The bioaccumulation of Molybdenum in the earthworm Eisenia andrei: influence of soil properties and ageing

Mo bioaccumulation in the earthworm Eisenia andrei was determined after 28 d exposure in ten different European field soils (pH 4.4-7.8) and an artificial soil, freshly spiked with Na₂MoO₄ at concentrations between 3.2 and 3200 mg Mo kg⁻¹ dry soil. Three field soils were also tested after ageing for 11 months. Earthworm Mo concentrations generally levelled off at high exposure levels but in most soils showed a (nearly) linear increase with increasing soil concentrations in the lower, non-toxic range (below EC10 or NOEC for reproduction effects). Bioaccumulation (BAF) and Bioconcentration factors (BCF) were calculated as the ratio of earthworm concentration to soil and estimated porewater concentrations, respectively. BAFs (0.35-3.44) and BCFs (1.31-276) did not seem much affected by soil concentration, suggesting that earthworms are not capable of regulating their internal Mo concentrations. BAF was best predicted by ammonium oxalate-extractable iron (Fe_ox) and phosphor (P_ox) contents of the soils.     

Influence of activated carbon amendment on the accumulation and elimination of PCBs in the earthworm Eisenia fetida

In this study we investigated the use of activated carbon (AC) as a soil amendment for reducing bioavailability of polychlorinated biphenyls (PCBs) to the earthworm Eisenia fetida. Artificial soil was contaminated with PCBs and used in bioaccumulation experiments fresh or after aging for 19 months. PCB bioaccumulation in earthworms was reduced by 68% when AC was placed as a layer without mixing and by 94% when AC was manually mixed into the soil. Aging of the same AC mixed soil for 19 months resulted in an overall reduction of 99% in PCB biouptake. AC-treated aged soil also showed two orders of magnitude lower equilibrium aqueous concentrations of PCBs compared to untreated aged soils. The findings from this study indicate that application of engineered sorbents like AC to PCB impacted soils may greatly reduce PCB uptake at the base of the terrestrial food chain.     

Effects of spatial and temporal variation in metal availability on earthworms in floodplain soils of the river Dommel, The Netherlands

Regarding impact on ecological soil functioning, metal pollution is often considered a constant factor for certain sampling sites. However, especially bioavailable concentrations may differ in space and time. This aspect was investigated on four sites along a metal-polluted river, differing in soil characteristics and metal concentrations. Every four weeks earthworm densities, soil characteristics, and metal concentrations in soil and earthworms were determined. Earthworm biomass and density fluctuated in time and increased with increasing metal contamination, indicating the presence of compensating factors. Multivariate analysis suggested organic matter and moisture content to be the main factors explaining earthworm biomass. Metal concentrations in the earthworms increased with increasing total or 0.01M CaCl(2) extractable soil concentrations, but no time-related trends were seen. Cadmium concentrations in the earthworms exceeded background values, suggesting a potential risk. The neutral red retention biomarker assay, however, did not show any signs of metal stress in the earthworms.     

Interactions between sewage sludge-amended soil and earthworms—comparison between <Emphasis Type="Italic">Eisenia fetida</Emphasis> and <Emphasis Type="Italic">Eisenia andrei</Emphasis> composting species

Vermicomposting is an eco-friendly technology, where earthworms are introduced in the waste, inter alia sewage sludge, to cooperate with microorganisms and enhance decomposition of organic matter. The main aims of the present study was to determine the influence of two different earthworm species, Eisenia fetida and Eisenia andrei, on the changes of selected metallic trace elements content in substratum during vermicomposting process using three different sewage sludge mainly differentiated by their metal contents. Final vermicompost has shown a slight reduction in Cd, Cu, Ni, and Pb, while the Zn concentration tends to increase. Accumulation of particular heavy metals in earthworms’ bodies was assessed. Both species revealed high tendency to accumulate Cd and Zn, but not Cu, Ni, and Pb, but E. andrei has higher capabilities to accumulate some metals. Riboflavin content, which content varies depending on metal pollution in several earthworms species, was measured supravitaly in extruded coelomocytes. Riboflavin content decreased slightly during the first 6 weeks of exposure and subsequently restored till the end of the 9-week experiment. Selected agronomic parameters have also been measured in the final product (vermicompost) to assess the influence of earthworms on substratum.     

