Biol Fertil Soils (2001) 33:328–330 DOI 10.1007/s003740000332

O R I G I N A L PA P E R

Jeffrey P. Obbard

Measurement of dehydrogenase activity using 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) in the presence of copper Received: 9 May 2000 / Published online: 4 January 2001 © Springer-Verlag 2001

Abstract This paper reports on the adverse effect of Cu on the use of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) as a substrate for measuring microbial dehydrogenase activity (DHA). The presence of Cu affected the absorbance of the reaction product 2p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium formazan. The experiment provides evidence that the use of INT is an unreliable measure of DHA activity in soils contaminated with Cu. Keywords Microbial dehydrogenase activity · Copper-contaminated soil · 2-p-Iodophenyl-3-pnitrophenyl-5-phenyltetrazolium chloride · 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium formazan · 2,3,5-Triphenyltetrazolium chloride

Introduction The measurement of the ecotoxicological impacts of soil contaminants has increased in prominence as it is increasingly recognised that potential impacts of soil contaminants cannot be evaluated by using measurement of physicochemical soil parameters alone (Domsch 1984; Doelman et al. 1994; Dahlin et al. 1997). The use of accurate biochemical methods to determine microbiological activity is essential, and various assays have been developed for this purpose (Nannipieri et al. 1990; Torstensson 1997). Enzyme activity is essential in both the mineralisation and transformation of organic C and plant nutrients. Hence, enzyme measurements have been used to determine effects of contaminants on soil microorganisms. The measurement of microbial dehydrogenase activity J.P. Obbard (✉) Department of Chemical and Environmental Engineering, 10 Kent Ridge Crescent, National University of Singapore, Singapore 119260 e-mail: [email protected] Fax: +65-779-1936

(DHA) in soils and sediments has been used extensively as dehydrogenases are intracellular to the microbial biomass, common throughout microbial species and are rapidly degraded following cell death (Somerville et al. 1987; Rossel and Tarradellas 1991). DHA in soil was first measured using the reduction of 2,3,5-triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) by Lenhard (1956). Benefield et al. (1977) reported that 2-p-iodophenyl-3-p-nitrophenyl5-phenyltetrazolium chloride (INT) was a more sensitive electron acceptor than TTC for dehydrogenase measurements, although both were less efficient than oxygen. Chander and Brookes (1991, 1995) showed that the measurement of DHA in soils contaminated with heavy metals from sewage sludge amendments were inherently flawed due to the interference of Cu ions with the method used, i.e. the reduction of TTC by dehydrogenase enzymes to TPF. It was demonstrated that Cu interfered with the absorbance of TPF, which could be incorrectly interpreted as a toxic effect of Cu on soil DHA. Despite this important finding, there have been a number of recent soil studies based on the measurement of DHA in metal-contaminated soils, including Cu (e.g. Prell-Swaid and Schwedt 1994; Aoyama and Nagumo 1996, 1997; Kelly and Tate 1998). The objective of this investigation was to determine if Cu, as well as other heavy metals, i.e. Zn, Ni, Cd, could adversely affect DHA measurements based on the use of the alternative substrate, INT. INT is reduced to 2p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium formazan (INTF) which, like TPF, can be measured spectrophotometrically. Both TTC and INT are the most commonly used tetrazolium salts used for dehydrogenase measurements and the use of INT is well established (Bitton and Koopman 1986). Similar to TTC, the use of INT has been shown to give good reproducibility and differentiation of DHA in various soil types, where INT may actually be the superior substrate due to greater enzyme reduction, shorter incubation times and greater extractability of INTF from soils (Friedel et al. 1994; Rossel et al. 1997).

329 Table 1 Effect of heavy metals on the absorbance of 2-piodophenyl-3-p-nitrophenyl5-phenyltetrazolium formazan

a Absorbance

values are means of duplicate samples. Repeated analysis (five replicates) was undertaken on the control and the 200 mg/l metal solution, where SDs about the mean absorbance value were <5%

Heavy metal concentration (mg/l)

0 1 5 10 20 50 100 200

Formazan absorbance at 485 nm [expressed as a percentage of the control (i.e. 0 mg/l=100% absorbance)]a Ni

Cd

Zn

Cu

100 99.7 100.5 99.6 100.6 100 99.4 99.4

100 99.7 99.9 99.8 99.3 99.6 100.1 100.1

100 100.1 100 100.4 99.9 99 100.4 100.9

100 95 79.9 53.1 34.2 21 14.6 13.5

Materials and methods INTF (100 mg) was dissolved in CH3OH and the final volume adjusted to 1 l. Aliquots (4 ml) of this solution were then pipetted into separate 25-ml volumetric flasks, followed by either 1 ml sterile autoclaved (121°C, 20 min) deionised water (control solution) or 1 ml of a stock metal sulphate solution for either Cu, Zn, Ni or Cd. Flasks were then made up to volume with sterile deionised water to give final concentrations of 0, 1, 5, 10, 20, 50, 100 and 200 mg/l for each of the four heavy metals. The flasks were then stoppered and incubated for 24 h at 37°C. The volumes were then adjusted to 25 ml with CH3OH, mixed thoroughly and absorbance of INTF measured at 485 nm on a calibrated Perkin Elmer UVVIS Lander 2000 spectrophotometer. Calibration was undertaken in the range of 0–100 mg/l of INTF in CH3OH. Absorbance was expressed as: 100 [(absorbance of INTF in metal-related solution)/(absorbance of INTF in control solution)]. Repeated analysis (five replicates) was undertaken for the control and the 200 mg/L solutions for each element.

Results and discussion Experimental results for INTF absorbance are presented in Table 1. Repeated analysis (five replicates) on the control and the 200 mg/l Cu solutions gave a high reproducibility with a SD of <5% for mean absorbance values. There was no discernible effect on INTF absorbance for Zn, Ni and Cd solutions, irrespective of concentration, but a sharp reduction in absorbance in Cu solutions. However, INTF absorbance was greater than TPF absorbance under identical conditions, i.e. approximately 35% of that in the control at a Cu concentration of 20 mg/l, versus 5% for TTF (unpublished data). Since no soil was present, this Cu-related effect must have been due to an abiological reaction. One potential explanation for this effect on the assay is the formation of chelate complexes between Cu2+ and the formazans (Altman 1976) resulting in a reduction of both TPF and INTF absorbance in solution. However, more detailed investigation on chemical speciation in the assay solutions would be needed to confirm this. Clearly, no such complex is formed between Ni2+, Cd2+, Zn2+ and INTF. In conclusion, it is clear that the presence of Cu has an adverse effect on the measurement of DHA, as determined by the absorbance of the assay product, i.e. INTF,

in the absence of biological activity. These experiments support the earlier work of Chander and Brookes (1991) based on the use of TTC as a substrate, and also provide evidence for a similar, albeit a slightly lesser effect using the alternative substrate INT for DHA measurements. Together, these findings have adverse implications for the measurement of DHA in Cu-contaminated soils when using both the TTC- and INT-based dehydrogenase assays, and such ecotoxicological data should be treated with caution.

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