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Background Biochar-based fertilizer products (BCF) have been reported to increase both crop yield and N-use efficiency. Such positive effects are often assumed to result from the slow-release of N adsorbed on BCF structures. However, a careful review of the literature suggests that actual mechanisms remain uncertain, which hampers the development of efficient BCF products. Scope Here, we aim at reviewing BCF mechanisms responsible for enhanced N uptake by plants, and evaluate the potential for further improvement. We review the capacity of biochar structures to adsorb and release N forms, the biochar properties supporting this effect, and the methods that have been proposed to enhance this effect. Conclusions Current biochar products show insufficient sorption capacity for the retention of N forms to support the production of slow-release BCFs of high enough N concentration. Substantial slow-release effects appear to require conventional coating technology. Sorption capacity can be improved through activation and additives, but currently not to the extent needed for concentrated BCFs. Positive effects of commercial BCFs containing small amount of biochar appear to result from pyrolysis-derived biostimulants. Our review highlights three prospects for improving N retention: 1) sorption of NH3 gas on specifically activated biochar, 2) synergies between biochar and clay porosities, which might provide economical sorption enhancement, and 3) physical loading of solid N forms within biochar. Beyond proof of concept, quantitative nutrient studies are needed to ascertain that potential future BCFs deliver expected effects on both slow-release and N use efficiency.

Biogas digestate is a nitrogen (N) rich waste product that has potential for application to soil as a fertilizer. Composting of digestate is recognized as an effective step to reduce potentially negative consequences of digestate application to soils. However, the structure of the digestate and the high N content can hinder effective composting. Biochar, which can be produced through the pyrolysis of waste biomass, has shown the potential to improve compost structure and increase N retention in soils. We studied how a high-temperature wood biochar affects the composting process, including greenhouse gas emissions, and the fertilizer value of the compost product including nutrient content, leachability and plant growth. The high Biochar dose (17% w/w) had a significantly positive effect on the maximum temperature (5°C increase vs. no biochar) and appeared to improve temperature stability during composting with less variability between replicates. Biochar addition reduced cumulative N2O emission by 65–70%, but had no significant effect on CO2 and CH4 emission. Biochar did not contribute to greater retention of nitrogen (N) contained in the digestate, but had a dilution effect on both N content and mineral nutrients. Fertilization with compost enhanced plant growth and nutrient retention in soil compared to mineral fertilization (NPK), but biochar had no additional effects on these parameters. Our results show that biochar improves the composting of digestate with no subsequent negative effects on plants.

Arbuscular mycorrhizal fungi (AMF) are important in plant nutrient uptake, but their function is prone to environmental constraints including soil factors that may suppress AMF transfer of phosphorus (P) from the soil to the plant. The objective of this study was to disentangle the biotic and abiotic components of AMF-suppressive soils. Suppression was measured in terms of AMF-mediated plant uptake of 33P mixed into a patch of soil and treatments included soil sterilization, soil mixing, pH manipulation and inoculation with isolated soil fungi. The degree of suppression was compared to volatile organic compound (VOC) production by isolated fungi and to multi-element analysis of soils. For a selected suppressive soil, sterilization and soil mixing experiments confirmed a biotic component of suppression. A Fusarium isolate from that soil suppressed the AMF activity and produced greater amounts than other fungal isolates of the antimicrobial VOC trichodiene (a trichothecene toxin precursor), beta-chamigrene, alpha-cuprenene and p-xylene. These metabolites deserve further attention when unravelling the chemical background behind the suppression of AMF activity by soil microorganisms. For the abiotic component of suppression, soil liming and acidification experiments confirmed that suppression was strongest at low pH. The pH effect might be associated with changed availability of specific suppressive elements. Indeed 33P uptake from the soil patches correlated negatively to Al levels and Al toxicity seems to play a major role in the AMF suppressiveness at pH below 5.0–5.2. However, the documentation of a biotic component of suppression for both low and high pH soils leads to the conclusion that biotic and abiotic components of suppression may act in parallel in some soils. The current insight into the components of soil suppressiveness of the AMF activity aids to develop management practices that allow for optimization of AMF functionality.

We investigated dissipation, earthworm and plant accumulation of organic contaminants in soil amended with three types of sewage sludge in the presence and absence of plants. After 3 months, soil, plants and earthworms were analyzed for their content of organic contaminants. The results showed that the presence of plant roots did not affect dissipation rates, except for galaxolide. Transfer of galaxolide and triclosan to earthworms was significant, with transfer factors of 10–60 for galaxolide and 140–620 for triclosan in the presence of plants. In the absence of plants, transfer factors were 2–9 times higher. The reduced transfer to worms in the presence of plants was most likely due to roots serving as an alternative food source. Nonylphenol monoethoxylate rapidly dissipated in soil, but initial exposure resulted in uptake in worms, which was detected even 3 months after sewage sludge application. These values were higher than the soil concentration at the start of the exposure period. This indicates that a chemical's short half-life in soil is no guarantee that it poses a minimal environmental risk, as even short-term exposure may cause bioaccumulation and risks for chronic or even transgenerational effects.

Heavy metals in soil pose a constant risk for animals and humans when entering their food chains, and limited means are available to reduce plant accumulation from more or less polluted soils. Biochar, which is made by pyrolysis of organic residues and sees increasing use as a soil amendment to mitigate anthropogenic C emissions and improve agronomic soil properties, has also been shown to reduce plant availability of heavy metals in soils. The cause for the reduction of metal uptake in plants when grown in soils enriched with biochar has generally been researched in terms of increased pH and alkalinity, while other potential mechanisms have been less studied. We conducted a pot experiment with barley using three soils differing in metal content and amended or not with 2% biochar made from Miscanthus x giganteus, and assessed plant contents and changes in bioavailability in bulk and rhizosphere soil by measuring extractability in acetic acid or ammonium nitrate. In spite of negligible pH changes upon biochar amendment, the results showed that biochar reduced extractability of Cu, Pb and Zn, but not of Cd. Rhizosphere soil contained more easily extractable Cu, Pb and Zn than bulk soil, while for Cd it did not. Generally, reduced plant uptake due to biochar was reflected in the amounts of metals extractable with ammonium nitrate, but not acetic acid.

