**4.2. Fertilization and pesticide amendment**

Organic and inorganic fertilizers are commonly used to increase nutrient availability (Macci et al., 2012). The balanced fertilization of major elements (N, P, K) for plant nutrient could be beneficial for the growth of plant aboveground parts and roots (Chu et al., 2007), and also for improvement of soil structure (Macci et al., 2012).

However, fertilization could affect on the population of soil microorganisms and consequently soil enzymatic activities. It is often assumed, that inorganic fertilizers had relatively less effect on soil enzymes activity than organic fertilizers (Chu et al., 2007; Xie et al., 2009; Romero et al., 2010). Macci et al. (2012) noted, that DHA usually reached higher level in the organic treatments.

As was suggested by Chu et al. (2007) and Xie et al. (2009) long-term balanced fertilization greatly increased DHA level in the soil environment, rather than nutrient-deficiency fertilization. Zhao et al. (2010) indicated, that soils with higher fertility are more capable of maintaining the original biological functions (i.e. have a higher functional stability).

On the other hand, Moeskops et al. (2010) compared the effect of organic and conventional farming practices on soil enzymatic activities. On the organic farms, soil fertility was maintained mainly with composted OM, in contrast to conventional farmers, who combined fresh manure and chemical fertilizers, and typically applied large amounts of pesticides. As a consequence, a strong negative impact of intensive fertilizer and also pesticide use on DHA was demonstrated (Moeskops et al., 2010).

Soil DHA is an indicator of soil quality and microbial activity and also is the most frequently used to determining the influence of various pollutants (like pesticides or excessive fertilization) on the microbiological quality of soils (Xie et al., 2009; Tejada et al., 2010).

Despite the fact that pesticides are important tools in agriculture that help to minimize economic losses caused by weeds, insects and pathogens, they also are recognized as a source of potential adverse environmental impacts (Tejada et al., 2010). It is often assumed that less than 0.3% of the pesticide reaches its target pest, the remaining 99.7% is released to the environment, representing a potential hazard for non-target organisms (Muñoz-Leoz et al., 2011).

196 Dehydrogenases

deeper part of the soil profile.

level in the organic treatments.

**4.2. Fertilization and pesticide amendment** 

for improvement of soil structure (Macci et al., 2012).

DHA was demonstrated (Moeskops et al., 2010).

whereas at the deepest part of the soil profile (40-60 cm) DHA was reduced by 95%, in relation to the surface layer. This trend is undoubtedly connected with presented in literature data and mentioned above spatial distribution of soil microorganisms (Agnelli et al., 2004; Levyk et al., 2007; Wolińska, 2010), and its preference to inhabiting the surface

Our results are in agreement and might be supported by the findings of Xiang et al. (2008), who observed that DHA was roughly 4-fold higher in surface (till to 5 cm depth), than in subsoil (90-100 cm). It was also suggested by study of Gajda (2008), that values of DHA noted in the anthropogenic soil, till depth to 4 cm were by c.a. 30% higher in relation to the

Brzezińska (2006), reported even 9-fold increase of DHA in the surface layer of the soil, than in the subsurface parts and 25-fold higher enzymatic activity in surface than in subsoil.

Generally it is possible to state, that both diversity, abundance as distribution of microorganisms are more even under oxic (surface layers) conditions, relative to anoxic

Organic and inorganic fertilizers are commonly used to increase nutrient availability (Macci et al., 2012). The balanced fertilization of major elements (N, P, K) for plant nutrient could be beneficial for the growth of plant aboveground parts and roots (Chu et al., 2007), and also

However, fertilization could affect on the population of soil microorganisms and consequently soil enzymatic activities. It is often assumed, that inorganic fertilizers had relatively less effect on soil enzymes activity than organic fertilizers (Chu et al., 2007; Xie et al., 2009; Romero et al., 2010). Macci et al. (2012) noted, that DHA usually reached higher

As was suggested by Chu et al. (2007) and Xie et al. (2009) long-term balanced fertilization greatly increased DHA level in the soil environment, rather than nutrient-deficiency fertilization. Zhao et al. (2010) indicated, that soils with higher fertility are more capable of

On the other hand, Moeskops et al. (2010) compared the effect of organic and conventional farming practices on soil enzymatic activities. On the organic farms, soil fertility was maintained mainly with composted OM, in contrast to conventional farmers, who combined fresh manure and chemical fertilizers, and typically applied large amounts of pesticides. As a consequence, a strong negative impact of intensive fertilizer and also pesticide use on

Soil DHA is an indicator of soil quality and microbial activity and also is the most frequently used to determining the influence of various pollutants (like pesticides or excessive fertilization) on the microbiological quality of soils (Xie et al., 2009; Tejada et al., 2010).

maintaining the original biological functions (i.e. have a higher functional stability).

