix.

Which soil Cu pool governs phytotoxicity in field-collected soils contaminated by copper smelting activities in central Chile?

Several studies have attempted to predict the so-called "phytoavailable" fraction by correlating plant responses with different soil metal pools. Most of the data derived from these studies tend to be inconsistent, making interpretations difficult. Thus, the main objective of this study was to determine which soil Cu pool (free Cu2+, salt-exchangeable Cu or total Cu) controls Cu phytotoxicity in soils near a Cu smelter in central Chile. We studied the following traits of the local plant community grown spontaneously on the study site: species richness, shoot biomass, and plant cover. The site was dominated by four early plant colonizers: Eschscholzia californica Cham., Hirschfeldia incana (L.) Lagr.-Fossat, Lolium perenne L., and Vulpia bromoides (L.) Gray. We determined exchangeable soil Cu and activity of free Cu2+ in 0.1 M KNO3 extracts using soil/solution ratio of 1/2.5. The effect of total soil Cu on plant responses was not significant (p > 0.05). In our field-collected soil series, exchangeable Cu was a better indicator of soil phytotoxicity than either total soil Cu or free Cu2+ in the soil solution. We determined upper critical threshold values for Cu exposure using the three plant traits cited above. The mean values of EC10, EC25, and EC50 (effective concentration at 10%, 25%, and 50%, respectively) of exchangeable soil Cu (in µg L-1) were 255, 391, and 533, respectively. The mean EC10, EC25 and EC50 values of pCu2+ were 7.5, 6.8, and 5.9, respectively. We highlight the importance of further studies on Cu phytotoxicity using actual field-contaminated soils.

Evaluation of connected clonal growth of Solidago chilensis as an avoidance mechanism in copper-polluted soils.

Plant resistance to metals can be achieved by two strategies, tolerance and avoidance. Although metal tolerance has been broadly studied in terrestrial plants, avoidance has been less considered as a strategy to cope with soil metal pollution. Avoidance may be an effective alternative in herbaceous plants with connected clonal growth in environments having high heterogeneity in soil micro-spatial distribution of available metals and other soil conditions (i.e. organic matter). In this study, we performed a laboratory experiment on clonal growth of Solidago chilensis when exposed to copper-spiked soils (800 mg kg-1) at different depths (0, 2, 5 and 8 cm depth), with (20%) and without addition of organic matter to mimic contrasting microhabitats found at smelter hinterlands (i.e. open bare ground and microhabitats below shrubs). Results showed that plants grown in the 2 cm-depth Cu-spiked soils were able to growth and produce ramets and rhizomes. However, increased Cu uptake of plants determined phytotoxic effects and a reduction in clonal spread in the 5 cm- and 8 cm-depth Cu-spiked soils. Addition of organic matter to the Cu-spiked soil layers allowed clonal spread. Considering that ramet and rhizome production is decreased but not inhibited when copper pollution is restricted to the uppermost soil layer (2 cm depth) and that organic matter eliminated soil copper toxicity allowing normal clonal spread, connected clonal growth may be an effective avoidance mechanism of Solidago chilensis, particularly in environments with high heterogeneity in micro-spatial distribution of metals and organic matter in the soil profile and between microhabitats.

Integrated Management of Pig Residues and Copper Mine Tailings for Aided Phytostabilization.

There is high demand for identifying socio-environmentally sound ways to dispose of large quantities of offensive odor-generating organic residues produced by intensive livestock farming. The use of these residues as amendments at large-scale mine tailings storage facilities for in situ plant-based reclamation may be a useful alternative. We performed a greenhouse assay to evaluate effectiveness of pig slurries and the solid organic fraction of aerobic-aerated pig slurries, both treated and not treated for odor emission, as amendments for copper tailings. Different doses of slurries or the solid organic fraction of the slurries, untreated and treated with either ozone or a commercial product used to manage strong odors (Just a Drop), were incorporated into tailings and stabilized (15 d) under greenhouse conditions. L. (ryegrass) was used as bioindicator for Cu and Zn toxicity. Plant performance (shoot and root dry biomass, Cu and Zn content in leaves) and general physicochemical characteristics of substrates were evaluated. Our results showed that odor management of pig residues was possible with commercial products, and their incorporation into tailings rapidly and effectively reduced odor emission. The solid organic fraction of the slurries neutralized acidic tailings, and both pig residues increased organic matter and nutrient content in tailings while reducing extractable Cu. As a result, ryegrass productivity was significantly improved by incorporation of either slurries or their solid organic fraction into tailings in a dose-dependent form. Foliar Cu content in ryegrass was not affected but foliar Zn content increased; however, Zn remained within acceptable ranges. Both the pig slurries and their soil organic fractions proved to be valuable residues for tailings reclamation.

