THE EFFECTS OF 2,4-D AND DICAMBA ON ISOPROTURON METABOLISM AND SELECTED BIOCHEMICAL PARAMETERS IN CLAY SOIL

Arkadiusz Telesiński
Department of Biochemistry, West Pomeranian Technological University

ABSTRACT

Laboratory experiment was carried out on light dusty clay, into which two herbicides: Izoturon 500 SC (composed of isoproturon only) and Rokituron D 470 SC (composed apart from isoproturon of 2,4-D and dicamba), were introduced in the following doses: recommended field dose (FD), tenfold higher dose (10FD) and one hundredfold higher dose (100FD). The amount of isoproturon introduced together with Izoturon 500 SC and Rokituron D 470 SC was the same, which allowed for assessing the effect of 2,4-D and dicamba on the rate of isoproturon decay in soil as well as for evaluating the modification of changes in soil biochemical parameters induced by isoproturon introduction into it by 2,4-D and dicamba. During the experiment, the content of isoproturon and its metabolites: MDIPU, DDIPU and 4-IA, and of dicamba and 2,4-D in soil as well as the activity of catalase, dehydrogenase and nitrate reductase. Isoproturon DT50 in soil ranged between 9.7 and 18.5 days and rose together with a dose increase. MDIPU, i.e. the metabolite produced in result of the N-demethylation of isoproturon, was detected as early as on day 1 of experiment. Other determined metabolites were detected in successive measurement times. Moreover, isoproturon addition to soil decreased the activity of examined enzymes as well as the value of AEC. It was also found that 2,4-D and dicamba addition slowed down the rate of isoproturon decay in soil as well as intensified the inhibition of nitrate reductase and dehydrogenase activity in soil and levelled the inhibition of catalase activity in soil induced by isoproturon.
Basing on the carried out research work, it can be stated that the best indicator of soil contamination with isoproturon appears to be the AEC value, which was negatively correlated with the content of isoproturon and MDIPU in soil, with the content of these compounds amounting respectively to 40 mg kg^-1_{d.w. soil} and 2.2 mg kg^-1_{d.w. soil} when the value of AEC decreased rapidly.

Key words: isoproturon, isoproturon metabolites, 2,4-D, dicamba, soil enzymes, adenylate energy charge.

INTRODUCTION

Soil contamination with xenobiotic chemicals has become a serious worldwide problem. Among anthropogenic factors, pesticides are of primary importance due to their continuous entry into soil environment. Pesticides may enter the soil by direct or indirect applications. The occurrence of pesticide interference on soil biochemical properties affecting the soil fertility is well known. Since the soil enzymes involved in nutrient mineralization and organic matter degradation are mostly of microbial origin, the response of soil microorganisms to the presence of pesticides is of particular interest [28].

Most frequently examined enzymes, due to their role in decomposition of organic matter and metabolism of nutrients, are oxidoreductases and hydrolases [8].

Another soil microbiological and biochemical indicator used in determination of soil quality is adenylate energy charge (AEC). The adenylate energy charge, defined as AEC = ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]), is a linear measure of metabolic energy stored in the adenine nucleotide pool, where [ATP], [ADP] and [AMP] are concentrations of adenosine tri-, di- and monophosphates, respectively [2]. Theoretically, AEC values can range from 0, corresponding to a totally dephosphorylated adenine nucleotide pool of heavily impaired microorganisms, to 1, corresponding to a completely phosphorylated adenine nucleotide pool of viable microorganisms under optimal growth conditions [40].

Isoproturon (3-(4-isopropylphenyl)-1,1-dimetylurea) – IPU, is a phenylurea herbicide used for pre- and postemergence control of annual grasses and broad spectrum of weeds in spring and winter cereals. It has relatively high water solubility and low tendency to soil sorption, resulting in its soil mobility [32]. Actually, by the reason of ending the registration, an amount of herbicides containing isoproturon in Poland decreased considerably. Now, only several preparations including this active substance, are used, for example: Isoguard 500 SC, Isoguard 83 WG, Bison 83 WG, Protugan 500 SC. Degradation of IPU in soil can involve biotic and abiotic processes, but the major process is microbially facilitated biodegradation [4]. Many earlier studies on the biodegradation of IPU in soil do not focus on the fate of degradation products, but these metabolites can be more toxic than IPU itself [19]. The chemical structure of IPU and its major metabolites is showed in Fig. 1.