PAH-sequestration capacity of granular and powder activated carbon amendments in soil, and their effects on earthworms and plants

A field lysimeter study was carried out to investigate whether the amendment of 2% powder and granular activated carbon (PAC and GAC) to a soil with moderate PAH contamination had an impact on the PAH bioaccumulation of earthworms and plants, since AC is known to be a strong sorbent for organic pollutants. Furthermore, secondary effects of AC on plants and earthworms were studied through growth and nutrient uptake, and survival and weight gain. Additionally, the effect of AC amendments on soil characteristics like pH, water holding capacity, and the water retention curve of the soil were investigated. Results show that the amendment of 2% PAC had a negative effect on plant growth while the GAC increased the growth rate of plants. PAC was toxic to earthworms, demonstrated by a significant weight loss, while the results for GAC were less clear due to ambiguous results of a field and a parallel laboratory study. Both kinds of AC significantly reduced biota to soil accumulation factors (BSAFs) of PAHs in earthworms and plants. The GAC reduced the BSAFs of earthworms by an average of 47 ± 44% and the PAC amendment reduced them by 72 ± 19%. For the investigated plants the BSAFs were reduced by 46 ± 36% and 53 ± 22% by the GAC and PAC, respectively.     

Responses of earthworm to aluminum toxicity in latosol

Excess aluminum (Al) in soils due to acid rain leaching is toxic to water resources and harmful to soil organisms and plants. This study investigated adverse impacts of Al levels upon earthworms (Eisenia fetida) from the latosol (acidic red soil). Laboratory experiments were performed to examine the survival and avoidance of earthworms from high Al concentrations and investigate the response of earthworms upon Al toxicity at seven different Al concentrations that ranged from 0 to 300 mg kg^?1 over a 28-day period. Our study showed that the rate of the earthworm survival was 100 % within the first 7 days and decreased as time elapsed, especially for the Al concentrations at 200 and 300 mg kg^?1. A very good linear correlation existed between the earthworm avoidance and the soil Al concentration. There was no Al toxicity to earthworms with the Al concentration ≤50 mg kg^?1, and the toxicity started with the Al concentration ≥100 mg kg^?1. Low Al concentration (i.e., <50 mg kg^?1) enhanced the growth of the earthworms, while high Al concentration (>100 mg kg^?1) retarded the growth of the earthworms. The weight of earthworms and the uptake of Al by earthworms increased with the Al concentrations from 0 to 50 mg kg^?1 and decreased with the Al concentrations from 50 to 300 mg kg^?1. The protein content in the earthworms decreased with the Al concentrations from 0 to 100 mg kg^?1 and increased from 100 to 300 mg kg^?1. In contrast, the catalase (CAT) and superoxide dismutase (SOD) activities in the earthworms increased with the Al concentrations from 0 to 100 mg kg^?1 and decreased from 100 to 300 mg kg^?1. The highest CAT and SOD activities and lowest protein content were found at the Al concentration of 100 mg kg^?1. Results suggest that a high level of Al content in latosol was harmful to earthworms.     

Lead and zinc bioavailability to Eisenia fetida after phosphorus amendment to repository soils

Four phosphorus forms were investigated as potential soil amendments to decrease the bioavailability of Pb and Zn in two repository soils to the earthworm, Eisenia fetida. Treatments were evaluated by examining differences in bioaccumulation factors between amended and non-amended soils. Triple super phosphate at 5000 mg P/kg decreased both Pb and Zn bioavailability in both soils. Rock phosphate at 5000 mg P/kg decreased Zn bioavailability, but not Pb bioavailability in both repository soils. Monocalcium phosphate and tricalcium phosphate at 5000 mg P/kg did not significantly decrease Pb or Zn bioavailability to earthworms in either repository soil. In order to optimize phosphorus amendments, additional phosphorus (up to 15,000 mg P/kg) and lowered pH were used in a series of tests. The combination of lowering the pH below 6.0 and increasing phosphorus concentrations caused complete mortality in all triple super phosphate amended soils and partial mortality in the highest rock phosphate amended soils. Results indicate that triple super phosphate and rock phosphate are viable soil amendments, but care should be taken when optimizing amendment quantity and pH so that adverse environmental effects are not a by-product.     