Effectiveness of pig sludge as organic amendment of different textural class mine tailings with different periods of amendment-contact time.

The present study assesses the effect of tailing texture (loamy sand (LT) and sandy loam (ST)), dose of pig sludge (0, 50, 100 and 200 t ha-1) and amendment-contact time (14, 28 and 42 days) on physicochemical quality of amended substrate using Lolium perenne var Nui as a bioindicator. The main properties of LT differed of ST in levels of total organic carbon (0.19 and 0.58%), in pH (4.6 and 8.5), total Cu (202 and 1647 mg kg-1) and Zn content (31 and 137 mg kg-1). Soil pore water of experimental substrates was characterized for pH, electrical conductivity (EC) and Cu2+ ion activity (pCu2+) while ammonium nitrogen (NH4+-N), infiltration rate and general physicochemical characteristics were measured in substrates. Shoot biomass (SB), root biomass (RB) and the RB:SB ratio was calculated for L. perenne. The results showed there to be a significant interaction (p < 0.05) between tailing texture, sludge dose and amendment-contact time for pCu2+, infiltration rate, SB, RB, and RB: SB ratio, but not for pH, EC, or NH4+-N. However, sludge dose and amendment-contact time significantly affected all variables. By increasing dosages of pig sludge, pore water pH increased, and this was associated with decreases in pCu2+ and the infiltration rate. High doses of pig sludge (100 and 200 t ha-1) impaired growth of L. perenne irrespective of tailing texture and amendment-contact time, likely because of the rise of EC (up to 14 mS cm-1). For both tailing textures, the highest biomass was obtained after incorporation of 50 t ha-1 of pig sludge, with increasing values as amendment-contact time rose. In conclusion, effective management of pig sludge for tailing reclamation should guarantee doses <50 t ha-1 and amendment-contact time >28 days, irrespective of tailing texture.

Comparison of exposure to trace elements through vegetable consumption between a mining area and an agricultural area in central Chile.

Human exposure to trace elements has been a large concern due to the potential health issues. Accordingly, this study aimed to compare the concentrations of arsenic, copper, and zinc in the edible parts of vegetables grown in a mining-agricultural area and in an exclusively agricultural area and to compare the potential human health risks of consuming vegetables from both areas. The consumption habits of the studied population were extracted from the 2010 National Alimentary Survey of Chile. In most cases, the concentrations of trace elements in the edible tissues of vegetables (lettuce, spinach, garlic, onion, carrot, potato, sweet corn, and tomato) were higher in the mining-agricultural area than those in the control area. This difference was most pronounced for leafy vegetables, with arsenic being the trace element of concern. Specifically, the arsenic concentrations in the edible tissues of lettuce and spinach were 8.2- and 5.4-fold higher, respectively, in the mining-agricultural area than in the control area. Lettuce was the vegetable of concern due to its relatively high consumption and relatively high concentration of trace elements. Nevertheless, there was no health risk associated with vegetable consumption in either the mining area or the control area because none of the HQ values surpassed 1.0.

Soil physicochemical factors as environmental filters for spontaneous plant colonization of abandoned tailing dumps.

Abandoned tailing dumps (ATDs) offer an opportunity to identify the main physicochemical filters that determine colonization of vegetation in solid mine wastes. The current study determined the soil physicochemical factors that explain the compositional variation of pioneer vegetal species on ATDs from surrounding areas in semiarid Mediterranean-climate type ecosystems of north-central Chile (Coquimbo Region). Geobotanical surveys-including physicochemical parameters of substrates (0-20 cm depth), plant richness, and coverage of plant species-were performed on 73 ATDs and surrounding areas. A total of 112 plant species were identified from which endemic/native species (67%) were more abundant than exotic species (33%) on ATDs. The distribution of sampling sites and plant species in canonical correspondence analysis (CCA) ordination diagrams indicated a gradual and progressive variation in species composition and abundance from surrounding areas to ATDs because of variations in total Cu concentration (1.3%) and the percentage of soil particles <2 µm (1.8%). According to the CCA, there were 10 plant species with greater abundance on sites with high total Cu concentrations and fine-textured substrates, which could be useful for developing plant-based stabilization programs of ATDs in semiarid Mediterranean-climate type ecosystems of north-central Chile.

Thresholds of copper phytotoxicity in field-collected agricultural soils exposed to copper mining activities in Chile.

