Temporal and spatial patterns in α- and γ-hexachlorocyclohexane concentrations in industrially contaminated rivers

Fluxes of HCH isomers α- and γ-HCH dynamics were determined in four industrial U.K. rivers feeding the North Sea. Sampling was conducted weekly basis over a 2-year period. This was complemented by discrete studies of events where two hourly sampling periods were used to investigate the fine time scale dynamics of fluxes. Two intensively industrialized rivers had average isomer concentrations of ~20 ng L<sup>-1sup> for both isomers, while average concentrations in the two less industrialized rivers ranged between 1.5 and 5.0 ng L<sup>-1sup>. α-HCH concentrations showed no strong temporal patterns on any river, which contrasts with γ-HCH levels that increased considerably during late summer/early autumn following sustained periods of low river flow. Sampling during high river flow events on rivers with differing HCH pollution histories both showed the same dynamics in HCH isomer concentrations. γ-HCH concentrations decreased 4-fold during events while α-HCH-concentrations stayed constant. The increases in γ-HCH concentrations under low flow conditions and the rapid dilution of this isomer during events indicate that γ-HCH has current inputs to these river systems. It was calculated that these four rivers export 30.8 kg yr<sup>-1sup> of γ-HCH and 14.8 kg yr<sup>-1sup> of α-HCH to the North Sea.

Contamination of Donana food-chains after the Aznalcollar mine disaster

On 25 April 1998 part of the tailings pond dike of the Aznalcollar Zn mine north of the Guadalquivir marshes (Donana) in southern Spain collapsed releasing an estimated 5 million m<sup>3sup> of acidic metal-rich waste. This event contaminated farmland and wetland up to >40 km downstream, including the 900-ha 'Entremuros', an important area for birds within the Donana world heritage site. In spite of the contamination, birds continued to feed in this area. Samples of two abundant macrophytes (Typha dominguensis and Scirpus maritimus) were taken from the Entremuros and nearby uncontaminated areas; these plants are important food items for several bird species. Analyses showed that in the Entremuros mean plant tissue concentrations of Cd were 3-40-fold (0.8-7.4 ppm) and Zn 20-100-fold (20-3384 ppm) greater than those from control areas. Comparable dietary concentrations of Zn have been reported to cause severe physiological damage to aquatic birds under experimental conditions. Elevated Cd concentrations are of concern as Cd bioconcentrates and is a cumulative poison. Metals released in this accident are moving into this food-chain and present a considerable risk to species feeding on Typha sp. and Scirpus sp. Many other food-webs exist in this area and require detailed examination to identify the species at risk, and to facilitate the management of these risks to minimise future impacts to the wildlife of Donana. Copyright (C) 1999 Elsevier Science Ltd.

Investigation of organic xenobiotic transfers, partitioning and processing in air-soil-plant systems using a microcosm apparatus. Part I:Microcosm development

A microcosm system was developed to investigate transfers of organic xenobiotics in air-soil-plant systems. This was validated using <sup>14sup>C labelled 1,2-dichlorobenzene (DCB) as a model compound. Trapping efficiency was 106 ± 3% for volatile compounds and 93.0 ± 2.2% for carbon dioxide in a blank microcosm arrangement. Recovery of 1,2-dichlorobenzene spiked to grassed and unplanted soils was > 90% after 1 week. The predominant DCB loss process was volatilisation with no evidence for mineralisation over 1 week and 20-30% of the added spike remained in soil. Although there was no evidence for root uptake and translocation of added label, foliar uptake of soil volatilised compound was detected. The microcosm showed good potential for study of <sup>14sup>C labelled and unlabelled organic xenobiotic transfers in air-soil-plant systems with single plants and also intact planted cores.

Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation studies

Autoclaved soil is commonly used for the study of xenobiotic sorption and as an abiotic control in biodegradation experiments. Autoclaving has been reported to alter soil physico-chemical and xenobiotic sorption characteristics such that comparison of autoclaved with non-autoclaved treatments in soil aging and bioavailability studies may yield misleading results. Experiments could be improved by using autoclaved soil re-inoculated with indigenous microorganisms as an additional or alternative non-sterile treatment for comparison with the sterile, autoclaved control. We examined the effect of autoclaving (3 x 1 h, 121°C, 103.5 KPa) on the physico-chemical properties of a silt loam soil (pH 7.2, 2.3% organic carbon) and the establishment of indigenous microorganisms reintroduced after autoclaving. Sterilisation by autoclaving significantly (p ≤ 0.05) decreased pH (0.6 of a unit) and increased concentrations of water-soluble organic carbon (WSOC; nontreated = 75 mg kg<sup>-1sup>; autoclaved = 1526 mg kg<sup>-1sup>). The initial first-order rate of <sup>14sup>C-2,4-dichloro-UL-phenol (2,4-DCP) adsorption to non-treated, autoclaved and re-inoculated soil was rapid (K₁ = 16.8-24.4 h<sup>-1sup>) followed by a slower linear phase (K₂). In comparison with autoclaved soil (0.038% day<sup>-1sup>), K₂ values were higher for re-inoculated (0.095% day<sup>-1sup>) and nontreated (0.181% day<sup>-1sup>) soil. This was attributed to a biological process. The Freundlich adsorption coefficient (K(f)) for autoclaved soil was significantly (p ≤ 0.05) higher than for re-inoculated or non-treated soil. Increased adsorption was attributed to autoclaving-induced changes to soil pH and solution composition. Glucose-induced respiration of autoclaved soil after re-inoculation was initially twice that in the non-treated control, but it decreased to control levels by day 4. This reduction corresponded to a depletion of WSOC. 2,4-DCP mineralisation experiments revealed that the inoculum of nonsterile soil (0.5 g) contained 2,4-DCP-degrading microorganisms capable of survival in autoclaved soil. The lag phase before detection of significant 2,4-DCP mineralisation was reduced (from 7 days to ≤3 days) by pre-incubation of re-inoculated soils for 7 and 14 days before 2,4-DCP addition. This was attributed to the preferential utilisation of WSOC prior to the onset of 2,4-DCP mineralisation. Cumulative <sup>14sup>CO₂ evolved after 21 days was significantly lower (p ≤ 0.05) from non-treated soil (25.3%) than re-inoculated soils (ca 45%). Experiments investigating sorption-biodegradation interactions of xenobiotics in soil require the physico-chemical properties of sterile and non-sterile treatments to be as comparable as possible. For fundamental studies, we suggest using re-inoculated autoclaved soil as an additional or alternative non-sterile treatment.

Degradation of 4-fluorobiphenyl by mycorrhizal fungi as determined by 19F nuclear magnetic resonance spectroscopy and 14C radiolabelling analysis

The pathways of biotransformation of 4-fluorobiphenyl (4FBP) by the ectomycorrhizal fungus Tylospora fibrilosa and several other mycorrhizal fungi were investigated by using <sup>19sup>F nuclear magnetic resonance (NMR) spectroscopy in combination with <sup>14sup>C radioisotope-detected high-performance liquid chromatography (<sup>14sup>C- HPLC). Under the conditions used in this study T. fibrillosa and some other species degraded 4FBP. <sup>14sup>C-HPLC profiles indicated that there were four major biotransformation products, whereas <sup>19sup>F NMR showed that there were six major fluorine-containing products. We confirmed that 4-fluorobiphen-4'-ol and 4-fluorobiphen-3'-ol were two of the major products formed, but no other products were conclusively identified. There was no evidence for the expected biotransformation pathway (namely, meta cleavage of the less halogenated ring), as none of the expected products of this route were found. To the best of our knowledge, this is the first report describing intermediates formed during mycorrhizal degradation of halogenated biphenyls.