Agro-industrial wastes as effective amendments for ecotoxicity reduction and soil health improvement in aided phytostabilization

Aided phytostabilization is a technology that uses metal tolerant plants and organic and/or inorganic amendments to reduce soil metal bioavailability, while improving soil health. Our objective was to determine the effects of the application of amendments [sheep manure (SHEEP), poultry litter (POULTRY), cow slurry (COW), and paper mill sludge mixed with poultry litter (PAPER)], together with the growth of a metallicolous Festuca rubra L. population, on (i) chemical and microbial indicators of soil health and (ii) soil ecotoxicity, during the aided phytostabilization of a Zn/Pb contaminated mine soil. Amendment application led to an increase in soil pH, organic matter content, and inorganic salts, resulting in a decrease in Pb and Zn CaCl₂-extractable concentrations in soil, which, in turn, contributed to lower ecotoxicity and a stimulation of plant growth and soil microbial communities. The factor most affecting the metal extractability was probably soil pH. POULTRY was the best amendment in terms of increasing plant growth, chlorophylls content, and soil microbial biomass and activity, but resulted in higher levels of phytoavailable Pb and Zn. SHEEP and PAPER were more effective at reducing metal CaCl₂-extractability and, consequently, led to lower values of metal accumulation in plant tissues, thereby reducing the risk of metals entering into the food chain. When combined with the application of organic amendments, the metallicolous F. rubra population studied here appears an excellent candidate for aided phytostabilization. Our results indicate that the application of organic amendments is essential for the short-term recovery of highly contaminated metalliferous soils during aided phytostabilization.     

Batch and column studies of the stabilization of toxic heavy metals in dredged marine sediments by hematite after bioremediation

The management of dredged sediments is an important issue in coastal regions where the marine sediments are highly polluted by metals and organic pollutants. In this paper, mineral-based amendments (hematite, zero-valent iron and zeolite) were used to stabilize metallic pollutants (As, Cd, Cu, Mo, Ni, Pb, and Zn) in a contaminated marine sediment sample. Mineral-based amendments were tested at three application rates (5 %, 10 %, and 15 %) in batch experiments in order to select the best amendment to perform column experiments. Batch tests have shown that hematite was the most efficient amendment to stabilize inorganic pollutants (As, Cd, Cu, Mo, Ni, Pb, and Zn) in the studied sediment. Based on batch tests, hematite was used at one application rate equal to 5 % to conduct column experiments. Column tests confirmed that hematite was able to decrease metal concentrations in leachates from stabilized sediment. The stabilization rates were particularly high for Cd (67 %), Mo (80 %), and Pb (90 %). The Microtox solid phase test showed that hematite could decrease significantly the toxicity of stabilized sediment. Based on batch and column experiments, it emerged that hematite could be a suitable adsorbent to stabilize metals in dredged marine sediment.     

Utilisation of chemically stabilized arsenic-contaminated soil in a landfill cover

The aim of the study was to determine if an As-contaminated soil, stabilized using zerovalent iron (Fe⁰) and its combination with gypsum waste, coal fly ash, peat, or sewage sludge, could be used as a construction material at the top layer of the landfill cover. A reproduction of 2 m thick protection/vegetation layer of a landfill cover using a column setup was used to determine the ability of the amendments to reduce As solubility and stimulate soil functionality along the soil profile. Soil amendment with Fe⁰ was highly efficient in reducing As in soil porewater reaching 99 % reduction, but only at the soil surface. In the deeper soil layers (below 0.5 m), the Fe treatment had a reverse effect, As solubility increased dramatically exceeding that of the untreated soil or any other treatment by one to two orders of magnitude. A slight bioluminescence inhibition of Vibrio fischeri was detected in the Fe⁰ treatment. Soil amendment with iron and peat showed no toxicity to bacteria and was the most efficient in reducing dissolved As in soil porewater throughout the 2 m soil profile followed by iron and gypsum treatment, most likely resulting from a low soil density and a good air diffusion to the soil. The least suitable combination of soil amendments for As immobilization was a mixture of iron with coal fly ash. An increase in all measured enzyme activities was observed in all treatments, particularly those receiving organic matter. For As to be stable in soil, a combination of amendments that can keep the soil porous and ensure the air diffusion through the entire soil layer of the landfill cover is required.     

