Metal residues in soils previously treated with sewage-sludge and their effects on growth and nitrogen fixation by blue-green algae

Nitrogen-fixation was measured in illuminated laboratory incubations of soils containing different concentrations of Zn, Cu, Ni and Cd. The soils were taken from field experiments that had received either farmyard manure (low-metal soils) between 1942–67 or metal-contaminated sewage-sludge (high-metal soils) between 1942–61 so that the last metal inputs from sewage-sludge were more than 20 years ago.Colonies of blue-green algae (cyanobacteria) and other microorganisms developed rapidly on the surface of low-metal soil and after a 14 day lag there was a rapid increase in rate of C₂H₂-reduction, indicative of N₂-fixation. C₂H₂-reduction reached a maximum rate after about 28 days then slowly declined. In contrast, colonisation of the surface of high-metal soil was very much poorer. C₂H₂-reduction only commenced after 50 days, reaching only about a third of the rate in the low-metal soil by 118 days, when the experiment ended.In another experiment the low-metal soil fixed about 10 times as much ¹⁵N-labelled N₂ as the high-metal soil. The ratio C₂H₂ reduced-to-N₂ fixed was 5.Measurements of total C₂H₂-reduction were made during a 60 day incubation on soils sampled on a transect from the middle of a low-metal farmyard manure (FYM) plot to the middle of a high-metal sludge plot. There was a negative correlation (r = −0.78) between total C₂H₂-reduction and increasing distance along the transect, corresponding to increasing soil concentrations of Zn, Cu, Ni and Cd with increasing distance. Total C₂H₂-reduction was decreased by 50% at about 50 μg EDTA-extractable Zn, 20 μg Cu, 2.5 μg Ni and 3 μg total Cd g⁻¹ soil. Because the amounts of metals in the soils were closely correlated with each other there was a similar relationship with Zinc Equivalent. At half the current permitted U.K. metal loading based on Zinc Equivalent, C₂H₂-reduction was also decreased by about 50%.     

Nitrogenase activity (Acetylene reduction) during the growth cycle of spring barley (Hordeum vulgare L.)

The nitrogenase activity (C₂H₂-reduction) was measured during the growth cycle of field grown spring barley in soil cores both with and without barley plants, and at two levels of nitrogen application, 30 and 120 kg N ha^?1 year^?1 respectively. The main purpose of the investigation was to study the effects of the growing barley plants on nitrogenase activity in the soil, and temperature and moisture contents were kept constant in all experiments. Therefore, the results cannot be used to calculate actual amounts of fixed nitrogen in the field, but should be considered rather as potential values. The nitrogenase activity was found to vary during the growth cycle, and seemed to be correlated to the photosynthetic activity of the plants. Relatively low nitrogenase activity was found in the early growth stages, and the activity increased up to a maximum in the late reproductive stage, followed by a rapid decrease during the grain filling stage. The mean values of nitrogenase activity in samples without barley plants and with barley plants were 40 and 78 nmoles C₂H₄ g soil dwt^?1 24 h^?1 respectively. The positive effect of barley plants on nitrogenase activity was stronger at 120 kg N than at 30 kg N fertilization. As a mean of the whole growth cycle the ratio between samples with and without barley plants was 1.7 with 30 kgN and 2.3 with 120 kg N fertilization. The inhibitory effect of nitrogen application on nitrogenase activity was measurable until 6–7 weeks after application, and it was strongest in cores without plants.     

Nitrogenase activity associated with soil cores of grasses in Brazil

The N₂-ase activities of field-grown Brazilian grasses were measured with C₂H₂ reduction by soil cores containing the plants. C₂H₂ and C₂H₄ were observed to diffuse at similar rates through soil and equilibrated across the Brazilian soil in 3 h, but could take up to 30 h or more with some British soils. The diurnal fluctuation in the rates of N₂-ase activity by Brachiaria mutica and Sorghum vulgare were similar and the variation in rate was correlated with soil temperature. Estimates of N₂-fixation by measurement of C₂H₂ reduction by soil cores ranged from 14.7 to 51.4 g N ha^?1 day^?1 and were much lower than with “pre-incubated” excised roots from the cores or taken directly from the field. The merits of the soil core and the “pre-incubated” excised root assays are discussed. ft1|Present address: U.S. Department of Agriculture, Science and Education Administration, Agricultural Research, Northeastern Region, Room 309, Building 001, Beltsville, MD 20705, U.S.A.     