In weed control as well as plant disease and pest control, complete protection systems for respective crops are being repeatedly applied, consisting of a number or several treatments carried out during vegetation period. In connection with this, different synergistic or antagonistic interactions can take place between the effects of pesticide interference on respective elements of natural environment [34]. The study of Sadowski and Kucharski [30] showed that the most often in surface waters are found residues of phenoxyacid and phenylurea herbicides, and it is related to using of these active substances.

The present study aims at determining the effect of isoproturon and its metabolites on soil biochemical processes as well as that of 2,4-D and dicamba on isoproturon metabolism in soil and on its changes in biochemical properties.

MATERIALS AND METHODS

Experiment was carried out on the black earth (Table 1) from the Gumieńce Plain. The soil sampled from arable-humus horizon was sifted through a 2 mm sieve and divided into 1 kg weighed samples, thereafter introducing into it water emulsions of herbicides Izoturon 500 SC (500 g of isoproturon in 1dm³ preparation) and Rokituron D 470 SC (250 g of isoproturon, 200 g of 2,4-D and 20 g of dicamba in 1 dm³ preparation) in the following doses: a manufacturer's recommended field dose (FD), a tenfold higher dose (10FD) and one hundredfold higher dose (100FD). The amount of isoproturon introduced together with Izoturon 500 SC and Rokituron D 470 SC preparations was the same (Table 2), which allowed for assessing the effect of 2,4-D and dicamba on the rate of isoproturon decay in soil as well as for evaluating the modification by 2,4-D and dicamba of changes in soil biochemical parameters induced by isoproturon introduction into it.

Respective preparation doses were converted into 1 kg soil, taking into account 10 cm depth, and are presented in Table 2. After application of preparation water emulsions, soil moisture was brought to 60% maximum soil water capacity. The soil was thoroughly mixed and stored in tightly-closed polyethylene bags at 20°C.

Isoproturon residues and metabolites were extracted by shaking 5 g of soil with 10 cm³ of methanol on a wrist-action shaker for 2 h. After shaking, the samples were centrifuged for 10 min at 5000 × g and supernatants were analysed for isoproturon residues and metabolites by HPLC.

The column used for analysing isoproturon residues was Adsorbosphere C18 5µ UHS (150 × 4.6 mm) and the solvent system was methanol/water (70/30 v/v) at a flow rate of 1 cm³ min^-1. Detection was done by UV absorbance at 240 nm, with a lower detection limit of 0.01 mg kg^-1. Retention time was 3.3 min. The recovery of isoproturon in the range from 0.65 to 65.00 mg kg^-1 varied from 97 to 101%.

The column used for analysing isoproturon metabolites (MDIPU, DDIPU and 4-IA) was Hypersil C18 5µ ODS (250 × 2.1 mm). The mobile phase was acetonitrile/water (40/60 v/v) at a flow rate of 0.7 cm³ min^-1. Isoproturon metabolites were detected by UV absorbance at 245 nm, with a lower detection limit of 0.001 mg kg^-1 for MDIPU and DDIPU, and 0.01 mg kg^-1 for 4-IA. Under these conditions, the retention time of MDIPU was 2.1 min, DDIPU – 2.7 min and 4-IA – 5.7 min, while recovery of MDIPU – 93–97%, DDIPU – 95–101% and 4-IA – 91–94%.

The residues of 2,4-D and dicamba were extracted from soil acetone/water/acetic acid mixture (80/19/1 v/v/v), following J.T. Baker EN-518 application. A SPE clean up was required prior to HPLC analysis. The residues of 2,4-D and dicamba from 1000 mg BEKERBOND SPE Polar Plus C₁₈-column were eluted using 3 cm³ of methanol.

The analyses of 2,4-D and dicamba residues were performed on Hypersil C18 5µ ODS (250 × 4.6 mm) column using an isocratic (1 cm^3-1) elution phase of acetonitrile/methanol/water/acetic acid (32/7,5/58,5/2 v/v/v/v). Detection was done by UV absorbance at 272 nm. The retention times of 2,4-D and dicamba were 11.1 min and 20.7 min. The recovery of 2,4-D varied from 92 to 95% and that of dicamba from 92 to 98%, with a lower quantitative limits of 0.005 mg kg^-1.