Remediation using nanoparticles depends on proper documentation of safety aspects, one of which is their ecotoxicology. Ecotoxicology of nanoparticles has some special features: while traditional ecotoxicology aims at measuring possible negative effects of more or less soluble chemicals or dissolved elements, nanoecotoxicology aims at measuring the toxicity of particles, and its main focus is on effects that are unique to nano-sized particles, as compared to larger particles or solutes. One of the main challenges when testing the ecotoxicity of nanoparticles lies in maintaining stable and reproducible exposure conditions, and adapt these to selected test organisms and endpoints. Another challenge is to use test media that are relevant to the matrices to be treated. Testing of nanoparticles used for remediation, particularly red-ox-active Fe-based nanoparticles, should also make sure to exclude confounding effects of altered red-ox potential which are not nanoparticle-specific. Yet another unique aspect of nanoparticles used for remediation is considerations of ageing of nanoparticles in soil or water, leading to reduced toxicity over field-relevant time scales. This review discusses these and other aspects of how to design and interpret appropriate tests and use these in hazard descriptions for subsequent risk assessments. 30.09.2021

The potential impact of silver nanoparticles (Ag NPs) on aquatic organisms is to a large extent determined by their bioavailability through different routes of exposure. In the present study juvenile Atlantic salmon (Salmo salar) were exposed to different sources of radiolabeled Ag (radiolabeled 110m Ag NPs and 110m AgNO3 ). After 48 h of waterborne exposure to 3 μg/L citrate stabilized 110m Ag NPs or 110m AgNO3 , or a dietary exposure to 0.6 mg Ag/kg fish (given as citrate stabilized or uncoated 110m Ag NPs, or 110m AgNO3 ), Ag had been taken up in fish regardless of route of exposure or source of Ag (Ag NPs or AgNO3 ). Waterborne exposure led to high Ag concentrations on the gills, and dietary exposure led to high concentrations in the gastrointestinal tract. Silver distribution to the target organs was similar for both dietary and waterborne exposure, with the liver as the main target organ. The accumulation level of Ag was 2 to 3 times higher for AgNO3 than for Ag NPs when exposure was through water, whereas no significant differences were seen after dietary exposure. The transfer (Bq/g liver/g food or water) from exposure through water was 4 orders of magnitude higher than from feed using the smallest, citrate-stabilized Ag NPs (4 nm). The smallest NPs had a 5 times higher bioavailability in food compared with the larger and uncoated Ag NPs (20 nm). Despite the relatively low transfer of Ag from diet to fish, the short lifetime of Ag NPs in water and their transfer to sediment, feed, or sediment-dwelling food sources such as larvae and worms could make diet a significant long-term exposure route. Environ Toxicol Chem 2018;37:2895-2903. © 2018 SETAC.

Arbuscular mycorrhizal fungi (AMF) colonise roots of most plants; their extra-radical mycelium (ERM) extends into the soil and acquires nutrients for the plant. The ERM coexists with soil microbial communities and it is unresolved whether these communities stimulate or suppress the ERM activity. This work studied the prevalence of suppressed ERM activity and identified main components behind the suppression. ERM activity was determined by quantifying ERM-mediated P uptake from radioisotope-labelled unsterile soil into plants, and compared to soil physicochemical characteristics and soil microbiome composition. ERM activity varied considerably and was greatly suppressed in 4 of 21 soils. Suppression was mitigated by soil pasteurisation and had a dominating biotic component. AMF-suppressive soils had high abundances of Acidobacteria, and other bacterial taxa being putative fungal antagonists. Suppression was also associated with low soil pH, but this effect was likely indirect, as the relative abundance of, e.g., Acidobacteria decreased after liming. Suppression could not be transferred by adding small amounts of suppressive soil to conducive soil, and thus appeared to involve the common action of several taxa. The presence of AMF antagonists resembles the phenomenon of disease-suppressive soils and implies that ecosystem services of AMF will depend strongly on the specific soil microbiome.

Green roofs are used increasingly to alleviate peaks of water discharge into the sewage systems in urban areas. Surface runoff from roofs contain pollutants from dry and wet deposition, and green roofs offer a possibility to reduce the amounts of pollutants in the water discharged from roofs by degradation and filtering. These pollutants would otherwise enter wastewater treatments plants and ultimately end up in sewage sludge that is spread on agricultural soils. The most common substrates used in green roofs have limited capacity for filtration and sorption. Also, more sustainable alternatives are sought, due to the high carbon footprint of these materials. Biochar is a carbon-rich material produced by pyrolysis of biomass, and several types of biochar have been described as good sorbents and filter materials. Biochar is also a light and carbon negative material, which may fulfill other desired criteria for new green roof substrates. We here report on an experiment where two types of biochar, produced from olive husks at 450 °C or from forest waste at 850 ° C were mixed with volcanic rock or peat, and tested for retention capacity of phenanthrene and six heavy metals in a column experiment with unsaturated gravimetric water flow lasting for 3 weeks. The results suggest that biochar as a component in green roof substrates perform better than traditional materials, concerning retention of the tested pollutants, and that different types of biochar have different properties in this respect. Electronic supplementary material The online version of this article (10.1007/s11356-017-0650-6) contains supplementary material, which is available to authorized users.