Soil algae: Effects of herbicides on growth and C2H2 reduction (nitrogenase) activity

The influence of the soil-applied herbicides chlortoluron, terbutryne, metabenzthiazuron, chloridazon, and dinosebacetate as well as the fungicide carbendazime on the growth and nitrogenase activity of soil algae was tested. The degree of algal cover on the soil surface was correlated with the measured C₂H₂-reduction (nitrogenase) activity. All the herbicides tested at recommended rates of application caused a total suppression of algal growth and C₂H₂-generation for several weeks. The fungicide had no detectable effect on algal populations or C₂H₂ reduction.     

Nitrogen fixation in association with the root systems of goldenrods (Solidago L.)

The roots of seven species of goldenrod (Asteraceae: Solidago) were assayed for associative nitrogen fixation. We used the acetylene reduction method to measure N₂ fixation rates in sampling jars that contained roots of sampled plants and soil. The rate of C₂H₂ reduction in jars with S. rigida (12.9 ± 1.2 nmol h⁻¹, mean ± SE) and S. canadensis (6.0 ± 2.0 nmol h⁻¹) was significantly higher than in soil-only control jars. There was measurable C₂H₂ reduction in jars of the other five species, but the rates were not significantly different from controls. The rates of C₂H₂ reduction per root dry weight and per total plant dry weight also were higher for S. rigida and S. canadensis than for the other species.     

Acetylene reduction in soil and litter from pine and eucalypt forests in south-eastern Australia

Rates of C₂H₂-reduction in surface soil and litter from pine and eucalypt forests were measured for 1 yr. Rates of reduction increased significantly with moisture content, and mean rates (nmol kg^?1 h^?1) decreased in the order pine litter (339), eucalypt litter (220), eucalypt soil (54), pine soil (7). Asymbiotic N₂-fixation in litter and surface soil was estimated to be 108 mg m^?2 yr^?1 in eucalypt forest and 64 mg m^?2 yr^?1 in pine forest. About 80% of total fixation in eucalypt was in the soil, while 80% of the total in pine was in the litter. N₂ase was active in rotting wood but not in fresh foliage.     

Acetylene reduction by roots and associated soil of New Zealand conifers

An extensive survey of native conifers (Dacrydium, Podocarpus, Libocedrus, Phyllocladus and Agathis spp.) showed that C₂H₂ reduction was often associated with their mycorrhizal short roots (‘nodules’). C₂H₂ reduction was associated with roots only if it also occurred in surrounding soil, but it could be found in soils and not in the root region. Over 50 per cent of the C₂H₂ reduction activity could be removed by washing roots in distilled water while complete loss of activity occurred when they were surface sterilized with hypochlorite solution. C₂H₂ reduction may also be associated with long roots of podocarps and roots of non-podocarp species. Fermenting and humifying horizons of forest soils showed much greater rates of C₂H₂ reduction than either mineral soil or roots. Results suggest that previous claims for nitrogen fixation by podocarp roots can be attributed to nitrogen fixation by bacteria in the root region rather than by endophytic organisms.     

Growth, Net Photosynthetic Rate, Nutrient Status and Secondary Xylem Anatomical Characteristics of Fagus Crenata Seedlings Grown in Brown Forest Soil Acidified with H2SO4 Solution

Dry matter production, net photosynthetic rate, leaf nutrient status and trunk anatomical characteristics of Fagus crenata seedlings grown in brown forest soil acidified by adding H₂SO₄ solution were investigated. The soil acidification leaded to decreased (Ca+Mg+K)/Al molar ratio in the soil solution. Dry mass per plant of the seedlings grown in the soil treated with H⁺ at 120 mg·L^?1 was significantly reduced compared with the control value at 0 mg·L^?1. When net photosynthetic rate was reduced in the seedlings grown in the soil treated with H⁺ at 120 mg·L^?1, the carboxylation efficiency and maximum net photosynthetic rate at saturated CO₂-concentration were lower than the control values. The addition of H⁺ to the soil at 120 mg·L^?1iinduced a reduction in the concentration of Ca in the leaf. By contrast, the concentration of Al in the leaf was increased with increasing the amount of H⁺ added to the soil. The annual ring formed in the seedlings grown in the soil treated with H⁺ at 120 mg·L^?1 was significantly narrower than that at 0 (control), 10, 30, 60 or 90 mg·L^?1. Based on the results obtained in the present study, we conclude that Fagus crenata is relatively sensitive to a reduction in the (Ca+Mg+K)/Al molar ratio of soil solution compared with Picea abies.     