Measurements of adenine nucleotides and calculations of the adenylate energy charge were made according to the procedure of Bai et al. [3] as described by Dyckmans and Raubuch [12]. Dimethylsulphoxide (DMSO), Na₃PO₄ (10 mM) buffer + EDTA (20 mM) at pH 12 were used as extractants. After derivatisation with chloracetaldehyde, the adenine nucleotides were determined by HPLC. The separation was carried out on a Hypersil C18 5µ ODS (250 × 4.6 mm) column. The chromatography was performed isocratically (2 cm³ min^-1) with 50 mM ammonium acetate buffer containing 1 mM EDTA and 0.4 mM TBAHS mixed with methanol (90/10 v/v) as a mobile phase. Fluorometric emission was measured at a wavelength of 410 nm with 280 nm as the excitation wavelength. The AEC was calculated as ([ATP] + 0.5[ADP])/([ATP] + [ADP] + [AMP]).

Enzyme activities were evaluated according to the release and the quantitative determination of products in the reaction mixtures, when soil samples were incubated with suitable substrate and buffer solutions.

Dehydrogenase activity was measured according to Thalmann [35], that of nitrate reductase according to Abdelmagid and Tabatabai [1], while that of catalase following Johnson and Temple procedure [9].

All analyses were made in three replications. In case of the content of isoproturon, its metabolites, 2,4-D and dicamba in soil, standard deviations were calculated. Non-linear regression equations for the logarithmic function of isoproturon decay rate in soil were also calculated and, basing on them, isoproturon half-live (DT50) values were determined.

In order to determine relationships between the content of isoproturon, its metabolites, 2,4-D and dicamba in soil and the assessed biochemical parameters, the obtained results were analysed statistically using Pearson's linear correlation coefficients, while by the method of calculating non-linear regression between the content of isporoturon and MDIPU in soil and the AEC value third degree polynomial curves were fitted to experimental data.

RESULTS

The amount of isoproturon residues in soil differed, depending on the applied dose of Izoturon 500 SC and Rokituron D 470 SC preparations. After adding these preparations to the soil in a recommended field dose (FD), no traces of isoproturon presence were found in it on day 112 of experiment (Table 3). After applying the preparation containing only isporoturon (Izoturon 500 SC) in a tenfold higher (10FD) and one hundredfold higher dose (100FD), the content of isoproturon in soil on the last day of experiment was, respectively, 11.23% (0.73 mg kg^-1) and 16.75% (10.89 mg kg^-1). On the other hand, in the soil containing isporoturon addition as well as that of 2,4-D and dicamba (Rokituron D 470 SC), the amount of isporoturon residues on day 112 of experiment was 12.25% (0.80 mg kg^-1) for 10FD and 12.77% (8.30 mg kg^-1) for 100FD. It was found during successive experiment days that only on day 1 of experiment the content of isoproturon was by about 1.42-3.24% higher in the soil with addition of isoproturon with 2,4-D and dicamba in all doses than in that containing isoproturon addition only (Fig. 2A). The same result was stated for FD on day 28 and 56 of experiment, but the observed differences were much higher – by about 20%. For the remaining doses, smaller isoproturon content was stated for the most part in the soil containing isproturon, 2,4-D and dicamba addition than in that with isoproturon only, this being noticeable to the extreme on day 7 and 14 of experiment.

Based on the regression curves, the formulae of which are given in Table 4, a 50% degradation time (DT50) of isoproturon in soil was calculated. The DT50 value increased together with an increase of isoproturon dose and ranged between 9.7 and 18.5 days. It was also found that addition of 2,4-D and dicamba in all doses induced a reduction of DT50. It was the largest for the recommended field dose, i.e. 5.8 days, decreasing together with a dose increase (Fig. 2B). For 10FD, it amounted to 5.0 days, whereas 0.9 day for 100FD.

3-(4-isopropylphenyl)-1-methylurea (monodemethyloisoproturon – MDIPU) is formed in result of isoproturon demethylation at the first nitrogen atom and its presence in soil was stated as early as after one day from introduction of examined preparations into it. For FD and 100FD, both in the soil with addition of isoproturon only and in that with isoproturon with 2,4-D and dicamba the largest content of that metabolite was on day 1 of experiment (Table 5). Similar situation was found in the soil with addition of isoproturon only in 10FD. On the other hand, in the soil with addition of isoproturon with 2,4-D and dicamba the heighest content of MDIPU was on day 7 of experiment. Moreover, for FD, the addition of 2,4-D and dicamba increased the content of MDIPU by 0.76–1.39% until day 28 of experiment, while decreasing it by about 0.2% in two last times (Fig. 3A). However, for 10FD and 100FD, the content of MDIPU in most times was lower in the soil with addition of isoproturon with 2,4-D and dicamba than in that with isoproturon only. The largest differences were on day 1 of experiment and amounted to about -1.67% for 10FD and -2.11% for 100FD.