Biosensing 2,4-dichlorophenol toxicity during biodegradation by Burkholderia sp. RASC c2 in soil

Biodegradation of the model pollutant, 2,4-dichlorophenol (2,4-DCP) by Burkholderia sp. RASC c2, in contaminated soil was assessed by combining chemical analysis with a toxicity test using Escherichia coli HB101 pUCD607. E. coli HB101 pUCD607 was previously marked with luxCDABE genes, encoding bacterial bioluminescence and was used as an alternative to Microtox. Mineralization of <sup>14sup>C-2,4-DCP (196.2 μg g<sup>-1sup> dry wt) in soil occurred rapidly after a 24 h lag. Correspondingly, 2,4-DCP concentrations in soil and soil water extracts decreased with time and concentrations in the latter were at background levels (<0.12 μg mL<sup>-1sup>) after day 2. Toxicity of soil water extracts to the lux-based biosensor also decreased with time. Mean light output of E. coli was stimulated by ~1.5 X control values in soil water extracts when concentrations of 2,4-DCP were approaching the limit of detection by HPLC but returned to values equivalent to those of controls when soil water 2,4-DCP concentrations were below the detection limit. No mineralization or microbial growth was detected in noninoculated microcosms. 2,4-DCP concentration in sterile controls decreased significantly with time as did toxicity to E. coli Lux-based E. coli was a sensitive biosensor of 2,4-DCP toxicity during biodegradation and results complemented chemical analysis.

Response of soil microbial biomass to 1,2-dichlorobenzene addition in the presence of plant residues

The impact of 1,2-dichlorobenzene on soil microbial biomass in the presence and absence of fresh plant residues (roots) was investigated by assaying total vital bacterial counts, vital fungel hyphal length, total culturable bacterial counts, and culturable fluorescent pseudomonads. Diversity of the fluorescent pseudomonads was investigated using fatty acid methyl ester (FAME) characterization in conjunction with metabolic profiling of the sampled culturable community (Biolog). Mineralization of [<sup>14sup>C]1,2- dichlorobenzene was also assayed. Addition of fresh roots stimulated 1,2- dichlorobenzene mineralization by over 100%, with nearly 20% of the label mineralized in root-amended treatments by the termination of the experiment. Presence of roots also buffered any impacts of 1,2-dichlorobenzene on microbial numbers. In the absence of roots, 1,2-dichlorobenzene greatly stimulated total culturable bacteria and culturable pseudomonads in a concentration-dependent manner. 1,2-Dichlorobenzene, up to concentrations of 50 μg/g soil dry weight had little or no deleterious effects on microbial counts. The phenotypic diversity of the fluorescent pseudomonad population was unaffected by the treatments, even though fluorescent pseudomonad numbers were greatly stimulated by both roots and 1,2-dichlorobenzene. The presence of roots had no detectable impact on the bacterial community composition. No phenotypic shifts in the natural population were required to benefit from the presence of roots and 1,2-dichlorobenzene. The metabolic capacity of the culturable bacterial community was altered in the presence of roots but not in the presence of 1,2-dichlorobenzene. It is argued that the increased microbial biomass and shifts in metabolic capacity of the microbial biomass are responsible for enhanced degradation of 1,2-dichlorobenzene in the presence of decaying plant roots.

Response of soil microbial communities to single and multiple doses of an organic pollutant