Physiological and molecular responses of the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> to polychlorinated biphenyl contamination in soil

Polychlorinated biphenyls (PCBs) are a class of man-made organic compounds ubiquitously present in the biosphere. In this study, we evaluated the toxic effects of different concentrations of PCBs in two natural soils (i.e. red soil and fluvo-aquic soil) on the earthworm Eisenia fetida. The parameters investigated included anti-oxidative response, genotoxic potential, weight variation and biochemical responses of the earthworm exposed to two different types of soils spiked with PCBs after 7 or 14 days of exposure. Earthworms had significantly lower weights in both soils after PCB exposure. PCBs significantly increased catalase (CAT), superoxide dismutase (SOD), and guaiacol peroxidase (POD) activity in earthworms exposed to either soil type for 7 or 14 days and decreased the malondialdehyde (MDA) content in earthworms exposed to red soil for 14 days. Of the enzymes examined, SOD activity was the most sensitive to PCB stress. In addition, PCB exposure triggered dose-dependent coelomocyte DNA damage, even at the lowest concentration tested. This response was relatively stable between different soils. Three-way analysis of variance (ANOVA) showed that the weight variation, anti-oxidant enzyme activities, and MDA contents were significantly correlated with exposure concentration or exposure duration (P < 0.01). Furthermore, weight variation, CAT activity, and SOD activity were significantly affected by soil type (P < 0.01). Therefore, the soil type and exposure time influence the toxic effects of PCBs, and these factors should be considered when selecting responsive biomarkers.     

Impact of the earthworm Lumbricus terrestris (L.) on As, Cu, Pb and Zn mobility and speciation in contaminated soils

To assess the risks that contaminated soils pose to the environment properly a greater understanding of how soil biota influence the mobility of metal(loid)s in soils is required. Lumbricus terrestris L. were incubated in three soils contaminated with As, Cu, Pb and Zn. The concentration and speciation of metal(loid)s in pore waters and the mobility and partitioning in casts were compared with earthworm-free soil. Generally the concentrations of water extractable metal(loid)s in earthworm casts were greater than in earthworm-free soil. The impact of the earthworms on concentration and speciation in pore waters was soil and metal specific and could be explained either by earthworm induced changes in soil pH or soluble organic carbon. The mobilisation of metal(loid)s in the environment by earthworm activity may allow for leaching or uptake into biota.     

Properties of biochars from conventional and alternative feedstocks and their suitability for metal immobilization in industrial soil

In contaminated soils, excessive concentrations of metals and their high mobility pose a serious environmental risk. A suitable soil amendment can minimize the negative effect of metals in soil. This study investigated the effect of different biochars on metal (Cu, Pb, Zn) immobilization in industrial soil. Biochars produced at 300 and 600 °C from conventional (MS, maize silage; WP, wooden pellets) and alternative (SC, sewage sludge compost; DR, digestate residue) feedstocks were used as soil amendments at a dosage of 10 % (w/w). The type of feedstock and pyrolysis temperature affected the properties of the biochars and their ability to immobilize metal in soil. Compared to production at 300 °C, all biochars produced at 600 °C had higher pH (6.2–10.7), content of ash (7.2–69.0 %) and fixed carbon (21.1–56.7 %), but lower content of volatile matter (9.7–37.2 %). All biochars except DR biochar had lower dissolved organic carbon (DOC) content (1.4–2.3 g C/L) when made at 600 °C. Only MS and SC biochars had higher cation exchange capacity (25.2 and 44.7 cmol/kg, respectively) after charring at 600 °C. All biochars contained low concentrations of Cd, Cu, Ni, Pb and Zn; Cd was volatilized to the greatest extent during pyrolysis. Based on FTIR analysis and molar ratios of H/C and O/C, biochars had a greater degree of carbonization and aromaticity after charring at 600 °C. The efficiency of the biochars in metal immobilization depended mainly on their pH, ash content, and concentration of DOC. SC and DR biochars were more effective for Cu and Zn immobilization than MS and WP biochars, which makes them attractive options for large-scale soil amendment.     

