RELATIONS BETWEEN FLUORINE CONTENT IN SOIL AND INHIBITION OF SOIL ENZYMES ACTIVITY

Janina Nowak¹, Beata Smolik², Helena Zakrzewska^1
1 Department of Biochemistry, Agriculture University of Szczecin, Poland
² Department of Biochemistry, West Pomeranian University of Technology

ABSTRACT

In this study the influence of various amounts of fluorine added to soil as NaF solutions (10, 30 and 50 mmol kg^-1) on the activity of enzymes (acid and alkaline phosphatase, β-glukosidase and dehydrogenases) in soils of various texture and organic carbon content was tested. Also the effect of aqueous solutions of fluoride added to samples of various kinds of soil on the content of fluorine soluble in 0.01 M CaCl₂ and in 2 M HClO₄ (potentially available for plants), has been studies.

The laboratory experiment was conducted at 20°C, and 60% water holding capacity, on three kinds of soil which differed in organic carbon content and mechanical composition. For three months the activity of enzymes and content of the two forms of fluorine were analyzed every few days. As reference served soil without any addition of fluoride.

The addition of various doses of NaF to the soil caused an appearance diverse amount of fluorine forms soluble in 0.01 M CaCl₂ and in 2 M HClO₄. The level of these fluorine forms was connected both with the kind of soil (the lightest soil contained the highest amount of water soluble fluorine), and the amount of the added NaF, and also duration of the experiment. The content of the fluorine forms was related to the inhibition of the tested enzymes. The highest inhibition pertained to the phosphatase, in the sequence: alkaline, acid then β-glukosidase and dehydrogenases. In the latter case a temporary increase of their activity also occurred. A high correlation between the content of the various forms of fluorine in soil and inhibition of the enzymes was found, the highest significance of that relation pertained to β-glukosidase.

Key words: soils, water soluble and insoluble fluorine content, inhibition of soil enzyme activities: phosphatases, β-glukosidase, dehydrogenases.

INTRODUCTION

In natural conditions the content of fluorine in soil is rather low, and is between 10-1500 mg F kg^-1 of soil [8]. Nevertheless in some areas the levels grow, due to the use of phosphorus fertilizers, pesticides, or local pollution caused by industry [28, 29]. The fluorine content in soil is connected also with its properties, such as organic matter and colloid content, pH, etc. The greatest sorption of this element occurs in mineral soils of acid or alkaline reaction [1, 11, 23]. Damage caused by fluorine in soil is connected mainly with the destabilization of natural soil structure due to changes of the soil humus-silt complex, and also mineral composition. The presence of alkaline metal fluorides decreases the content of organic matter soluble in water, also the mobility of mineral-organic complexes [4]. As result of such changes the biological activity of soil can be decreased. A good indicator of soil fertility is its enzymatic activity [13] which depends on organic matter content [21, 27]. Elevated fluorine content in soil may change the taxonomic structure of fungi [6]. The Fˉ ions can be bound directly to some enzymes, e.g. those containing heme groups, or with other metal enzymes thus changing the metabolism. Fluorides can form complexes with aluminum or beryllium, thus enhance or depress the activity of phosphatases which results in an influence on the content of phosphates in soil. Fluorides inhibit urease activity, which action increases in acid environment [18]. In soils which had received large doses of phosphorus fertilizers, an elevated level of fluorine had been found (5300 mg F kg^-1 dw). Such soil contained also lesser amount of microbial biomass, and its activity of dehydrogenases was also lower [14].

Fluorine is rather hard to be taken up by plants, though in case of soil pollution it can be absorbed excessively [11]. Plants which took up large amount of fluorine can suffer damage, thus give smaller crops. The total fluorine content in soil is not of great importance for agriculture. For that reason the content of available forms should be assessed [19]. According to [9] as form fluorine potentially available to the plant is that soluble in 2 M HClO_4. However, water or 0.01 M CaCl₂ – soluble form of fluorine was those most frequently determined as sole source of the element to plants [15]. The content of fluorine in soil solution differs independently of its total content. This phenomenon is connected with rapid processes of fluorine ions sorption by soil colloids and with the formation of low – solubility salts, particularly CaF₂ [2 according to 24].

Taking into consideration the properties of organic matter and of silt material we investigate the influence of increasing NaF concentration on the activity of some soil enzymes (acid and alkaline phosphatase, b-glukosidase and dehydrogenase) in soils of various granulometric composition and organic carbon content, considering also transformation of water soluble fluorine forms into insoluble but potentially available for plants.