The effect of 100 μg 1,2-dichlorobenzene (1,2-DCB) g<sup>-1sup> dry weight (dw) of soil introduced either as a single dose or multiple (10 fortnightly) doses of 10 μg g<sup>-1sup> dw, on the microbial biomass, diversity of culturable bacterial community and the rate of 1,2-DCB mineralisation, were compared. After 22 weeks exposure both application regimes significantly reduced total bacterial counts and viable fungal hyphal length. The single dose had the greatest overall inhibitory effect, although the extent of inhibition varied throughout the study. Total culturable bacterial counts, determined after 22 weeks exposure showed little response to 1,2-DCB, but pseudomonad counts in single and multiple treatments were reduced to 9.7 and 0.147%, respectively, of the numbers detected in the control soil. The effect of 1,2-DCB application on the taxonomic composition of the culturable bacteria community was determined by fatty acid methyl ester (FAME) analysis. Compared to control soils, the single dose treatment had a lower percentage of Arthrobacter and Micrococcus. Multiple applications had a significant effect upon pseudomonad abundance, which represented only 2% of the identified community, compared to 45.6% in the control. The multi-dosed soils contained a high percentage of bacilli (> 25%). The effects of 1,2-DCB applications on the metabolic potential of the soil microbial community was determined by BIOLOG profiling. The number of carbon compounds utilised by the community in the multi-dosed soils (49 positives) was significantly less (P < 0.05) than detected in the single dose treatment (76) and control (66). The rate of 1,2-DCB mineralisation, determined by <sup>14sup>CO₂ production from radiolabelled [UL-<sup>14sup>C] 1,2-DCB, declined throughout the study, and after 22 weeks was slightly but significantly (P < 0.05) lower in the multiply- than the singly-dosed soils. The differential response to 1,2-DCB treatments was attributed to its reduced bioavailability in soils after a single exposure, compared to multiple applications.

Toxic interactions of metal ions (Cd2+, Pb2+, Zn2+ and Sb3- on in vitro biomass production of ectomycorrhizal fungi

A number of ectomycorrhizal (ECM) fungi, from sites uncontaminated by toxic metals, were investigated to determine their sensitivity to Cd<sup>2-sup>, Pb<sup>2+sup>, Zn<sup>2+sup> and Sb<sup>3-sup>, measured as an inhibition of fungal biomass production. Isolates were grown in liquid media amended with the metals, individually (over a range of concentrations) and in combination (at single concentrations) to determine any significant interactions between the metals. Significant interspecific variation in sensitivity to Cd<sup>2+sup> and Zn<sup>2+sup> was recorded, while Pb<sup>2+sup> and Sb<sup>3-sup> individually had little effect. The presence of Pb<sup>2+sup> and Sb<sup>3-sup> in the media did however, ameliorate Cd<sup>2+sup> and Zn<sup>2+sup> toxicity in some circumstances. Interactions between Cd<sup>2+sup> and Zn<sup>2+sup> were investigated further over a range of concentrations. Zn<sup>2+sup> was found to significantly ameliorate the toxicity of Cd<sup>2+sup> to three of the four isolates tested. The influence of Zn<sup>2+sup> varied between ECM species and with the concentrations of metals tested.

High-affinity NO3 --H+ cotransport in the fungus Neurospora:Induction and control by pH and membrane voltate