It has been argued that the identification of the phytotoxic metal thresholds in soil should be based on field-collected soil rather than on artificially-contaminated soils. However, the use of field-collected soils presents several difficulties for interpretation because of mixed contamination and unavoidable covariance of metal contamination with other soil properties that affect plant growth. The objective of this study was to estimate thresholds of copper phytotoxicity in topsoils of 27 agricultural areas historically contaminated by mining activities in Chile. We performed emergence and early growth (21 days) tests (OECD 208 and ISO 11269-2) with perennial ryegrass (Lolium perenne L.). The total Cu content in soils was the best predictor of plant growth and shoot Cu concentrations, while soluble Cu and pCu(2+) did not well correlate with these biological responses. The effects of Pb, Zn, and As on plant responses were not significant, suggesting that Cu is a metal of prime concern for plant growth in soils exposed to copper mining activities in Chile. The effects of soil nutrient availability and shoot nutrient concentrations on ryegrass response were not significant. It was possible to determine EC10, EC25 and EC50 of total Cu in the soil of 327 mg kg(-1), 735 mg kg(-1) and 1144 mg kg(-1), respectively, using the shoot length as a response variable. However, the derived 95% confidence intervals for EC10, EC25 and EC50 values of total soil Cu were wide, and thus not allowing a robust assessment of metal toxicity for agricultural crops, based on total soil Cu concentrations. Thus, plant tests might need to be performed for metal toxicity assessment. This study suggests shoot length of ryegrass as a robust response variable for metal toxicity assessment in contaminated soils with different nutrient availability.

Lime and compost promote plant re-colonization of metal-polluted, acidic soils.

The revegetation of soils affected by historic depositions of an industrial complex in Central Chile was studied. The plant re-colonization from the existing soil seed bank and changes in the physico-chemical properties of the soil were evaluated in field plots amended with lime and/or compost. We found that the application of lime and/or compost decreased the Cu2+ ion activity in the soil solution and the exchangeable Cu in the soil, showing an effective Cu immobilization in the topsoil. Whereas lime application had no effect on plant productivity in comparison with the unamended control, the application of compost and lime+compost increased the plant cover and aboveground biomass due to the higher nutrient availability and water-holding capacity of the compost-amended soils. Although the Cu2+ activity and the exchangeable Cu were markedly lower in the amended soils than in the unamended control, the shoot Cu concentrations of Lolium spp. and Eschscholzia californica did not differ between the treatments.

Simultaneous immobilization of metals and arsenic in acidic polluted soils near a copper smelter in central Chile.

INTRODUCTION: Acidic and metal(oid)-rich topsoils resulted after 34 years of continuous operations of a copper smelter in the Puchuncaví valley, central Chile. Currently, large-scale remediation actions for simultaneous in situ immobilization of metals and As are needed to reduce environmental risks of polluted soils. Aided phytostabilization is a cost-effective alternative, but adequate local available soil amendments have to be identified and management options have to be defined. MATERIALS AND METHODS: Efficacy of seashell grit (SG), biosolids (B), natural zeolite (Z), and iron-activated zeolite (AZ), either alone or in mixtures, was evaluated for reducing metal (Cu and Zn) and As solubilization in polluted soils under laboratory conditions. Perennial ryegrass was used to test phytotoxicity of experimental substrates. RESULTS: Soil neutralization to a pH of 6.5 with SG, with or without incorporation of AZ, significantly reduces metal (Cu and Zn) solubilization without affecting As solubilization in soil pore water; furthermore, it eliminates phytotoxicity and excessive metal(oid) accumulation in aerial plant tissues. Addition of B or Z to SG-amended soil does not further reduce metal solubilization into soil pore water, but increase As solubilization due to excessive soil neutralization (pH > 6.5); however, no significant As increase occurs in aerial plant tissues. CONCLUSION: Simultaneous in situ immobilization of metal(oid) in acidic topsoils is possible through aided phytostabilization.

Amendments promote the development of Lolium perenne in soils affected by historical copper smelting operations.

The Puchuncaví valley, central Chile, has been exposed to aerial emissions from a copper smelter. Nowadays, soils in the surroundings are sparsely-vegetated, acidic, and metal-contaminated, and their remediation is needed to reduce environmental risks. We assessed effectiveness of lime, fly ash, compost, and iron grit as amendments to immobilize Cu in soils and promote plant growth. Amended soils were cultivated with Lolium perenne for 60 days under controlled conditions. Total dissolved Cu and Cu2+ activity in the soil solution, ryegrass biomass, and Cu accumulation in plant tissues were measured. Addition of lime and fly ash decreased Cu concentrations and Cu2+ activity in the soil solution, increased plant biomass, and reduced shoot Cu concentration below 22 mg kg(-1) (the phytotoxicity threshold for the species). The most effective amendment with respect to the shoot biomass yield was a combination of lime and compost. Water content of the substrate and the K accumulation were positively correlated with the compost application rate. Compost combined with iron grit decreased dissolved Cu concentrations during the period of highest solubility, i.e., during the first 60 days after the compost application. However, iron grit incorporation into soils amended with lime and compost decreased the shoot biomass of ryegrass.