Nanoremediation with iron (Fe) nanomaterials opens new doors for treating contaminated soil and groundwater, but is also accompanied by new potential risks as large quantities of engineered nanomaterials are introduced into the environment. In this study, we have assessed the ecotoxicity of four engineered Fe nanomaterials, specifically, Nano-Goethite, Trap-Ox Fe-zeolites, Carbo-Iron® and FerMEG12, developed within the European FP7 project NanoRem for sub-surface remediation towards a test battery consisting of eight ecotoxicity tests on bacteria (V. fisheri, E. coli), algae (P. subcapitata, Chlamydomonas sp.), crustaceans (D. magna), worms (E. fetida, L. variegatus) and plants (R. sativus, L. multiflorum). The tested materials are commercially available and include Fe oxide and nanoscale zero valent iron (nZVI), but also hybrid products with Fe loaded into a matrix. All but one material, a ball milled nZVI (FerMEG12), showed no toxicity in the test battery when tested in concentrations up to 100 mg/L, which is the cutoff for hazard labeling in chemicals regulation in Europe. However it should be noted that Fe nanomaterials proved challenging to test adequately due to their turbidity, aggregation and sedimentation behavior in aqueous media. This paper provides a number of recommendations concerning future testing of Fe nanomaterials and discusses environmental risk assessment considerations related to these.

Sewage sludge is an important amendment that enriches soils with organic matter and provides plants with nutrients such as nitrogen and phosphorus. However, knowledge on the fate and effects of organic pollutants present in the sludge on soil organisms is limited. In the present study, the uptake of triclosan, galaxolide, and tonalide in the earthworm Dendrobaena veneta was measured 1 wk after amendment of agricultural soil with sewage sludge, while elimination kinetics were assessed over a 21-d period after transferring worms to clean soil. After 1-wk exposure, earthworms had accumulated 2.6 ± 0.6 μg g-1 galaxolide, 0.04 ± 0.02 μg g-1 tonalide, and 0.6 ± 0.2 μg g-1 triclosan. Both synthetic musks were efficiently excreted and below the limit of quantification after 3 and 14 d of depuration for tonalide and galaxolide, respectively. Triclosan concentrations, on the other hand, did not decrease significantly over the depuration period, which may lead to the transfer of triclosan in the food web. Environ Toxicol Chem 2017;36:2068-2073. © 2017 SETAC.

The growing number of nanotechnology products on the market will inevitably lead to the release of engineered nanomaterials with potential risk to humans and environment. This study set out to investigate the exposure of soil biota to engineered nanoparticles (NPs). Cerium dioxide (CeO2 NPs) and tin dioxide nanoparticles (SnO2 NPs) were radiolabelled using neutron activation, and employed to assess the uptake and excretion kinetics in the earthworm Eisenia fetida. Through sequential extraction, NPs bioavailability in two contrasting soils and in earthworm feed was also investigated. Neither CeO2 NPs nor SnO2 NPs bioaccumulated in earthworms, and both were rapidly excreted when worms were transferred to clean soil. Low bioavailability was also indicated by low amounts of NPs recovered during extraction with non-stringent extractants. CeO2 NPs showed increasing mobility in organic soil over time (28 days), indicating that organic matter has a strong influence on the fate of CeO2 NPs in soil.

The potential impact of silver nanoparticles (Ag NPs) on aquatic organisms is to a large extent determined by their bioavailability through different routes of exposure. In the present study juvenile Atlantic salmon (Salmo salar) were exposed to different sources of radiolabeled Ag (radiolabeled

Two types of nano-scale zero-valent iron (nZVI-B prepared by borohydride reduction and nZVI-T produced by thermal reduction of iron oxide nanoparticles in H2) and a micro-scale ZVI (mZVI) were compared for PCB degradation efficiency in water and soil. In addition, the ecotoxicity of nZVI-B and nZVI-T particles in treated water and soil was evaluated on bacteria, plants, earthworms, and ostracods. All types of nZVI and mZVI were highly efficient in degradation of PCBs in water, but had little degradation effect on PCBs in soil. Although nZVI-B had a significant negative impact on the organisms tested, treatment with nZVI-T showed no negative effect, probably due to surface passivation through controlled oxidation of the nanoparticles.

Sewage sludge is an important amendment that enriches soils with organic matter and provides plants with nutrients such as nitrogen and phosphorus. However, knowledge on the fate and effects of organic pollutants present in the sludge on soil organisms is limited. In the present study, the uptake of triclosan, galaxolide, and tonalide in the earthworm Dendrobaena veneta was measured 1 wk after amendment of agricultural soil with sewage sludge, while elimination kinetics were assessed over a 21-d period after transferring worms to clean soil. After 1-wk exposure, earthworms had accumulated 2.6 ± 0.6 μg g

Sewage sludge application on soils represents an important potential source of silver nanoparticles (Ag NPs) to terrestrial ecosystems, and it is thus important to understand the fate of Ag NPs once in contact with soil components. Our aim was to compare the behavior of three different forms of silver, namely silver nitrate, citrate stabilized Ag NPs (5 nm) and uncoated Ag NPs (19 nm), in two soils with contrasting organic matter content, and to follow changes in binding strength over time. Soil samples were spiked with silver and left to age for 2 h, 2 days, 5 weeks or 10 weeks before they were submitted to sequential extraction. The ionic silver solution and the two Ag NP types were radiolabeled so that silver could be quantified by gamma spectrometry by measuring the 110m Ag tracer in the different sequential extraction fractions. Different patterns of partitioning of silver were observed for the three forms of silver. All types of silver were more mobile in the mineral soil than in the soil rich in organic matter, although the fractionation patterns were very different for the three silver forms in both cases. Over 20% of citrate stabilized Ag NPs was extractible with water in both soils the first two days after spiking (compared to 1–3% for AgNO 3 and uncoated Ag NPs), but the fraction decreased to trace levels thereafter. Regarding the 19 nm uncoated Ag NPs, 80% was not extractible at all, but contrary to AgNO 3 and citrate stabilized Ag NPs, the bioaccessible fraction increased over time, and by day 70 was between 8 and 9 times greater than that seen in the other two treatments. This new and unexpected finding demonstrates that some Ag NPs can act as a continuous source of bioaccessible Ag, while AgNO 3 is rapidly immobilized in soil.