3-(4-isopropylphenyl)-urea (didemethyloisoproturon – DDIPU) is formed in result of next demethylation at the first nitrogen atom and it was detected for the first time in all applied doses on day 7 of experiment. The largest percentage of that metabolite on that day was stated for FD and it decreased together with a dose increase (Table 6). In the soil with addition of isoproturon only, the content of DDIPU in all doses increased until day 14 of experiment and then gradually decreased during the experiment. The content of DDIPU differed after appliaction of isoproturon with 2,4-D and dicamba. For FD and 100FD, the content of DDIPU increased up to day 56 of experiment, whereas up to day 28 of experiment for 10FD.

Moreover, the addition of 2,4-D and dicamba in FD decreased the content of DDIPU until day 14 of experiment, increasing its amount in successive experiment days, even by about 2.5% on day 56 of experiment (Fig. 3B). For 10FD, in general a larger content of that metabolite was found in the soil with addition of isoproturon with 2,4-D and dicamba, by about 0.33–0.69%. Only on day 14 of experiment, the addition of 2,4-D and dicamba decreased the amount of DDIPU by about 0.35%. On the other hand, for 100FD, the addition of 2,4-D and dicamba decreased the content of DDIPU by about 0.05–0.63% until day 28 of experiment, while increasing the content of that metabolite on day 56 and 112 of experiment by 1.53% and 0.73%, respectively.

After application of the herbicide containing isoproturon only (Izoturon 500 SC) in FD, the content of 4-isopropylaniline (4-IA) was stated for the first time on day 28 of experiment. On the other hand, in higher doses of that preparation as well as in all doses of Rokituron D 470 SC preparation 4-IA was detected for the first time in soil on day 14 of experiment. When analysing the percentage of that metabolite, it was observed to be the highest in FD both after application of isoproturon only and of isoproturon with 2,4-D and dicamba, in particular on day 56 of experiment, when it reached 21.41% (Table 7).

Moreover, it was showed that in all measurement times when 4-IA was detected the amount of that metabolite was higher after application of isoproturon with 2,4-D and dicamba than after that of isoporoturon only, even by 6.44% (Fig. 3C). Only in 10FD on day 56 of experiment, the amount of 40IA was one and the same both after application of Izoturon 500 SC and Rokituron D 470 SC.

The presence of 2,4-D in soil after application of Rokituron D 470 SC in FD was found on day 28 of experiment and it was at the level of about 5.63% (Table 8). On the other hand, in larger doses the content of 2,4-D amounted on the same day to about 11.35–15.58%. However, it decreased until day 56 of experiment to 2.34% for 10FD and 4.33% for 100FD, this day being the last when the presence of 2,4-D was detected in soil. On the other hand, the presence of dicamba in soil for all doses was detected for the last time on day 28 of experiment, its amount amounting to 14.99% for FD, 16.45% for 10FD and 20.56% for 100FD.

The activity of soil enzymes, i.e. catalase, dehydrogenase and nitrate reductase, was converted and given as the percentage of activity in control soil (without herbicide addition), assuming its activity to be 100%; in most measurement times it was lower in soil with herbicide addition than in the control one. Enzyme stimulation was noticed several times, which most frequently occurred in the first two weeks of experiment duration (Table 9).

When analysing percentual differences in the activity of examined enzymes in the soil with addition of isoproturon with 2,4-D and dicamba and that in the soil with isoproturon only, it was found that in case of catalase they were positive for all doses in most measurement times, which is evidence of stimulating effect of 2,4-D and dicamba on the activity of that enzyme (Fig. 4A). Only on day 28 of experiment for FD, the activity of catalase in the soil with addition of isoproturon mixture with 2,4-D and dicamba was decidedly lower, as low as by 66%, than that in the soil with addition of isoproturon only.

In case of dehydrogenase, it was found that for FD the addition of 2,4-D and dicamba increased its activity until day 14 of experiment, as well as on day 56 of experiment, when compared with the interference of isoproturon only (Fig. 4B). For 10FD and 100FD, the activity of dehydrogenase in most measurement times was lower in the soil with isoproturon, 2,4-D and dicamba addition than in that with isoproturon only. The positive effect of 2,4-D and dicamba was observed on the interference of isoproturon in 10FD on day 14 of experiment, not exceeding several percent on 14 and 28 day of experiment for 100FD.