(deeper layers) conditions (Fierer et al., 2003; Wolińska & Stępniewska, 2011).

layers, where optimum conditions for its growth and development are guaranteed.

Stepniewska et al. (2007) noted the relationship between soil DHA and Fonofos (Stauffer Chemical Co., Westport, USA) concentration in the *Mollic Gleysol*. In the investigated samples influence of pesticide on soil enzymatic activity started to be observed after one week of incubation, but since 14th day to the end of experiment this effect was significant and noticeable (Fig. 10). Generally, 1µg g-1 dose of Fonofos was responsible for about 26% inhibition of soil DHA, whereas ten times higher factor reduced activity for 46.6% at 21st day of incubation time, later fall of enzymatic activity ranged from 22.5% to 30% in relation to the control samples was considered.

**Figure 10.** Dynamic of DHA during incubation at 20ºC (0 - control, 1 - 1µg g-1 Fonofos supplement; 10 - 10 µg g-1 Fonofos supplement), according to Stępniewska et al., 2007. Averaged values of three replicates with standard deviations are presented

Our results suggest a negative effect of Fonofos on soil DHA in the first stage after application (1-7 day), later an initial, almost linear growth of DHA was observed and the final day of incubation resulted in significant extension of DHA, presumably because the process of Fonofos decomposition in the soil environment was almost completely finished.

Tejada et al. (2010) indicated that MCPA herbicide rate of 1.5 l ha-1 (manufactures rate recommended) was the reason of 39.3% soil DHA inhibition, what suggest that the MCPA caused toxic effect on soil enzymatic activity. A field half-life of MCPA ranged from 14 days to 1 month, dependently on soil moisture, pH and microorganisms abundance. The most important soil factor in predicting MCPA effect on soil enzyme activities is pH, as at acidic conditions persistence of pesticide may last even 5 years, whereas at alkaline pH only 6 days. Moreover, decrease of soil moisture and reduction of microbial abundance influence on elongation of MCPA decomposition process.

Dehydrogenase Activity in the Soil Environment 199

is reported between 1 to 174 days (Bennicelli et al., 2009). Glyphosate in soil is transformed to aminomethylphosphonic acid (AMPA), which is non-persistent metabolite. As a effect of mentioned transformations and in presence of dehydrogenases (microorganisms), Glyphosate give CO2 and H2O (Forlani et al., 1999). Glyphosate degradation in soil is mainly the reason of microbial activity, while the chemical decomposition and photolysis play a minor role (Bennicelli et al., 2009). As was reported by Zabaloy et al. (2008), Glyphosate, as an organophosphonate can be used as a source of P, C or N by either gram-positive as gram

The purpose of our study was to research the influence of Glyphosate on soil DHA in the *Mollic Gleysol* (from Wieprz river valley), *Eutric Fluvisol* (from Vistula river valley) and *Terric Histosol* (from Bystrzyca river valley), taken from surface layer (0-20 cm). Soil samples were enriched with Glyphosate, as follows: with 1 µg (first combination), and 10 µg (next version), and 0 µg (control) of pesticide per 1g of soil. Thus prepared samples were

incubated in thermostatic chamber at 20ºC. Received results are presented in Fig. 12.

**Figure 12.** Mean DHA in *Terric Histosols*, *Eutric Fluvisols* and *Mollic Gleysols,* in three combinations of

We found that both 1 and 10 µg of Glyphosate additions to soils caused a decrease of DHA, dependently on the pesticide doses. The strongest effect of Glyphosate was observed in *Terric Histosols* and *Eutric Fluvisol* (10 µg g-1 of soil)*,*where reduction of DHA by 33-47%, relative to control soils (non-amended with Glyphosate), was noted. The most resistant to

Glyphosate supplement seemed to be *Mollic Gleysol*, in 10 µg g-1 of soil dose, where DHA

Glyphosate doses: 0-control; 1-1µg g-1; 10-10µg g-1 (according to Bennicelli et al., 2009)

negative bacteria.

dropped by c.a. 24%.

**DHA (µg TPF g-1 min-1**

**\*10-6)** 

We also studied effect posed by MCPA (Organika Sarzyna, Poland) on soil DHA behavior (Fig. 11). The following MCPA dosage were introduced into the soil samples: 0.165; 0.30 and 3.3 mg MCPA per g of soil. Non-amended with pesticide soil sample was marked as 0 and used as a control. As a result of realized experiment we found linear inhibition of DHA by increasing MCPA doses (R2=0.99). Decrease of DHA level at 3.3. mg g-1 MCPA dose by c.a. 38.5%, in comparison to the control sample, was noted. However, registered inhibition was not significant (*P*>0.05). Our conclusions are comparable with results presented by Tejada et al. (2010).