Due to difficulties in tracing engineered nanoparticles (ENPs) in complex media, there are few data on the exposure of soil biota to ENPs. This study used neutron activated cobalt (Co NPs) and silver (Ag NPs) nanoparticles, as well as soluble cobalt and silver salts, to assess the uptake, excretion and biodistribution in the earthworm Eisenia fetida. Concentrations of cobalt in worms after four weeks exposure reached 88% and 69% of the Co ions and Co NPs concentrations in food, respectively, while corresponding values for Ag ions and Ag NPs were 2.3% and 0.4%. Both Ag ions and Ag NPs in earthworms were excreted rapidly, while only 32% of the cobalt accumulated from Co ions and Co NPs were excreted within four months. High accumulation of cobalt was found in blood and in the digestive tract. Metal characterization in the exposure medium was assessed by sequential extraction and ultrafiltration. The Co NPs showed significant dissolution and release of ions, while Ag ions and particularly Ag NPs were more inert.

In terrestrial ecotoxicology there is a serious lack of data for potential hazards posed by engineered nanoparticles (ENPs). This is partly due to complex interactions between ENPs and the soil matrix, but also to the lack of suitable toxicological end points in organisms that are exposed to ENPs in a relevant manner. Earthworms are key organisms in terrestrial ecosystems, but so far only physiological end points of low sensitivity have been used in ecotoxicity studies with ENPs. We exposed the earthworm Lumbricus terrestris to silver nanoparticles and measured their impact on apoptosis in different tissues. Increased apoptotic activity was detected in a range of tissues both at acute and sublethal concentrations (down to 4 mg/kg soil). Comparing exposure in water and soil showed reduced bioavailability in soil reflected in the apoptotic response. Apoptosis appears to be a sensitive end point and potentially a powerful tool for quantifying environmental hazards of ENPs.

The potential environmental toxicity of zero-valent iron nanoparticles (nZVI) and three types of nanosilver differing in average particle size from 1 to 20 nm was evaluated using seed germination tests with ryegrass, barley, and flax exposed to 0-5000 mg L(-1) nZVI or 0-100 mg L(-1) Ag. For nZVI, germination tests were conducted both in water and in two contrasting soils to test the impact of assumed differences in bioavailability of nanoparticles. Inhibitory effects were observed in aqueous suspensions at 250 mg L(-1) for nZVI and 10 mg L(-1) for Ag. Reduction in shoot growth was a more sensitive endpoint than germination percentage. Complete inhibition of germination was observed at 1000-2000 mg L(-1) for nZVI. For Ag, complete inhibition was not achieved. The presence of soil had a modest influence on toxicity, and inhibitory effects were observed at 300 mg nZVI L(-1) water in soil (equivalent to 1000 mg nZVI kg(-1) soil). Complete inhibition was observed at 750 and 1500 mg L(-1) in sandy soil for flax and ryegrass, respectively, while for barley 13% germination still occurred at 1500 mg L(-1) . In clay soil, inhibition was less pronounced. Our results indicate that nZVI at low concentrations can be used without detrimental effects on plants and thus be suitable for combined remediation where plants are involved. Silver nanoparticles inhibited seed germination at lower concentrations, but showed no clear size-dependent effects, and never completely impeded germination. Thus, seed germination tests seem less suited for estimation of environmental impact of

The study aimed at investigating effects of three differently acting biocides; the insecticide esfenvalerate, the fungicide picoxystrobin and the bactericide triclosan, applied individually and as a mixture, on an earthworm community in the field. A concentration-response design was chosen and results were analyzed using univariate and multivariate approaches. Effects on juvenile proportions were less pronounced and more variable than effects on abundance, but effects in general were species- and chemical-specific, and temporal variations distinct. Esfenvalerate and picoxystrobin appeared to elicit stronger effects than triclosan at laboratory-based ECx values, which is in accordance with our previous laboratory study on Eisenia fetida. The mixture affected abundance and juvenile proportions, but the latter only at high mixture concentrations. Esfenvalerate and picoxystrobin appeared to be the main drivers for the mixture's toxicity. Species-specific toxicity patterns question the reliability of mixture toxicity predictions derived on E. fetida for field earthworms. Biocide concentrations equaling EC50s (reproduction) for E. fetida provoked effects on the field earthworms mainly exceeding 50%, indicating effect intensification from the laboratory to field as well as the influence of indirect effects produced by species interactions. The differing results of the present field study and the previous laboratory study imply that lower- and higher-tier studies may not be mutually exclusive, but to be used in complementary.

Exploitation of mycorrhizas to enhance phytoremediation of organic pollutants has received attention recently due to their positive effects on establishment of plants in polluted soils. Some evidence exist that ectomycorrhizas enhance the degradation of pollutants of low recalcitrance, while less easily degradable polyaromatic molecules have been degraded only by some of these fungi in vitro. Natural polyaromatic (humic) substances are degraded more slowly in soil where ectomycorrhizal fungi are present, thus phytoremediation of recalcitrant pollutants may not benefit from the presence of these fungi. Using a soil spiked with three polycyclic aromatic hydrocarbons (PAHs) and an industrially polluted soil (1 g kg(-1) of summation operator12 PAHs), we show that the ectomycorrhizal fungus Suillus bovinus, forming hydrophobic mycelium in soil that would easily enter into contact with hydrophobic pollutants, impedes rather than promotes PAH degradation. This result is likely to be a nutrient depletion effect caused by fungal scavenging of mineral nutrients.