Also the activity of nitrate reductase was lower in most measurement times after application of isoproturon with 2,4-D and dicamba than after that with isoproturon only (Fig. 4C). Only on day 1 and 28 of experiment, a rise in the activity of nitrate reductase was observed under the influence of 2,4-D and dicamba in all doses when compared with the interference of isoproturon.

Also the mean activity of enzymes was calculated from all measurement times and it was found that application of isoproturon only induced a decrease in the mean activity of examined enzymes, deepening together with a dose increase. In case of catalase, its activity decreased from 88% for FD to 59% for 100FD, whereas in case of dehydrogenase from 97% to 76% and of nitrate reductase from 97% to 78%, respectively (Fig. 5). The addition of 2,4-D and dicamba in all doses induced an increase in the activity of catalase when compared to the interference of isoproturon only, the mean activity of that enzyme approximating 100%. However, only for FD the mean activity of that enzyme in the soil with addition of isoproturon, 2,4-D and dicamba was higher by about 3%, whereas by about 22% lower for 10FD and 100FD, when compared with that with addition of isoproturon only. The mean activity of nitrate reductase in soil for all doses was lower by about 5–14% after application of isoproturan with 2,4-D and dicamba than after that with isoproturon only, with the highest difference being found for FD.

The value of adenylate energy charge (AEC) in the soil with addition of herbicide containing isoproturon only was lower during the whole experiment than in the control soil (Table 10). The largest drop in the value of adenylate energy charge was observed in the first two weeks of experiment, in particular in the soil containing Izoturon 500 SC in 100FD. On day 1 of experiment, the application of Izoturon 500 SC in that dose induced a decrease in the value of AEC as low as 0.46.

After addition of isoproturon into soil with 2,4-D and dicamba, a decrease in the value of adenylate energy charge was also observed during the whole experiment. The lowest value of AEC, amounting to 0.73, was however found on day 28 of experiment in the soil with addition of that herbicide in 100FD.

In order to determine relationships between the content of isoproturon, its metabolites, 2,4-D and dicamba in soil and the assessed biochemical parameters, the coefficients of Pearson's linear correlation were calculated and a negative correlation was found between the content of isoproturon and MDIPU in soil and the adenylate energy charge (Table 11). This was confirmed both after application of isoproturon only and after that of its mixture with 2,4-D and dicamba. However, the calculated coefficients of correlation were lower in the latter variant. Also significant negative correlation was noticed between the AEC value and the content of 2,4-D and dicamba in soil.

Thus, it results from the above that from among the assessed soil biochemical parameters the value of adenylate energy charge appears to be the best indicator of soil contamination with isoproturon and its metabolite – MDIPU. Therefore, in order to determine better the relationships between the content of these compounds in soil and the value of AEC, third degree polynomial curves were fitted to experimental data by the method of calculating non-linear regression. Based on them, it can be seen that the value of AEC underwent a very small drop together with an increase in the content of IPU to 6 mg kg^-1_{d.w. soil} (Fig. 6). Then, the value of AEC remained more and less at the similar level (0.75), undergoing another clear decrease after exceeding the IPU amount of 40 mg kg^-1_{d.w. soil}. Similar tendency was found when analysing the relationship between the content of MDIPU in soil – a small decrease in the AEC value was observed at the MDIPU content reaching 0.5 mg kg^-1_{d.w. soil}, thereafter the value of AEC being almost unchanged in relation to the MDIPU content of 2.2 mg kg^-1_{d.w. soil}, then the value of AEC undergoing a substantial decrease after exceeding the latter amount of that metabolite.

DISCUSSION

Both degradation of isoproturon and that of 2,4-D and dicamba in soil is microbially facilitated [7,20,29,32,36]. Based on the carried out research work, it was found that isoproturon half-life in soil ranged from 9.7 to 18.5 days. Similar values (6–30 days) were stated by many author in their works [5,11,15,26,37,39]. On the other hand, Mosleh et al. [21] showed that after adding isoproturon into soil in doses much larger than the recommended ones (0.5–1.4 g kg^-1), its content after 60 days decreased merely to about 40%. The own results showed that both retention of isoproturon and that of 2,4-D and dicamba in soil increased together with a dose increase.

It was found in the present study that the content of 2,4-D in soil was detected until day 28–56 of experiment, depending on dose, while that of dicamba until day 28 of experiment. McGhee and Burns [20] report that the half-life of 2,4-D amounts to 7–42 days. On the other hand, the half-life of dicamba ranges, depending of the type of soil and the presence of microorganisms in it, from 13 to 160 days [10,14]. Pavel et al. [25] report however that the half-life of dicamba can even amount to 300 days.

