**4.3. Heavy metals presence**

Heavy metals, even though they are natural constituents of soil, could have long-term hazardous impacts on the health of soil ecosystems, and adverse influences on soil biological processes (Pan & Yu, 2011). Generally, it was assumed that heavy metals can reduce enzyme activity by interacting with the enzyme-substrate complex, denaturing the enzyme protein or interacting with the protein-active groups, they could also affect the synthesis of enzyme microbial cells (Pan & Yu, 2011).

Xie et al. (2009) noted that Cu of 100 mg kg-1 could suppress DHA significantly, while Cd of 5 mg kg-1 had relative greater influence on soil microbial diversity, what suggest that the effect of each soil pollutant on soil microbes and their enzymatic activities was specific. On the contrary, a study by Fernandez-Calviño et al. (2010) indicated adverse effect of Cu on DHA (r=-0.24, *P*<0.01). Threshold Cu concentrations at which changes in the enzyme activities became evident were 150-200 mg total Cu kg-1 and 60-80 mg bioavailable Cu kg-1.

A study by Pan & Yu (2011) undertaken with brown soil, showed that DHA was significantly lower by 37.8% and by 51.1% in Cd and Pb treatments, than in control. Moreover, mentioned researchers noted that the effect of Cd and Pb combined on DHA were higher, than Cd or Pb alone.

We also investigated effect posed by Cd (2 and 20 mg kg-1) on soil DHA (Fig. 13). Incubation of soil material with mentioned Cd doses lasted 42 days. After that DHA was determined via Casida et al. (1964) method.

We observed that Cd presence at concentration of 2 mg kg-1 had stimulating effect on soil DHA level, and we noted increase of DHA by 8.8%, in comparison with control sample (without Cd contamination). However, 10-fold higher Cd amendment (20 mg kg-1) consequence with strong DHA inhibition, by as follows: 29.4% and 35%, in relation to control and 2 mg kg-1 sample, respectively. Observed inhibition effect was probably caused by Cd interaction with enzyme-substrate complex, what resulted with strong decrease of DHA level.

200 Dehydrogenases

processes.

**4.3. Heavy metals presence** 

were higher, than Cd or Pb alone.

via Casida et al. (1964) method.

synthesis of enzyme microbial cells (Pan & Yu, 2011).

Suggested by us inhibition effect, may be supported by founding's of Zabaloy et al. (2008), who in typical Ardiudoll from Argentina observed reduction of DHA for about 48%, as an

Results, suggesting inhibitory pesticide effect on DHA level are also in agreement with those obtained by other plaguicides such as: chlorpyrifos (Kadian et al., 2012), or vermicompost (Romero et al., 2010). Moreover, Muñoz-Leoz et al. (2011) noted that DHA was inhibited by 14%, as a effect of application 5 mg kg-1 tebuconazole fungicide dosage.

Conversely, others have found also different results. For example, Tejada et al. (2011) noted insignificant (by 10%) growth of DHA, when the Prochloraz fungicide applied to the soil increased, possibly because the fungicide is commonly used by bacterial communities, as a source of energy and nutrients. Also Andreá et al. (2003), noted that DHA was slight higher after month from Glyphosate application. In that case authors reported, that Glyphosate stimulated DHA, which means that the herbicide might stimulate the soil oxidative

Heavy metals, even though they are natural constituents of soil, could have long-term hazardous impacts on the health of soil ecosystems, and adverse influences on soil biological processes (Pan & Yu, 2011). Generally, it was assumed that heavy metals can reduce enzyme activity by interacting with the enzyme-substrate complex, denaturing the enzyme protein or interacting with the protein-active groups, they could also affect the

Xie et al. (2009) noted that Cu of 100 mg kg-1 could suppress DHA significantly, while Cd of 5 mg kg-1 had relative greater influence on soil microbial diversity, what suggest that the effect of each soil pollutant on soil microbes and their enzymatic activities was specific. On the contrary, a study by Fernandez-Calviño et al. (2010) indicated adverse effect of Cu on DHA (r=-0.24, *P*<0.01). Threshold Cu concentrations at which changes in the enzyme activities became evident were 150-200 mg total Cu kg-1 and 60-80 mg bioavailable Cu kg-1.

A study by Pan & Yu (2011) undertaken with brown soil, showed that DHA was significantly lower by 37.8% and by 51.1% in Cd and Pb treatments, than in control. Moreover, mentioned researchers noted that the effect of Cd and Pb combined on DHA

We also investigated effect posed by Cd (2 and 20 mg kg-1) on soil DHA (Fig. 13). Incubation of soil material with mentioned Cd doses lasted 42 days. After that DHA was determined

We observed that Cd presence at concentration of 2 mg kg-1 had stimulating effect on soil DHA level, and we noted increase of DHA by 8.8%, in comparison with control sample (without Cd contamination). However, 10-fold higher Cd amendment (20 mg kg-1)

effect of Glyphosate contamination, in comparison to control sample.