Because plants significantly affect radionuclides (RN) cycling and further dispersion into the biosphere, it is important to understand the biological factors influencing RN plant uptake, accumulation and redistribution. In this respect, mycorrhizal fungi are of particular interest. The effects of ecto-mycorrhizal (ECM) and arbuscular mycorrhizal (AM) fungi on the transport of uranium (U) or radiocaesium (Cs) were investigated both under pot and in vitro culture conditions. Results obtained in vitro demonstrated that AM hyphae can take up and translocate U and Cs towards roots, while this uptake and translocation were not perceptible using pot culture systems with soil. These contrasting results could be due to different experimental conditions, including the K level in the external solution and the bio-availability of Cs. The in vitro studies also indicated that root colonisation by AM fungi might limit U and Cs root transport. Under pot culture conditions, they appeared to significantly reduce root to shoot translocation of U. Under the same conditions, ECM transport of Cs was demonstrated, and appeared to be dependent on fungal species. A better estimation of the potential use of mycorrhizal fungi for the phytoremediation of RN-contaminated areas is now available and will be further discussed.

Phytoremediation is a promising technology for the cleanup of polluted environments. The technology has so far been used mainly to remove toxic heavy metals from contaminated soil, but there is a growing interest in broadening its applications to remove/degrade organic pollutants in the environment. Both plants and soil microorganisms have certain limitations with respect to their individual abilities to remove/breakdown organic compounds. A synergistic action by both rhizosphere microorganisms that leads to increased availability of hydrophobic compounds, and plants that leads to their removal and/or degradation, may overcome many of the limitations, and thus provide a useful basis for enhancing remediation of contaminated environments. The review of literature presented in this article provides an insight to the nature of plant-microbial interactions in the rhizosphere, with a focus on those processes that are relevant to the breakdown and/or removal of organic pollutants. Due consideration has been given to identify opportunities for utilising the plant-microbial synergy in the rhizosphere to enhance remediation of contaminated environments. The literature review has highlighted the existence of a synergistic interaction between plants and microbial communities in the rhizosphere. This interaction benefits both microorganisms through provision of nutrients by root exudates, and plants through enhanced nutrient uptake and reduced toxicity of soil contaminants. The ability of the plant-microbial interaction to tackle some of the most recalcitrant organic chemicals is of particular interest with regard to enhancing and extending the scope of remediation technologies. Plant-microbial interactions in the rhizosphere offer very useful means for remediating environments contaminated with recalcitrant organic compounds. A better knowledge of plant-microbial interactions will provide a basis for improving the efficacy of biological remediations. Further research is, however, needed to investigate different feedback mechanisms that select and regulate microbial activity in the rhizosphere.

Rhizodegradation of recalcitrant organic pollutants, such as polycyclic aromatic hydrocarbons (PAH), may benefit from the major role that root exudates have on rhizospheric microbial processes. We investigated the influence of the proximity to ryegrass (Lolium perenne L.) roots on microbial populations and their biodegradation of phenanthrene (PHE) using compartmented pots. PAH degrading bacteria, total heterotrophic bacteria and PHE biodegradation were quantified in three consecutive sections at different distance (0–3, 3–6, 6–9 mm) from a mat of actively exuding roots. A bacterial gradient was observed with higher numbers of heterotrophs and PAH degrading bacteria closest to the roots. In parallel, a PHE biodegradation gradient was evident in the presence of roots. The biodegradation reached 86%, 48% and 36% of initially added PHE, respectively, in the layers 0–3 mm, 3–6 and 6–9 mm from the roots. The biodegradation rate was similar throughout the three layers of the non planted control. The present experimental system seems well suited for spatial and dynamic studies of PAH rhizoremediation.

Phytoremediation as a means of cleaning up polluted soils has gained popularity during the last decade due to its convenience and low costs of installation and maintenance. When the target pollutant is biodegradable, this technology exploits the stimulating effect that roots have on microbial processes and physical/chemical modifications in the rhizosphere. Among the microorganisms that affect rhizosphere processes, symbiotic fungi forming mycorrhizas induce a series of changes in plant physiology, nutrient availability and microbial composition that may determine the outcome of a phytoremediation attempt. Beyond the rhizosphere, mycorrhizal hyphae act as the roots of the roots, and may thus extend the rhizosphere into the bulk soil by creating a new interface of soil-plant interactions; the hyphosphere. We here discuss some of the recent results on phytoremediation of organic pollutants with emphasis on processes in the mycorrhizosphere, and highlight future research priorities.

Phytoremediation of organic pollutants depends on plant-microbe interactions in the rhizosphere, but the extent and intensity of such rhizosphere effects are likely to decrease with increasing distance from the root surface. We conducted a time-course pot experiment to measure dissipation of polycyclic aromatic hydrocarbons (PAHs) in the rhizosphere of clover and ryegrass grown together on two industrially polluted soils (containing 0.4 and 2 g kg(-1) of 12 PAHs). The impact of the fungal root symbiosis arbuscular mycorrhiza (AM) on PAH degradation was also assessed, as these fungi have previously improved plant establishment on PAH-polluted soils and enhanced PAH degradation in spiked soil. The two soils behaved differently with respect to the time-course of PAH dissipation. The less polluted and more highly organic soil showed low initial PAH dissipation rates, with small positive effects of plants after 13 weeks. At the final harvest (26 weeks), the amounts of PAHs extracted from nonplanted pots were higher than the initial concentrations. In parallel planted pots, PAH concentrations decreased as a function of proximity to roots. The most polluted soil showed higher initial PAH dissipation (25% during 13 weeks), but at the final harvest PAH concentrations had increased to values between the initial concentration and those at 13 weeks. An effect of root proximity was observed for the last harvest only. The presence of mycorrhiza generally enhanced plant growth and favored growth of clover at the expense of ryegrass. Mycorrhiza enhanced PAH dissipation when plant effects were observed.