The first step in isoproturon biodegradation in soil is its N-demethylation to MDIPU or dehydroxylation to 3-(4-(2-hydroxyisopropyl)-phenyl)-1,1-dimethylurea [16,18,31]. These metabolites can be then degraded to many other metabolites, including DDIPU and 4-IA. In result of the carried out study, it was found that as early as on day 1 after introduction of isoproturon into soil the presence of MDIPU was observed. Earlier studies of other authors showed that it is a main metabolite accumulated during isoproturon degradation in different soils [11,16,29]. Furthermore, Juhler et al. [16] observed the presence of 4-isopropylaniline (4-IA) after two days from isoproturon application into soil. However, they did not observe the occurrence of 3-(4-isopropylphenyl)-urea (DDIPU). Moreover, Pieuchot et al. [29] showed also the presence of 3-[4-(2-hydroxyisopropylphenyl)]-methylurea (MDIPU-OH), apart from MDIPU, as early as on the first day of their experiment. The amount of the latter metabolite however was clearly smaller. On successive days of their experiment, they detected in soil next isoproturon metabolites, i.e. DDIPU and 3-[4-(2-hydroxyisopropylphenyl)]-urea (DDIPU-OH). The same isoproturon metabolites in soil were found after 60 days of experiment by Perrin-Ganier et al. [27]. On the other hand, the presence of DDIPU in the own study was stated on day 7 of experiment, while that of 4-IA on day 14 or 28 of experiment, depending on dose.

Furthermore, it was found in the present study that addition of 2,4-D and dicamba increased the content of 4-IA and accelerated the decay of isoproturon in soil. Perrin-Ganier et al. [27] and Suhadolc et al. [33] also stated that isoproturon decayed faster in the soil contaminated with heavy metals. On the other hand, Krutz et al. [17] found that Roundup Ultra addition slowed down degradation of atrazine in soil. Also the study of Gebendinger and Radosevich [13] showed that cyanazine inhibited atrazine degradation in soil.

In result of the carried out experiment, it was found that addition of 2,4-D and dicamba into the preparation containing isoproturon affected significantly the activity of enzymes and the value of cell energy charge in soil. In most cases, isoproturon decreased the activity of examined soil enzymes as well as the value of AEC. Also Berger and Heitefuss [6] stated a drop in the activity of dehydrogenase in soil after isoproturon application. On the other hand, previous own studies showed that isoproturon stimulated the activity of peroxidase [22] as well as that of acid and alkaline phosphatase in soil [23].

Furthermore, the activity of nitrate reductase and degydrogenase was smaller, while that of catalase higher, after application of isoproturon with 2,4-D and dicamba when compared with that of isoproturon only. In the previous study, it was also showed that after application of 2,4-D and dicamba with isoproturon the activity of peroxidase in soil was lower than after that of isoproturon only [22]. On the other hand, addition of metribuzin and amidosulfuron increased the activity of soil catalase and proxidase when compared to the interference of isoproturon only [24].

The content of isoproturon and MDIPU in soil, as well as that of 2,4-D and dicamba, was significantly negatively correlated with the value of cell energy charge in soil. The value of energy charge is a very good indicator for evaluation of cell physiological stress [40] and depends on many factors, including the presence of pollutants, with pesticides among others [18]. It can be thus concluded that the value of AEC is a very good indicator of the microflora state of soil contaminated with isoproturon. Walker et al. [38] also showed a significant negative correlation between isoproturon DT50 in soil and microbial biomass volume in it.

CONCLUSIONS

The presented findings show that isoproturon and its metabolites clearly affected biochemical parameters in soil, i.e. the activity of catalase, dehydrogenase and nitrate reductase and the value of AEC. The main metabolite accumulating in soil is MDIPU. The addition of 2,4-D and dicamba slowed down the rate of isoproturon decay in soil as well as intensified the inhibition of nitrate reductase and dehydrogenase activity in soil and levelled the inhibition of catalase activity in soil induced by isoproturon. The best indicator of soil contamination with isoproturon appears to be the value of AEC, which was significantly negatively correlated with the content of isoproturon and MDIPU in soil, with the content of these compounds amounting respectively to 40 mg kg^-1_{d.w. soil} and 2.2 mg kg^-1_{d.w. soil} when the value of AEC decreased rapidly.

ACKNOWLEDGEMENTS

This work was financially supported by Polish Committee of Sciences.

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Accepted for print: 01.03.2010

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