Nitrogen fixation by biological soil crusts and heterotrophic bacteria in an intact Mojave Desert ecosystem with elevated CO2 and added soil carbon

Fixation of N by biological soil crusts and free-living heterotrophic soil microbes provides a significant proportion of ecosystem N in arid lands. To gain a better understanding of how elevated CO₂ may affect N₂-fixation in aridland ecosystems, we measured C₂H₂ reduction as a proxy for nitrogenase activity in biological soil crusts for 2 yr, and in soils either with or without dextrose-C additions for 1 yr, in an intact Mojave Desert ecosystem exposed to elevated CO₂. We also measured crust and soil δ¹⁵N and total N to assess changes in N sources, and δ¹³C of crusts to determine a functional shift in crust species, with elevated CO₂. The mean rate of C₂H₂ reduction by biological soil crusts was 76.9±5.6 μmol C₂H₄ m⁻² h⁻¹. There was no significant CO₂ effect, but crusts from plant interspaces showed high variability in nitrogenase activity with elevated CO₂. Additions of dextrose-C had a positive effect on rates of C₂H₂ reduction in soil. There was no elevated CO₂ effect on soil nitrogenase activity. Plant cover affected soil response to C addition, with the largest response in plant interspaces. The mean rate of C₂H₂ reduction in soils either with or without C additions were 8.5±3.6 μmol C₂H₄ m⁻² h⁻¹ and 4.8±2.1 μmol m⁻² h⁻¹, respectively. Crust and soil δ¹⁵N and δ¹³C values were not affected by CO₂ treatment, but did show an effect of cover type. Crust and soil samples in plant interspaces had the lowest values for both measurements. Analysis of soil and crust [N] and δ¹⁵N data with the Rayleigh distillation model suggests that any plant community changes with elevated CO₂ and concomitant changes in litter composition likely will overwhelm any physiological changes in N₂-fixation.     

Denitrification potentials: Measurement of seasonal variation using a short-term anaerobic incubation technique

A short-term anaerobic incubation technique using the C₂H₂ inhibition of N₂O-reductase for comparing denitrification potentials of soils is described. Twenty grams of soil with added NO^?1₃ are incubated in the presence of He and 0.1 atm C₂H₂ at 25°C and 0 soil matric potential for 8 h. N₂O evolution is linear within 60 to 120 min. The denitrification potential of soils stored at 4°C decreased markedly over 21 days of storage in accordance with changes in the available C. Denitrification under an anaerobic atmosphere was observed at 4 C. Denitrification potentials were independent of NO^?3₃ concentrations above 25 μg NO^?₃-N g^?1 soil. Biphasic linear rates of N₂O evolution were observed in one soil. Incubation of this soil with chloramphenicol suggested the first linear phase is attributable to the in situ enzyme activity at the time of sampling. The second linear phase is indicative of the dentrification potential and is attributed to the full induction of denitrifying enzymes. The denitrification potential of a soil was maintained at or close to the maximum for 8 months of the year. During midsummer months the denitrification potential decreased markedly and the soil demonstrated a biphasic rate of denitrification suggesting an in situ denitrification activity less than the maximum potential. Results indicate that the maximum denitrification potential of this soil may often be limited not by NO^?₃ but by available C.     

Effect of long-term field exposure of soil to acetylene on nitrification,denitrification, and acetylene consumption