**Figure 11.** Effect of MCPA pesticide on DHA level in *Eutric Fluvisol*, according to Stępniewska founding (unpublished data). Averaged values of three replicates with standard deviations are presented

Other pesticide, which we take into account in our investigations was Glyphosate – commonly used by Polish farmers (in the form of RUNDUP), a broad spectrum, nonselective, systemic and post-emergence herbicide, widely popular in soil cultivation, forestry, rights-of-way and aquatic systems to prevent grass and weeds competition with plant seedlings (Bennicelli et al., 2009). At low doses it is used as a plant growth regulator.

Glyphosate (Monsanto Co., USA) is a polar substance that is highly soluble in water (12 g l-1 at 25°C), and insoluble in most organic solvents. In soil is moderately persistent; its half-life is reported between 1 to 174 days (Bennicelli et al., 2009). Glyphosate in soil is transformed to aminomethylphosphonic acid (AMPA), which is non-persistent metabolite. As a effect of mentioned transformations and in presence of dehydrogenases (microorganisms), Glyphosate give CO2 and H2O (Forlani et al., 1999). Glyphosate degradation in soil is mainly the reason of microbial activity, while the chemical decomposition and photolysis play a minor role (Bennicelli et al., 2009). As was reported by Zabaloy et al. (2008), Glyphosate, as an organophosphonate can be used as a source of P, C or N by either gram-positive as gram negative bacteria.

198 Dehydrogenases

presented

on elongation of MCPA decomposition process.

presented by Tejada et al. (2010).

caused toxic effect on soil enzymatic activity. A field half-life of MCPA ranged from 14 days to 1 month, dependently on soil moisture, pH and microorganisms abundance. The most important soil factor in predicting MCPA effect on soil enzyme activities is pH, as at acidic conditions persistence of pesticide may last even 5 years, whereas at alkaline pH only 6 days. Moreover, decrease of soil moisture and reduction of microbial abundance influence

We also studied effect posed by MCPA (Organika Sarzyna, Poland) on soil DHA behavior (Fig. 11). The following MCPA dosage were introduced into the soil samples: 0.165; 0.30 and 3.3 mg MCPA per g of soil. Non-amended with pesticide soil sample was marked as 0 and used as a control. As a result of realized experiment we found linear inhibition of DHA by increasing MCPA doses (R2=0.99). Decrease of DHA level at 3.3. mg g-1 MCPA dose by c.a. 38.5%, in comparison to the control sample, was noted. However, registered inhibition was not significant (*P*>0.05). Our conclusions are comparable with results

**Figure 11.** Effect of MCPA pesticide on DHA level in *Eutric Fluvisol*, according to Stępniewska founding (unpublished data). Averaged values of three replicates with standard deviations are

Other pesticide, which we take into account in our investigations was Glyphosate – commonly used by Polish farmers (in the form of RUNDUP), a broad spectrum, nonselective, systemic and post-emergence herbicide, widely popular in soil cultivation, forestry, rights-of-way and aquatic systems to prevent grass and weeds competition with plant seedlings (Bennicelli et al., 2009). At low doses it is used as a plant growth regulator.

Glyphosate (Monsanto Co., USA) is a polar substance that is highly soluble in water (12 g l-1 at 25°C), and insoluble in most organic solvents. In soil is moderately persistent; its half-life The purpose of our study was to research the influence of Glyphosate on soil DHA in the *Mollic Gleysol* (from Wieprz river valley), *Eutric Fluvisol* (from Vistula river valley) and *Terric Histosol* (from Bystrzyca river valley), taken from surface layer (0-20 cm). Soil samples were enriched with Glyphosate, as follows: with 1 µg (first combination), and 10 µg (next version), and 0 µg (control) of pesticide per 1g of soil. Thus prepared samples were incubated in thermostatic chamber at 20ºC. Received results are presented in Fig. 12.

**Figure 12.** Mean DHA in *Terric Histosols*, *Eutric Fluvisols* and *Mollic Gleysols,* in three combinations of Glyphosate doses: 0-control; 1-1µg g-1; 10-10µg g-1 (according to Bennicelli et al., 2009)

We found that both 1 and 10 µg of Glyphosate additions to soils caused a decrease of DHA, dependently on the pesticide doses. The strongest effect of Glyphosate was observed in *Terric Histosols* and *Eutric Fluvisol* (10 µg g-1 of soil)*,*where reduction of DHA by 33-47%, relative to control soils (non-amended with Glyphosate), was noted. The most resistant to

Glyphosate supplement seemed to be *Mollic Gleysol*, in 10 µg g-1 of soil dose, where DHA dropped by c.a. 24%.

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 effect of Glyphosate contamination, in comparison to control sample.

Dehydrogenase Activity in the Soil Environment 201

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

**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

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

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

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

activity, and this effect is noticeable even after 3 hours.

DHA level.

are presented

displayed by Hg and Cd.

on living organisms, than Cr (III).

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 processes.