A new method is described for monitoring hyphal P-32 transport in compartmented, monoxenic mycorrhizal root cultures. Nondestructive time-course measurements of P transport in hyphae were obtained by capturing digital autoradiograms on P-imaging screens, and comparing with growth observed by optical scanning. P-32 distribution measured by densitometry on the day of harvest closely agreed with values obtained by liquid scintillation counting after destructive harvest. Virtually all labeled PO4 was absorbed by arbuscular mycorrhizal (AM) hyphae, but transfer to the roots appeared to be incomplete. P transport was not unidirectional towards the roots, as P-32 was also transported from the root compartment to the hyphal compartment. Net P flux rates were calculated for hyphae crossing between compartments, taking bidirectional flow into account. Amounts of transported P were poorly correlated with extra-radical hyphal length and root d. wt, but highly correlated with the number of hyphae crossing the barrier separating the two compartments. Such correlations were highest when only hyphae with detectable protoplasmic streaming were considered. The method was tested using radiolabeled P sources, H2PO4- and cytidine triphosphate (CTP), and the AM fungi, Glomus intraradices and G. proliferum. Fungal transport of P-32 from CTP was much slower than from PO4 for both fungi. (C) New Phytologist (2002) 154: 809-819.

An industrially polluted soil (3.1 g PAH kg−1) was used to study C, N and P limitations to dissipation of polycyclic aromatic hydrocarbons (PAHs) in a model rhizosphere. Soil columns (50 cm3) were packed with soil and percolated daily with artificial root exudates (ARE) at 100 μg C g−1, 1 mM NH4NO3 or 1 mM NaH2PO4, or any double or triple combination of these for 30 days. At harvest, soil was analyzed for PAHs, and the numbers of total heterotrophic and PAH degrading bacteria estimated by an MPN technique. Dissipation of 3 and 4 ring PAHs was highest in soil receiving ARE+N, followed by the treatments with N+P and ARE+N+P. The complete treatment ARE+N+P had the highest dissipation of 5-ring PAHs. For both 5 and 6 ring PAHs most treatments resulted in increased concentrations at the end of the experiment, which was attributed to desorption of initially unextractable molecules. PAH degrading bacteria reached the highest numbers in the ARE+N+P treatment, followed by the treatment receiving only P, whereas total number of heterotrophs was elevated in the treatments ARE, P, ARE+N and ARE+N+P. Net PAH dissipation in soil was seemingly due to the two counteracting processes biodegradation and desorption. In the presence of ARE, the dissipation of higher molecular weight PAHs apparently involved microbial degradation and/or biotransformation through co-metabolism.

The 2,7-diaminofluorene (DAF) was a far more sensitive substrate for lucerne root peroxidases than were guaiacol or pyrogallol. All the peroxidase assays had an optimum pH of 6. The optimum temperature for the activity of DAF-peroxidase was 35 °C, far lower than those of guaiacol- and pyrogallol-peroxidases (60 °C). Electrophoresis of lucerne root peroxidases revealed the presence of eight isoenzymes using DAF as substrate, and only two with guaiacol. The influence of four organic solvents on peroxidase activity was also assessed.

A pot experiment was conducted to test the effect of three microbial regimes on the time course of heavy metal uptake in clover and maize from an industrially polluted soil. The three treatments included: (1) an intact flora of bacteria and fungi, including indigenous arbuscular mycorrhizal (AM) fungi together with soil microfauna; (2) the indigenous bacterial/fungal flora except AM fungi, reintroduced into sterilized soil; or (3) the same bacterial/fungal flora plus an AM fungus. For the final harvest, two pot sizes were included to assess the effect of root density. Plant uptake of P and heavy metals varied according to plant species, harvest time and soil treatment. For both plant species, shoot concentration of Zn, Cd and Cu decreased and Ni increased with plant age. Plants growing in sterilized soil with reintroduced AM fungi generally grew better, but contained higher concentrations of heavy metals than those colonized by indigenous AM fungi. Plants with mycorrhiza frequently contained more P, Zn, Cd, Cu and Pb in roots and shoots compared to nonmycorrhizal plants. Elevated root/shoot concentration ratios of P and metals indicate a sequestration of metal phosphates in mycorrhizal roots. Mycorrhizal performance was influenced by root density. At low root densities, metal concentrations in mycorrhizal plants were reduced, whereas it had no effect at high root densities when the entire soil volume was efficiently exploited by roots. We conclude that root density data are essential for interpretations of the influence of AM on metal uptake in plants.

The effect of arbuscular mycorrhiza (AM) on white clover and ryegrass grown together in a soil spiked with polycyclic aromatic hydrocarbons (PAH) was assessed in a pot experiment. The soil was spiked with 500 mg kg–1 anthracene, 500 mg kg–1 chrysene and 50 mg kg–1 dibenz(a,h)anthracene, representing common PAH compounds with three, four and five aromatic rings, respectively. Three treatments and two harvest times (8 and 16 weeks) were imposed on plants grown in spiked soil: no mycorrhizal inoculation, mycorrhizal inoculation (Glomus mosseae P2, BEG 69) and mycorrhizal inoculation and surfactant addition (Triton X-100). Pots without PAH were also included as a control of plant growth and mycorrhizal colonization as affected by PAH additions. The competitive ability of clover vis-a-vis ryegrass regarding shoot and root growth was enhanced by AM, but reduced by PAH and the added surfactant. This was reflected by mycorrhizal root colonization which was moderate for clover (20–40% of total root length) and very low for ryegrass (0.5–5% of total root length). Colonization of either plant was similar in spiked soil with and without the added surfactant, but the PAH reduced colonization of clover to half that in non-spiked soil. P uptake was maintained in mycorrhizal clover when PAH were added, but was reduced in non-mycorrhizal clover and in mycorrhizal clover that received surfactant. Similar effects were not observed on ryegrass. These results are discussed in the context of the natural attenuation of organic pollutants in soils.