Coated CaC₂ is a newly developed product which can supply nitrification-inhibiting quantities of C₂H₂ (1–10 Pa) to the soil, throughout a cropping season. This method of applying C₂H₂ to the soil maintains C₂H₂ in the soil continuously for several months. It is not know whether these low C₂H₂ concentrations alter soil microbial processes. A field study was initiated to determine the effect of supplying C₂H₂ to a clay soil, using coated CaC₂, on soil respiration, denitrification, nitrification, and C₂H₂ consumption. The C₂H₂ consumption rate increased with length of soil exposure to C₂H₂ (r ²=0.59). The rates of CO₂ production (r ²=0.88) and denitrification (r ²=0.86) were both highly correlated with the C₂H₂ consumption rates. The nitrifier potential decreased to a minimum of 21% of the control after 3 months of C₂H₂ treatment. After this time, nitrifier activity increased to 41% of the control after 11 months of treatment. This increase was due to increased C₂H₂ consumption in the soil. After 3 months of continuous application of C₂H₂ to the soil, the C₂H₂ concentrations were generally below that necessary to inhibit nitrification. No adaptation to the C₂H₂ by nitrifiers was found. Repeating these measurements 1 year later showed that soils previously exposed to C₂H₂ retained their enhanced C₂H₂ oxidation capacity and the capacity to use C₂H₂ to increase denitrification. Nitrification potentials remained about 50% lower in soils exposed to C₂H₂ a year earlier compared to soils not previously exposed to C₂H₂.     

Effect of acetylene and ethylene gases released from encapsulated calcium carbide on growth and yield of wheat and cotton

Calcium carbide (CaC₂) is a rich source of the nitrification inhibitor acetylene (C₂H₂) and plant hormone ethylene (C₂H₄). C₂H₄ formed from biotic reduction of C₂H₂ released from CaC₂ may accumulate in soil at physiologically active concentrations. Laboratory studies were conducted to evaluate the potential of encapsulated CaC₂ for gradually releasing C₂H₂ and its product C₂H₄ in soil. The GC-FID analysis revealed that encapsulated CaC₂ released a copious amount of C₂H₂ (up to 23700 nmol kg⁻¹ soil), which was gradually reduced to C₂H₄ over a period of time via a strictly biotic reaction as no C₂H₄ was detected in CaC₂-amended sterilized soil. Ammonium oxidation was suppressed by the encapsulated CaC₂ indicating that C₂H₂ acted as a nitrification inhibitor. Results of pot trials conducted in the net house indicated that encapsulated CaC₂ applied at 30 mg kg⁻¹ soil significantly increased the number of tillers (up to 45.5%), root weight (up to 14.9%), straw (up to 32.8%) and grain yield (up to 37.3%) of wheat over the fertilizer application alone. In the case of cotton, the number of bolls, root, shoot and seed weight were also significantly increased in response to the application of encapsulated CaC₂. Moreover, application of encapsulated CaC₂ resulted in greater N-use efficiency (NUE) (up to 61.1%) by both wheat and cotton crops than that observed at the same rates of N fertilizer alone. These findings imply that CaC₂ affects plant growth through hormonal action of C₂H₄ as well as improved NUE; however, the latter factor might be a relatively more contributing. It is desirable that CaC₂ is formulated for gradually slow release of C₂H₂ and C₂H₄ in soil air.     

Partitioning root and microbial contributions to soil respiration in Leymus chinensis populations

Based on the enclosed chamber method, soil respiration measurements of Leymus chinensis populations with four planting densities (30, 60, 90 and 120 plants/0.25 m²) and blank control were made from July 31 to November 24, 2003. In terms of soil respiration rates of L. chinensis populations with four planting densities and their corresponding root biomass, linear regressive equations between soil respiration rates and dry root weights were obtained at different observation times. Thus, soil respiration rates attributed to soil microbial activity could be estimated by extrapolating the regressive equations to zero root biomass. The soil microbial respiration rates of L. chinensis populations during the growing season ranged from 52.08 to 256.35 mg CO₂ m⁻² h⁻¹. Soil microbial respiration rates in blank control plots were also observed directly, ranging from 65.00 to 267.40 mg CO₂ m⁻² h⁻¹. The difference of soil microbial respiration rates between the inferred and the observed methods ranged from −26.09 to 9.35 mg CO₂ m⁻² h⁻¹. Some assumptions associated with these two approaches were not completely valid, which might result in this discrepancy. However, these two methods' application could provide new insights into separating root respiration from soil microbial respiration. The root respiration rates of L. chinensis populations with four planting densities could be estimated based on measured soil respiration rates, soil microbial respiration rates and corresponding mean dry root weight, and the highest values appeared at the early stage, then dropped off rapidly and tended to be constant after September 10. The mean proportions of soil respiration rates of L. chinensis populations attributable to the inferred and the observed root respiration rates were 36.8% (ranging from 9.7 to 52.9%) and 30.0% (ranging from 5.8 to 41.2%), respectively. Although root respiration rates of L. chinensis populations declined rapidly, the proportion of root respiration to soil respiration still increased gradually with the increase of root biomass.     