High-affinity nitrate transport was examined in intact hyphae of Neurospora crassa using electrophysiological recordings to characterize the response of the plasma membrane to NO₃ <sup>-sup> challenge and to quantify transport activity. The NO₃ <sup>-sup>-associated membrane current was determined using a three electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in hyphae transferred to NO₃ <sup>-sup>-free, N-limited medium for 15 hr, and in hyphae grown in the absence of a nitrogen source after a single 2-min exposure to 100 μM NO₃ <sup>-sup>. In the latter, induction showed a latency of 40-80 min and rose in scalar fashion with full transport activity mensurable approx. 100 min after first exposure to NO₃ <sup>-sup>; it was marked by the appearance of a pronounced sensitivity of membrane voltage to extracellular NO₃ <sup>-sup> additions which, after induction, resulted in reversible membrane depolarizations of (+)54-85 mV in the presence of 50 μM NO₃ <sup>-sup>; and it was suppressed when NH₄ <sup>+sup>, was present during the first, inductive exposure to NO₃ <sup>-sup>. Voltage clamp measurements carried out immediately before and following NO₃ <sup>-sup> additions showed that the NO₃ <sup>-sup>-evoked depolarizations were the consequence of an inward-directed current that appeared in parallel with the depolarizations across the entire range of accessible voltages -400 to +100 mV). Measurements of NO₃ <sup>-sup> uptake using NO₃ <sup>-sup>-selective macroelectrodes indicated a charge stoichiometry for NO₃ <sup>-sup> transport of 1(+):1(NO₃ <sup>-sup>) with common K(m) and J(max) values around 25 μM and 75 pmol NO₃ <sup>-sup> cm<sup>-2sup>sec<sup>-1sup>, respectively, and combined measurements of pH(o) and [NO₃ <sup>-sup>](o) showed a net uptake of approx. 1 H<sup>+sup> with each NO₃ <sup>-sup> anion. Analysis of the NO₃ <sup>-sup> current demonstrated a pronounced voltage sensitivity within the normal physiological range between -300 and -100 mV as well as interactions between the kinetic parameters of membrane voltage, pH(o) and [NO₃ <sup>-sup>](o). Increasing the bathing pH from 5.5 to 8.0 reduced the current and the associated membrane depolarizations 2- to 4-fold. At a constant pH(o) of 6.1, driving the membrane voltage from -350 to -150 mV resulted in an approx. 3-fold reduction in the maximum current and a 5-fold rise in the apparent affinity for NO₃ <sup>-sup>. By contrast, the same depolarization effected an approx. 20% fall in the K(m) for transport as a function in [H<sup>+sup>](o). These, and additional results are consistent with a charge-coupling stoichiometry of 2(H<sup>+sup>) per NO anion transported across the membrane, and implicate a carrier cycle in which NO binding is kinetically adjacent to the rate-limiting step of membrane charge transit. The data concur with previous studies demonstrating a pronounced voltage-dependence to high-affinity NO₃ <sup>-sup> transport system in Arabidopsis, and underline the importance of voltage as a kinetic factor controlling NO₃ <sup>-sup> transport; finally, they distinguish metabolite repression of NO₃ <sup>-sup> transport induction from its sensitivity to metabolic blockade and competition with the uptake of other substrates that draw on membrane voltage as a kinetic substrate.

Biotransformation of 2,4,6-trinitrotoluene (TNT) by ectomycorrhizal basidiomycetes

The ability of four ectomycorrhizal basidiomycetes to biotransform 2,4,6-trinitrotoluene (TNT) in axenic culture was tested. All species were capable of TNT biotransformation to a greater or lesser extent. When biotransformation was expressed on a biomass basis 4 out of the 5 isolates tested were equally efficient at transforming TNT. The factors regulating TNT biotransformation were investigated in detail for one fungus, Suillus variegatus. When the fungus was grown under nitrogen limiting conditions the rate of biotransformation decreased relative to nitrogen sufficient conditions, but no decrease was observed under short term carbon starvation. Extracellular enzymes of S. variegatus could transform TNT, but transformation was greater in intact cells. The mycelial cell wall fraction did not degrade TNT. The TNT concentration that caused 50% reduction in biomass (EC₅₀) for S. variegatus was within the range observed for other basidiomycete fungi being between 2-10 μg mL<sup>-1sup>. The potential use of ectomycorrhizal basidiomycetes as in-situ bioremediation agents for TNT contaminated soils is discussed.

Bioavailability of atrazine to soil microbes in the presence of the earthworm Lumbricus terrestrius (L.)

Experiments were conducted to investigate the interactions between an earthworm species (Lumbricus terrestrius) and soil microflora with respect to the bioavailability and mineralisation of <sup>14sup>C ring-labelled atrazine. Presence of earthworms had no affect on atrazine in soil solution (assayed by soil centrifugation). This soil solution pool was highly time dependent, decreasing considerably as the experiment proceeded. KCl-extractable label was, however, affected by the presence of earthworms, with this pool initially increasing in the presence of the worms. This pool was also highly time-dependent although, the pattern of this dependence did not follow that for label in soil solution. Mineralisation of the atrazine closely followed the KCl exchangeable pool and not that of the soil solution pool. However, label sorbed to the surface of the worms was closely correlated to the soil solution pool. Mineralisation in the presence of earthworms was double that of the controls. By the end of the experiment 6% of added radioactivity was present in the earthworm biomass.