Bioremediation trial on aged PCB-polluted soils—a bench study in Iceland

Polychlorinated biphenyls (PCBs) pose a threat to the environment due to their high adsorption capacity to soil organic matter, stability and low reactivity, low water solubility, toxicity and ability to bioaccumulate. With Icelandic soils, research on contamination issues has been very limited and no data has been reported either on PCB degradation potential or rate. The goals of this research were to assess the bioavailability of aged PCBs in the soils of the old North Atlantic Treaty Organization facility in Keflavík, Iceland and to find the best biostimulation method to decrease the pollution. The effectiveness of different biostimulation additives (N fertiliser, white clover and pine needles) at different temperatures (10 and 30 °C) and oxygen levels (aerobic and anaerobic) were tested. PCB bioavailability to soil fauna was assessed with earthworms (Eisenia foetida). PCBs were bioavailable to earthworms (bioaccumulation factor 0.89 and 0.82 for earthworms in 12.5 ppm PCB soil and in 25 ppm PCB soil, respectively), with less chlorinated congeners showing higher bioaccumulation factors than highly chlorinated congeners. Biostimulation with pine needles at 10 °C under aerobic conditions resulted in nearly 38 % degradation of total PCBs after 2 months of incubation. Detection of the aerobic PCB degrading bphA gene supports the indigenous capability of the soils to aerobically degrade PCBs. Further research on field scale biostimulation trials with pine needles in cold environments is recommended in order to optimise the method for onsite remediation.     

Contrasting effects of silicates on cadmium uptake by three dicotyledonous crops grown in contaminated soil

The effects of several silicates (talcum powder (TP), calcium silicate (CS), sodium silicate (SS), and potassium silicate (PS)), in comparison with other amendments (quicklime (QL) and potassium dihydrogen phosphate (PDP)) on cadmium (Cd) uptake by three dicotyledonous crops (Amaranthus hypochondriacus L. Cv. ‘K112’, Amaranthus tricolor L., and Brassica oleracea var. albiflora Kuntze) were investigated in Cd–contaminated soil. The effects of both application methods of amendments (singly and combined) and timing of application were also evaluated. Sodium silicate was the most effective in reducing crop Cd uptake and translocation, which was diminished by 51 % in roots, 53 % in stems, and 72 % in leaves on average. Application of CS amendment showed greater efficiency than PDP amendment in decreasing Cd uptake by crops and resulted in increased biomass. Potassium silicate only slightly decreased shoot Cd concentration. Combination of PDP and SS was able to overcome the inhibitory effect of SS on crop yield while decreasing Cd concentrations in roots, stems and leaves of the tested crops by average rates of 52, 65, and 68 % respectively. Applications of SS and PS significantly reduced the root-to-shoot Cd transfer factor. We found that Si accumulation in crops was not associated with lower Cd concentration, indicating that Si in crops may play a major role in alleviating metal stress rather than inhibiting crop Cd accumulation. We suggested that the inhibitive effect of silicates on crops Cd uptake was majorly attributed to the properties of the silicates, those were their specific effects on soil pH and cations, which increased Cd adsorption by soil and suppressed Cd uptake from soil solution by increasing the relative dissolved concentrations of competing cations.     

Earthworms as biological monitors of cadmium, copper, lead and zinc in metalliferous soils

Earthworms (Lumbricus rebellus and Dendrodrilus rubidus) were sampled from one uncontaminated and fifteen metal-contaminated sites. Significant positive correlations were found between the earthworm and 'total' (conc. nitric acid-extractable) soil Cd, Cu, Pb and Zn concentrations (data log1) transformed). The relationships were linear, and the accumulation patterns for both species were similar when a single metal was considered, even though there were species difference in mean metal concentrations. Generally, the earthworm Cd concentration exceeded that of the soil; by contrast, the worm Pb concentration was lower than the soil Pb concentration in all but one (acidic, low soil Ca) site. Our observations suggest that Cu and Zn accumulation may be physiologically regulated by both species. Total-soil Cd explained 82-86% of the variability (V2) in earthworm Cd concentration; 52-58% of worm Pb and worm Zn concentrations were explained by the total-soil concentrations of the respective metals. Total-soil Cu explained only 11-32% of the worm Cu concentration. The effect of soil pH, total Ca concentration, cation-exchange capacity (CEC) and organic carbon on metal accumulation by L. rubellus and D. rubidus was investigated by multiple regression analysis. Soil pH (coupled with CEC) and soil Ca had a major influence on Pb accumulation (V2 of worm Pb increased to 77-83%), and there was some evidence that Cd accumulation may be suppressed in extremely organic soils. The edaphic factors investigated had no effect on Cu or Zn accumulation by earthworms. In the context of biomonitoring, it is proposed that earthworms have a potential in a dual role: (1) as 'quantitative' monitors of total-soil metal concentrations (as shown for Cd); and (2) as estimators of 'ecologically significant' soil metal, integrating the effects of edaphic factors (as shown for Pb).     