Microbial ethylene production and inhibition of methanotrophic activity in a deciduous forest soil

Production of C₂H₄, but not of CH₄, was observed in anoxically incubated soil samples (cambisol on loamy sand) from a deciduous forest. Ethylene production was prevented by autoclaving, indicating its microbial origin. Ethylene production gradually decreased from 4 to 12 cm soil depth and was not affected by moisture or addition of methionine, a possible precursor of C₂H₄. Oxidation of atmospheric CH₄ in soil samples was inhibited by C₂H₄. Ethylene concentrations of 3, 6 and 10 μl l⁻¹ decreased CH₄ uptake by 21, 63 and 98%, respectively. Methionine and methanethiol, a possible product of methionine degradation, also inhibited CH₄ oxidation. Under oxic conditions, C₂H₄ was consumed in the soil samples. Ethylene oxidation kinetics exhibited two apparent K_m values of 40 μl l⁻¹ and 12,600 μl l⁻¹ suggesting the presence of two different types of C₂H₄-oxidizing microorganisms. Methanotrophic bacteria were most probably not responsible for C₂H₄ oxidation, since the maximum of C₂H₄ oxidation activity was localized in soil layers (2-8 cm depth) above those (8-10 cm depth) of CH₄ oxidation activity. Our observations suggest that C₂H₄ production in the upper soil layers inhibits CH₄ oxidation, thus being one reason for the localization of methanotrophic activity in deeper soil layers.     

Critical evaluation of the acetylene reduction test for estimating the activity of nitrogen-fixing bacteria associated with the roots of wheat and barley

The reliability of the C₂H₂ reduction test for estimating the activity of N₂-fixing bacteria associated with the roots of cereals has been evaluated in Scottish soils. Six wheat cultivars, including two chromosome substitution lines, and five barley cultivars were grown in a glasshouse in nine soils from the North East of Scotland. All the soils exhibited C₂H₄ oxidase activity which was completely inhibited by 0.0001–0.1 atm C₂H₂. Over-estimation of C₂H₂ reduction, resulting from the accumulation of endogenous C₂H₄, could, therefore, occur in assays of undisturbed plants, with the real possibility of deducing the existence of N₂-fixation where none existed. However, radiolabelled C₂H₂ reduction tests on undisturbed plants producing 2.4–18.0 μmol C₂H₄ day^?1, showed that all the C₂H₄ had been derived from the C₂H₂. With less active plants, the source of the C₂H₄ could not be accurately determined by this tracer method. These low rates of C₂H₄ production (< 2.4 μmol C₂H₄ day^?1), referred to as apparent C₂H₂ reduction, should, therefore, not be considered proof of N₂-fixation.The highest C₂H₂ reduction activities were observed in soils at maximum water holding capacity (MWHC). Roots removed from these soils reduced C₂H₂ immediately, if the initial partial pressure of O₂ (pO₂) was < 0.1 atm. Roots washed free of soil did not oxidize C₂H₄ during the 8 h assay. The C₂H₂ reduction activities of these excised roots could not be related to the activity of plants in soil for three reasons. (1) Development of C₂H₂ reduction was dependent on protein synthesis (inhibited by chloramphenicol), indicating re-establishment of activity destroyed by exposure to atmospheric pO₂, rather than continuation of the activity of undisturbed roots. (2) A lag period, dependent on the volume of the incubation vessel, was observed, indicating the involvement of root respiration in the assay. (3) Growth of N₂-fixing bacteria on compounds released from the roots (reducing sugars, amino acids and α-keto acids) occurred during the assay.Even with the possibility of over-estimation of N₂-fixation, the C₂H₂ reduction activities measured were considered to be too low to contribute significantly to the nitrogen requirement of the cereals grown under field conditions in Scotland.Some guidelines for screening programmes of N₂-fixation associated with the roots of grasses are suggested.     