Loss of exudates from the roots of perennial ryegrass inoculated with a range of micro-organisms

To determine the effect of microbial metabolites on the release of root exudates from perennial ryegrass, seedlings were pulse labelled with [<sup>14sup>C]-CO₂ in the presence of a range of soil micro-organisms. Microbial inoculants were spatially separated from roots by Millipore membranes so that root infection did not occur. Using this technique, only microbial metabolites affected root exudation. The effect of microbial metabolites on carbon assimilation and distribution and root exudation was determined for 15 microbial species. Assimilation of a pulse label varied by over 3.5 fold, dependent on inoculant. Distribution of the label between roots and shoots also varied with inoculant, but the carbon pool that was most sensitive to inoculation was root exudation. In the absence of a microbial inoculant only 1% of assimilated label was exuded. Inoculation of the microcosms always caused an increase in exudation but the percentage exuded varied greatly, within the range of 3-34%. © 1995 Kluwer Academic Publishers.

Phosphorus nutrition of arsenate-tolerant and nontolerant phenotypes of velvetgrass

Velvetgrass (Holcus lanatus L.), also known as Yorkshire fog grass, has evolved tolerance to high levels of arsenate, and this adaptation involves reduced accumulation of arsenate through the suppression of the high affinity phosphate-arsenate uptake system. To determine the role of P nutrition in arsenate tolerance, inhibition kinetics of arsenate influx by phosphate were determined. The concentration of inhibitor required to reduce maximum influx (V(max)) by 50%, K₁, of phosphate inhibition of arsenate influx was 0.02 mol m<sup>-3sup> in both tolerant and nontolerant clones. This was compared with the concentration where influx is 50% of maximum, a K(m), for arsenate influx of 0.6 mol m<sup>-3sup> for tolerants and 0.025 mol m<sup>-3sup> for nontolerants and, therefore, phosphate was much more effective at inhibiting arsenate influx in tolerant genotypes. The high affinity phosphate uptake system is inducible under low plant phosphate status, this increasing plant phosphate status should increase tolerance by decreasing arsenate influx. Root extension in arsenate solutions of tolerant and nontolerant tillers grown under differing phosphate nutritional regimes showed that indeed, increased plant P status increased the tolerance to arsenate of both tolerant and nontolerant clones. That plant P status increased tolerance again argues that P nutrition has a critical role in arsenate tolerance. To determine if short term flux and solution culture studies were relevant to As and P accumulation in soils, soil and plant material from a range of As contaminated sites were analyzed. As predicted from the short-term competition studies, P was accumulated preferentially to As in arsenate tolerant clones growing on mine spoil soils even when acid extractable arsenate in the soils was much greater than acid extractable phosphate. Though phosphate was much more efficient at competing with arsenate for uptake, plants growing on arsenate contaminated land still accumulated considerable amounts of As. Plants from the differing habitats showed large variation in plant phosphate status, pasture plants having much higher P levels than plants growing on the most contaminated mine spoil soils. The selectivity of the phosphate-arsenate uptake system for phosphate compared with arsenate, coupled with the suppression of this uptake system enabled tolerant clones of the grass velvetgrass to grow on soils that were highly contaminated with arsenate and deficient in phosphate.

A critical review of labelling techniques used to quantify rhizosphere carbon-flow

The rhizosphere is a major sink for photo-assimilated carbon and quantifying inputs into this sink is one of the main goals of rhizosphere biology as organic carbon lost from plant roots supports a higher microbial population in the rhizosphere compared to bulk soil. Two fundamentally different<sup>14sup>CO₂ labelling strategies have been developed to estimate carbon fluxes through the rhizosphere - continuous feeding of shoots with labelled carbon dioxide and pulse-chase experiments. The biological interpretation that can be placed on the results of labelling experiments is greatly biased by the technique used. It is the purpose of this paper to assess the advantages, disadvantages and the biological interpretation of both continuous and pulse labelling and to consider how to partition carbon fluxes within the rhizosphere. © 1994 Kluwer Academic Publishers.