The ability of arbuscular mycorrhizal (AM) fungi from a metal-tolerant plant (Viola calaminaria, violet) to colonise and reduce metal uptake by a non-tolerant plant (Trifolium subterraneum, subterranean clover) in comparison to a metal-tolerant AM fungus isolated from a non-tolerant plant was studied. AM spores from the violet rhizosphere and from violet roots were characterised by polymerase chain reaction (PCR) amplification of the SSU rDNA, and sequencing. Subterranean clover was grown in pots containing a soil supplemented with Cd and Zn salts and inoculated either with a mixture of spores extracted from the violet rhizosphere or with spores of a Cd-tolerant Glomus mosseae P2 (BEG 69), or non-inoculated. The diversity of fungi, including AM fungi, colonising clover roots was assessed and analysed using terminal-restriction fragment length polymorphism. At least four different Glomus species were found in the violet rhizosphere. After 8 weeks in a growth chamber, colonisation of clover roots with spores from the violet rhizosphere increased Cd and Zn concentrations in clover roots without significantly affecting the concentrations of metals in the shoot and plant growth. G. mosseae P2 reduced plant growth and slightly increased the Cd concentration. Only one AM fungus (Glomus b) from the violet rhizosphere colonised clover roots, but other fungi were present. AM fungi from heavy metal-contaminated soils and associated with metal-tolerant plants may be effective in accumulating heavy metals in roots in a non-toxic form.

A pot experiment was conducted with soil from a long-term (74-year) fertilization field experiment to compare the effects of organic and mineral fertilizers on mycorrhiza formation in clover, and mycorrhiza-mediated plant P uptake. Five treatments were selected from the field experiment representing different forms and levels of P. Mycorrhizal effects on plant growth and P uptake were estimated by comparing plants grown in untreated soil containing indigenous arbuscular mycorrhizal (AM) fungi, with plants grown in pasteurized soil. Short-term versus residual effects of fertilizer/manure were also measured by comparing treatments with or without fertilizers added at the start of the pot experiment. Mycorrhiza formation was greatest in soil that had received no P for 74 years, followed by soil having received 30 or 60 Mg ha–1 farmyard manure (FYM), and soil having received 25 or 44 kg P ha–1 in NPK fertilizers. Plant growth and P uptake were severely reduced in the absence of AM fungi for all mineral fertilizer treatments. In contrast, plants growing in soil that had received FYM grew equally well or better when non-mycorrhizal. Recent additions of NK fertilizer and FYM had no effect on mycorrhiza formation, while additions of NPK led to reduced colonization. It thus seems that moderate quantities of FYM have less adverse effects on AM than equivalent amounts of nutrients in NPK fertilizers, a phenomenon that is most likely due to a temporal difference in P availability and its gradual release that balance plant demand.

DAF (2,7-diaminofluorene)-peroxidases were the principal oxidative system in roots and rhizosphere of lucerne. The expression of these enzymes was stimulated by mycorrhiza as well as by anthracene (500 mg kg−1 added to the growth medium). Inversely, infection by mycorrhizal fungi repressed laccase activities. Electrophoretic analysis of the DAF-peroxidases revealed similar isoenzyme patterns for all treatments but in presence of anthracene some of the isoenzymes had more intensive bands.

Compartmented monoxenic cultures of Ri T-DNA transformed carrot roots and a symbiotic arbuscular mycorrhizal fungus demonstrated for the first time that phosphate in an organic form (32P-labelled AMP) may be hydrolysed by extra-radical mycorrhizal hyphae in the absence of other organisms, and subsequently utilized as a mineral nutrient source by the host plant after fungal transport.

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (Po) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of Po seems to be far more important than Po hydrolysis for utilization of Po by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity.

Extraradical hyphae of Glomus intraradices or G. claroideum were extracted from root free sand of two-compartment pot cultures and used to determine fungal phosphatase activity [p-nitrophenyl phosphate (p-NPP) hydrolysis]. Enzyme activity was assayed with respect to pH, temperature and different fractions of the hyphae (external soluble, wall-bound and internal phosphatases). The results showed an overall maximum enzyme activity at pH 5·2–5·6 for both fungi, with a possible secondary maximum at pH ≥ 8·8 for G. claroideum. Of the two fungi tested, G. intraradices had the highest external phosphatase activity in two experiments and the same activity in one experiment, reaching 184 μmol p-NPP hydrolysed mg−1 D.W. h−1. Phosphatase activity at pH 5·2 decreased sharply with temperature, with 4·5 and 10·5% of the enzyme activity remaining at 5 °C relative to that at 37° for G. intraradices and G. claroideum, respectively. Separation of the phosphatase activity into external soluble, wall-bound and internal fractions revealed that up to 70% of the measured activity was associated with the hyphal wall, and the rest with internal structures. Exuded phosphatases were not found in measurable amounts. The implications of these results on possible hyphal utilization of organic P in soil are discussed.

Experiments with excised mycelium of several Glomus spp. with different histories of exposure to heavy metals were carried out to measure their capacities to bind Cd and Zn. Cd sorption was followed for up to 6 h of incubation to determine its time course relationships. Controls treated with a metabolic inhibitor were included to evaluate whether sorption was due to active uptake or passive adsorption. The effect of ion competition (effects of Ca or Zn on Cd sorption) and general measurements of cation exchange capacity (CEC) of roots and hyphae were also performed. The results showed that AM mycelium has a high metal sorption capacity relative to other microorganisms, and a CEC comparable to other fungi. Metal sorption was rapid (

SUMMARY Subterranean clover (Trifolium subterraneum L.) in symbiosis with Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe was grown in compartmented pots. Side compartments were filled with soil where recently added (1 wk) radio-labelled cadmium (Cd) at three levels of non-labelled Cd could be accessed by either roots or arbuscular mycorrhizal hyphae. All treatments were replicated with non-mycorrhizal plants. After a growth period of 52 d roots and shoots were analysed for l09Cd, and lengths of roots and hyphae in respective labelled compartments determined. Cadmium uptake by roots was not significantly influenced by the mycorrhizal status of the plant. Uptake of Cd from hyphal compartments was higher in mycorrhizal than in non-mycorrhizal plants, corresponding to 96, 127 and 131 % of that in non-mycorrhizal plants when 1, 10 and 100 mg Cd kg-i was added, respectively. A large proportion of the increased Cd content of mycorrhizal plants was sequestered in the roots. It is concluded that extraradical hyphae of AM fungi can transport Cd from soil to plants, but that transfer from fungus to plant is restricted due to fungal immobilization. No reduction of hyphal growth into soil with up to 20 mg extractable Cd kg-' was observed.