Influence of plant-earthworm interactions on SOM chemistry and p,p'-DDE bioaccumulation

Laboratory experiments assessed how bioaccumulation of weathered p,p′-DDE from soil and humic acid (HA) chemistry are affected by interactions between the plants Cucurbita pepo ssp. pepo and ssp. ovifera and the earthworms Eisenia fetida, Lumbricus terrestris, and Apporectodea caliginosa. Total organochlorine phytoextraction by ssp. pepo increased at least 25% in the presence of any of the earthworm species (relative to plants grown in isolation). Uptake of the compound by ssp. ovifera was unaffected by earthworms. Plants influenced earthworm bioaccumulation as well. When combined with pepo, p,p′-DDE levels in E. fetida decreased by 50%, whereas, in the presence of ovifera, bioconcentration by L. terrestris increased by more than 2-fold. Spectral analysis indicated a decrease in hydrophobicity of HA in each of the soils in which both pepo and earthworms were present. However, HA chemistry from ovifera treatments was largely unaffected by earthworms. Risk assessments of contaminated soils should account for species interactions, and SOM chemistry may be a useful indictor of pollutant bioaccumulation.     

Evaluation of epoxiconazole bioavailability in soil to the earthworm <Emphasis Type="Italic">Aporrectodea icterica</Emphasis>

In soil, the determination of total concentration using an exhaustive extraction method has little relevance to evaluate the exposure of an organism to a chemical, because of sorption processes. This study aims to propose a mild extraction method to evaluate the bioavailability of the fungicide epoxiconazole to the earthworm Aporrectodea icterica. Experiments were conducted in soils presenting various textures and organic carbon contents, spiked with formulated epoxiconazole 7 to 56 days prior to their extraction. In parallel, the epoxiconazole concentration was determined in exposed earthworms and the fungicide’s effects were evaluated by measuring weight gain, enzymatic activities and total protein contents. Among the various mild chemical solvents tested to evaluate the environmental availability of the fungicide, the 50 mM hydroxypropyl-β-cyclodextrin solution allowed to extract around 30 % of epoxiconazole. This percentage corresponded to the ratio determined in exposed A. icterica under similar soil conditions. Furthermore, this mild method was demonstrated to be sensitive to soil sorption capacities and to ageing. The mild extraction method was then applied to explore the relationship between total and (bio)available concentrations in soil and in A. icterica, over 7- or 28-day exposure time. This demonstrated the proportionality between epoxiconazole concentration in earthworm and available in soil (up to 96 %, with regression coefficient R ²?=?0.98). Sublethal effects on earthworm remained not significant.     

Assessing the chemical and biological accessibility of the herbicide isoproturon in soil amended with biochar

Decamethylcyclopentasiloxane (D5) is a cyclic volatile methyl siloxane (cVMS) commonly found in commercially available products. D5 is expected to enter the terrestrial environment through the deposit of biosolids from sewage treatment plants onto agricultural fields for nutrient enrichment. Little to no information currently exists as to the risks of D5 to the terrestrial environment. In order to evaluate the potential risk to terrestrial organisms, the toxicity of a D5 contaminated biosolid in an agricultural soil was assessed with a battery of standardized soil toxicity tests.D5 was spiked into a surrogate biosolid and then mixed with a sandy loam soil to create test concentrations ranging from 0 to 4074 mg kg⁻¹. Plant (Hordeum vulgare (barley) and Trifolium pratense (red clover)) and soil invertebrates (Eisenia andrei (earthworm) and Folsomia candida (springtail)) toxicity tests were completed to assess for lethal and sub-lethal effects. Plant testing evaluated the effects on seedling emergence, shoot and root length, and shoot and root dry mass. Invertebrate test endpoints included adult lethality, juvenile production, and individual juvenile dry mass (earthworms only). Soil samples were collected over time to confirm test concentrations and evaluate the loss of chemical over the duration of a test. The toxicity of the D5 was species and endpoint dependent, such that no significant adverse effects were observed for T. pratense or E. andrei test endpoints, however, toxicity was observed for H. vulgare plant growth and F. candida survival and reproduction. Chemical losses of up to 50% were observed throughout the tests, most significantly at high concentrations.