Is the dehydrogenase assay invalid as a method to estimate microbial activity in copper-contaminated soils?

Abstract

Soil dehydrogenase activity is commonly estimated from the conversion of triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF). There are many reports of larger proportional decreases in dehydrogenase activity than in other indices of microbial activity in Cu-contaminated soils or in soils recently amended with Cu-contaminated sewage sludge. We describe four experiments which measured the effects of Cu on the dehydrogenase assay. In the first, soil microbial biomass, soil dehydrogenase activity and biomass specific dehydrogenase activity were compared in a soil contaminated with heavy metals, including Cu, and in an uncontaminated soil. The specific dehydrogenase activity of the biomass was about 35% less in the metal-contaminated soil than in the uncontaminated soil. In contrast, previous work showed that the biomass specific respiration rate was about 1.5 times faster in the metal-contaminated soil. In the second experiment, sewage sludges (uncontaminated, contaminated singly with Cu, Ni, Cd or Zn and a sludge containing the metals in combination) were added separately to an uncontaminated soil and incubalcd for 7 days at 25°C. Large, similar increases in biomass C, CO 2 evolution and biomass specific respiration were measured within all sludge-amended soils at 1 and 7 days after sludge addition. Soil dehydrogenase activity increased similarly in the soils amended with sludges which contained no added Cu. Thus the specific dehydrogenase activity of the biomass was very similar in all these treatments. In contrast, soil dehydrogenase activity and specific dehydrogenase activity of the biomass in the soils amended with Cu-rich sludge or with the sludge containing all the metals, including Cu, were 2–3 times smaller than in the other treatments. In the third experiment, TPF was incubated with separate solutions (0–100 mg l −1) of heavy metals in the absence of soil. Absorbance of TPF was virtually unaffected at any concentration of Ni, Cd or Zn but declined almost to zero between 0 and 20 mg Cu l −1. If this abiological reaction occurred in soil it would be reported, incorrectly, as decreased dehydrogenase activity. This was tested in the fourth experiment by adding TPF to soils previously incubated with the various metal-rich sludges. Absorbance due to TPF was significantly decreased in the soils containing Cu-rich sludges, but was unaffected in the other treatments. It thus appears that the main reason why soils contaminated with Cu apparently have significantly decreased dehydrogenase activities is the abiological reaction between TPF and Cu. Previous research on the effects of Cu on soil dehydrogenase activity has not considered this phenomenon and much of it, therefore, is probably invalid.