Mycorrhizae are ubiquitous symbiosis which can mediate uptake of some plant nutrients. In polluted soils they could be of great importance in heavy metal availability and toxicity to plants. Mycorrhizae have also been reported to protect plants against toxic metals. We investigated the occurrence and infectivity of arbuscular mycorrhizal (AM) spores as affected by heavy metal levels and other soil properties in Norwegian soils collected from heavy metal polluted, high natural background and non-polluted areas. Spore numbers, mycorrhizal infectivity and spore germination of indigenous mycorrhizal fungi and of a reference strain (Glomus mosseae) in soils showed lower values in two soils with high metal concentrations and in one soil with a low pH. Mycorrhizal infectivity was negatively correlated with extractable metals. Spore number and mycorrhizal infectivity in a soil with naturally high heavy metal content were not different to in non-polluted soils, and indigenous AM fungi appeared more tolerant to metals than those in non-polluted soils. Mycorrhizal infectivity, expressed as MSI50 values, was significantly correlated (r′=0.89, P< 0.05) with the percentage of germinating G. mosseae spores in the soils. However, the number of spores per volume of soil was not significantly correlated with infectivity or spore germination of the reference strain. The spore germination method is discussed as a bioassay of heavy metal toxicity in soil.

An experiment was set up to test the ability of arbuscular mycorrhizal (AM) roots and hyphae to produce extracellular phosphatases and to study the relationship between phosphatase activity and soil organic P (Po). Non-mycorrhizal cucumber and cucumber in symbiosis with either of two mycorrhizal fungi were grown in a sandy loam-sand mixture in three-compartment pots. Plant roots were separated from two consecutively adjoining compartments, first by a 37 m mesh excluding roots and subsequently by a 0.45 m membrane excluding mycorrhizal hyphae. Soil from the two root-free compartments was sectioned in a freezing microtome and analyzed for extracellular acid (pH 5.2) and alkaline (pH 8.5) phosphatase activity as well as depletion of NaHCO3-extractable inorganic P (Pi) and Po. Roots and mycorrhizal hyphae depleted the soil of Pi but did not influence the concentration of Po in spite of increased phosphatase activity in soil influenced by roots. Phosphatase activity at both pH values was highest in soil influenced by uncolonized roots, but this was attributed to higher root length densities as compared to mycorrhizal roots. Mycorrhizal hyphae showed no influence on soil phosphatase activity in spite of high hyphal length densities (> 22 m cm−3). Hyphae were also able to deplete soil of Pi beyond the membrane interface.

Two experiments were set up to investigate the influence of soil organic matter on growth of arbuscular mycorrhizal (AM) hyphae and concurrent changes in soil inorganic P, organic P and phosphatase activity. A sandy loam soil was kept for 14 months under two regimes (outdoor where surplus precipitation leached through the soil, or indoor at constant moisture) with or without 9% (w/w) chopped wheat straw plus mineral N. Then the soils were partially sterilized and placed in two-compartment pots where mycorrhizal or non-mycorrhizal cucumber plants were grown in one root compartment (RC), and soils differing in organic matter were placed in six parallel hyphal compartments (HC) separated from the RC with a 37 μm mesh. In the first experiment, using Glomus caledonium, hyphal length densities were measured in the HC after 31 days. Added straw increased hyphal length densities by 34 and 62% for soil kept outdoors and indoors, respectively. In the second experiment, using G. invermaium and only soil kept outdoors, three treatments were included: soil with no added straw with or without a new addition of 0.5% (w/w) of ground clover leaves, and soil with 9% straw plus mineral N. After 41 days hyphal length density was twice as high in soil with added straw compared to the two other treatments. Mycorrhizal colonization resulted in lower activity of acid phosphatase in the HC for two out of three treatments. Alkaline phosphatase activity was only decreased by mycorrhiza in soil without organic matter additions. In soil with added clover alkaline phosphatase activity increased due to the presence of mycorrhizal hyphae. We suggest that mycorrhizas may influence the exudation of acid phosphatase by roots. Hyphae of G. invermaium did apparently not excrete extracellular phosphatases, but their presence may have influenced alkaline phosphatase excreted by other microorganisms, probably through competition for nutrients. Phosphatase activity was not correlated with the concentration of labile organic P in soil extracts.

An experiment was designed to study whether hyphae and colonized roots of arbuscular mycorrhiza have more direct access to P in organic matter than roots of non-mycorrhizal plants. Soil supplied with 0, 15 or 45 mg P kg−1 was uniformly mixed with 32P-labelled organic matter at four levels (0, 1, 2 and 5 g kg−1) and inoculated with a mycorrhizal fungus or left uninoculated. Pots were incubated at 60% of field capacity for one week prior to sowing of clover, and plants were harvested after a growth period of 23 days. Mycorrhizal colonization increased shoot dry weight, P concentration and 32P uptake at all P levels. Specific activity in plants was consistently higher than in corresponding soil. This indicates that the added 32P never reached an equilibrium with inorganic P in the soil. P mineralized from organic matter thus had a residence time in the soil solution sh ort enought to partially avoid isotopic exchange and adsorption. Mycorrhizal colonization influenced specific activity of 32P in plants from three of the nine combinations of P and labelled organic matter: At the lowest level of P the specific activity was highest in non-mycorrhizal plants, and at the intermediate level of P there was one treatment where mycorrhizal plants had the highest specific activity. These differences are discussed. Plant dry weight and P concentration did not respond to addition of organic matter, though soil extracts consistently contained higher amounts of inorganic P as a result of organic matter addition. The results suggest that mycorrhizal plants at an early growth stage utilize a substantially higher amount of P released from organic matter than non-mycorrhizal plants. This mycorrhizal advantage does not seem to be related to a mycorrhizal influence on mineralization.