Nitrogenase activity associated with three tropical grasses growing in undisturbed soil cores

Nitrogenase activity associated with the root system of three tropical grasses Axonopus compressus, Digitaria decumbens var. pangolaand Paspalum notatum was measured by C₂H₂ reduction assay of soil-plant cores. The cores were incubated in perspex chambers in which 10% of the air was replaced with C₂H₂. Gas samples were taken at 7, 24, 31, 48, 55 and 72 h. No lag before onset of C₂H₄ production was evident and good agreement was obtained between replicates. Cumulative C₂H₄ production maintained a linear trend during the 72 h incubation.The largest increase in N₂-ase activity was detected in the A. compressus—gleyed podzolic system while D. decumbens (lateritic podzolic) and P. notatum (sandy yellow podzolic) had smaller activities. Variation between sampling sites in the second year of sampling of the experiment was associated with large variations in soil moisture.N₂ fixation estimated from N₂-ase activity in soil-plant cores was similar to the amount of N accumulated in the above-ground herbage in the field during 12 weeks.Response curves relating N₂-ase activity to soil moisture and soil temperature were established for all species. P. notatum and D. decumbens responded similarly to changes in both soil temperature and soil moisture while A. compressus contrasted sharply to the other two species in its reaction to both.     

Production and stability of ethylene in soil

Summary One of the major factors affecting the production and stability of ethylene (C₂H₄) in soil is its water content. This study was conducted to determine the effect of unsaturated vs. saturated conditions on the production and stability of C₂H₄ in soil. L-Methionine and D-glucose were added alone and in combination at 1.0 and 5.0 g kg^-1 soil, respectively. The addition of l-methionine significantly promoted C₂H₄ production at field capacity to a much greater extent than under waterlogged conditions. Glucose was equally effective under both moisture regimes, while the combined application of both amendments (l-methionine and d-glucose) led to the release of significantly higher amounts of C₂H₄ under saturated conditions. Antibiotic experiments revealed that under aerobic conditions, l-methionine may be more efficiently converted to C₂H₄ by soil fungi, while in glucose-amended soil, both bacteria and fungi are active in generating C₂H₄. C₂H₄ was more stable under saturated conditions. The magnitude of C₂H₄ removal from the headspace after 3 days of incubation under unsaturated conditions (25.7%) was comparable to that after 6 days under saturated conditions (24.1%). The loss of C₂H₄ was approximately 10-fold greater in non-sterilized soil than in sterilized (autoclaved) soil, both maintained at field capacity, indicating that a biotic component has a major influence on C₂H₄ stability. Kinetic analysis revealed that the C₂H₄ loss/degradation in nonautoclaved soil under aerobic conditions followed a firstorder reaction, with a rate constant (k) of 0.115 day^-1 and a half-life (t _1/2) of 6.0 days.     

Dinoseb as a specific inhibitor of nitrogen fixation in soil

N₂ fixation, estimated by reduction of C₂H₂, by a loamy soil amended with glucose was highly sensitive to the pesticide, 4,6-dinitro-o-sec-butylphenol (DNBP).At a concentration of 6 parts/10⁶ DNBP inhibited reduction of C₂H₂ by 90% in an anaerobic artificial soil system. Studies of the nitrogen-fixing enzyme system isolated from Azotobacter suggested that DNBP may compete with ATP for the same site on the enzyme.     

Kinetics of nitrogen fixation in a glucose-amended,anaerobically incubated soil

Nitrogen fixation and acetylene reduction activities were studied in a sandy loam soil amended with glucose (2% w/w) at field moisture content and incubated anaerobically. Optimum temperature for C₂H₂ reduction was about 37°C and the maximum was 45°C. Q₁₀ values were 1.6–3.7 in the range 10–35°C. Calculated activation energies were lower than those reported for Clostridium whole cells. Apparent K_m (C₂H₂) averaged 0.006 atm pC₂H₂ and the apparent K_m (N₂) was 0.095 atm pN₂. Low concentrations of C₂H₂ competed strongly with N₂ for the soil N₂ase (apparent K[ini] was 0.0003 atm pC₂H₂ with 0.8 atm pN₂). A relatively high concentration of ethylene (0.22 atm pC₂H₄) caused 30–40 per cent inhibition of N₂ase activity (measured as ¹⁵N₂ fixation) but the lower concentrations likely to be encountered in C₂H₂ assays had no significant effect. Conversion factors (C₂H₄/N₂ molar ratios) determined under various conditions ranged from 0.75 to 3.6. A value of 2.6 was obtained using the most favourable short-term C₂H₂ assays.