**Figure 13.** Effect of Cd on DHA in *Eutric Fluvisol* (according to Stępniewska & Wolińska, unpublished data) from Cd introduction into the soil. Averaged values of three replicates with standard deviations are presented

Our results, may be supported by findings of Moreno et al. (2001), who by investigating the influence of Cd on DHA stated, that Cd content strongly affected on DHA, by reducing its activity, and this effect is noticeable even after 3 hours.

Negative effect of heavy metals on DHA was reported also by Kizilkaya et al. (2004), who organized the following order of this inhibition: Cu > Cd > Co. Analogically, strong reduction of DHA by Cd contamination was indicated by Welp (1999), who tested the effect of Co, As, Hg, Cd, Pb and Cu on soil DHA, and demonstrated that the strongest effect was displayed by Hg and Cd.

Stępniewska & Wolińska (2005) found that the application of trivalent and hexavalent chromium compounds had a noticeable negative effect on soil DHA (Fig. 14). The soil sample (*Haplic Luvisol*) was amended with Cr (III), as a CrCl3 and with Cr (VI), as a K2Cr2O7 in the concentration range from 0 to 20 mg kg−1 and 0-100 µg kg-1, for Cr (III) and Cr (VI), respectively. The differences in Cr (III) and Cr (VI) doses resulted from the fact that Cr (VI) is highly toxic and much mobile form of Cr, and is considered to cause much stronger effect on living organisms, than Cr (III).

Dehydrogenase Activity in the Soil Environment 203

**Figure 15.** The variations of DHA in *Eutric Cambisol* at different Cr (III) and Cr (VI) concentrations (according to Stępniewska & Wolińska, 2004). Averaged values of three replicates with standard

Soil is a part of the terrestrial compartment, and supports all terrestrial life forms. Thus, without proper soil protection policies, numerous problems may arise, like reduction of soil fertility, erosion, groundwater contamination, insufficient water holding capacity and loss of biodiversity. To asses soil quality, it is essential to measure all potential changes in biological soil properties, because they are highly sensitive to any environmental perturbations and stresses. A usual approach to diagnose soil quality, is to use a soil microbial indicators, which are very sensitive and respond quickly to environmental

Among different soil indicators, DHA is one of the most adequate, important and one of the most sensitive bioindicators, relating to soil quality and fertility. Moreover, their routine measurement is simple and low-cost under laboratory condition. However, we should not remind about limitations, resulting from laboratory conditions, when we are able to measure and estimate only potential DHA, similarly like we are able to cultivate only small

Soil enzymes are strongly associated with microorganisms. Soil enzymatic activity plays an important role in catalyzing reactions indispensable in life processes of soil microorganisms, decomposition of organic residues, circulation of nutrients, as well as forming organic matter and soil structure. Thus, it is possible to say that without proper soil enzymes

percentage of soil microorganisms, on artificial media.

system, soil life processes will be disturbed.

deviations are presented

**5. Conclusions** 

alterations.

**Figure 14.** The variations of DHA in *Haplic Luvisol* at different Cr (III) and Cr (VI) concentrations (according to Stępniewska & Wolińska, 2005). Averaged values of three replicates with standard deviations are presented

Non-amended soil samples were used as a control, and their enzymatic activity were estimated as 100%. Effect posed by Cr content was calculated as a decrease of its level, in relation to the control value. We found that the lowest values of DHA were the effect of increasing Cr(III) and Cr(VI) doses. *Haplic Luvisol* seemed to be very sensitive on Cr contamination. DHA was reduced to 18-20% in the samples enriched in Cr (III) forms. Surprisingly, the more dangerous form of Cr (VI) was less harmful for DHA in the *Haplic Luvisol*, because enzymatic activity remained on the level of 84%, with a 1µg kg-1 addition and decreased to the value of 14% with the highest supplement of Cr (VI). One possible explanation for this fact is that. the more dangerous form of Cr (VI) was reduced to the less toxic form of Cr (III) by microorganisms, living in the soil (Stępniewska & Wolińska, 2005).

In the same way we investigated effect of Cr forms on *Eutric Cambisol* (Stępniewska & Wolińska, 2004). Received results are shown in Fig. 15. We stated that excess of Cr forms in soil disturb homeostatic metabolism of microbes, what reflect their enzymatic activity. DHA demonstrated a tendency to decrease with increase of Cr concentration. The lowest content of both Cr (III) and Cr (VI), at the level of 2 mg kg-1 reduced soil DHA to 51-66%, respectively. But at the same time the highest Cr (III) and Cr (VI) supplement at the level of 20 mg kg-1 limited DHA to 6-15%, in relation to the control.

Inhibition of DHA by applied Cr compounds was also reported by Wyszkowska et al. (2001), who noted that decrease of enzymatic activity in soil should be considered as very unfavourable in terms of soil fertility, because soils of good quality and high content of soil OM show high enzymatic activity.

The decrease of soil DHA by several metallic elements (Al, Be, Cu, U) was also discussed by Antunes et al. (2011), whereas a study by Nowak et al. (2002) found that DHA decreased by up to 85% at 5 mM selenic acid (IV) presence.

**Figure 15.** The variations of DHA in *Eutric Cambisol* at different Cr (III) and Cr (VI) concentrations (according to Stępniewska & Wolińska, 2004). Averaged values of three replicates with standard deviations are presented