MATERIALS AND METHODS

For the laboratory analyses, soil samples from 0 – 30 cm depth, from soils differing in particle size and organic carbon content from three agricultural fields of Zachodniopomorskie District (Poland) were collected. As shown in table 1 these are: rust-brown soil (soil 1) and black earth (soil 2 and 3). Air dry soil samples were passed through a 2 mm sieve. To 1 kg samples aqueous solutions of NaF were added at three doses: 10 mmol kg ^–1 (dose I), 30 mmol kg ^–1 (dose II) and 50 mmol kg ^–1 (dose III) which was 190, 570 and 950 mg Fˉ kg ^–1 soil d.m. The first concentration is within the permissible limit in soils [11], the third equaled the solubility in water, the second was between the first and third. After addition of the NaF solution the soil moisture was adjusted to 60% water holding capacity, and the soil thoroughly mixed. The samples were stored in airtight plastic containers at 20°C. As control, soil with no NaF addition was used. The activity of enzymes (acid and alkaline phosphatase, β-glukosidase and dehydrogenase) was assessed in the soil 1, 3, 6, 12, 24 and 48 days after treatment. On day 1, 12 and 48 the content of F soluble in 0.01M CaCl₂ [15] and in 2 M HClO₄ [22] modified by [19] was assessed. All analyses were done in three replications. Results pertaining to the transformation of F forms from water soluble into non-soluble and the soil of pH were published by [20]. In this paper they are going to be referred to for a better depiction of the relation between the content of the various F forms and the activity of soil enzymes. The fluorine content and pH_H2O i pH_KCl were analyzed using ionic-selective electrode (pHmeter/jonometer Orion 920A). Sensitivity of the applied F assessment method was 10^-6 M F.

Activity of acid and alkaline phosphatase was analyzed by the method [25] and [5] modified by [17] with the substrate p-Nitrophenyl Phosphate, activity of b-glukosidase was assessed accordingly to [10] using b-glucosido-saligenin (salicin) as substrate and that of dehydrogenase after [26] modified by [16] with the substrate TTC. All analyses were done in three replications. For statistical analysis the Tukey test was used, at 0.05 significance (LSD_0.05). The results obtained were calculated against the control soil (no NaF added). In cases when no inhibition but increased activity had been found, the results were given as a negative % of inhibition.

RESULTS

This paper presents results pertaining to the relation between the inhibition of soil enzyme and the content of fluorine soluble in 0.01 M CaCl₂ and soluble in 2 M HClO₄, in three kinds of soil. Data presented in table 2 have shown that after the incorporation of an aqueous solution of NaF into soil, a major part of it was transformed into not soluble forms, and the level of that transformation depended on soil kind, the applied dose, and the extent of this process was slightly smaller in the first part of the experiment (day 1 to 12). In soil No 1 (light dusty clay) in the first day, at the first dose there was 21.91 mg Fˉ kg^-1, at the second dose 71.47 mg Fˉ kg^-1, at the third dose 159.32 mg Fˉ kg^-1, which amounted to 11.5%, 12.5% and 16.8%, in the 12 day these values were 9.8, 10.9 and 16.5%, respectively. At last day of the experiment (48 day) the levels did not differ much from those at day 12. In soil No 2 black soil – dusty light clay) the lowest level of soluble (in 0.01 M CaCl₂) fluorine was found in soil which had been treated with 570 mg Fˉ kg^-1 dry soil (dose II) – these having been ca 7.5%, at day 24 and 48 day these values were slightly lower, ca 6.2%. If the lowest dose (190 mg Fˉ kg^-1 dry soil – dose I) had been used, at the first day the content percentage was higher – ca 15.2%, and in the following days 11.9%. When the highest NaF dose had been added to soil (950 mg Fˉ kg^-1 dry soil), in the first day the level of fluorine soluble in 0.01 M CaCl₂ was 10.9% and in the next days 9.7 and 9.6%, respectively. In soil No 3 the content of soluble fluorine was slightly lower, but similar to those in soil No 2. Fluorine form soluble in 2 M HClO₄ was different from the above described. Soils No 2 and 3 contained less fluorine at the 12^th day as compared to day one, and this continued to the 48^th day. Contrary, in soil No 1 these forms of fluorine, after addition of 950 mg Fˉ kg^-1 d. m. soil (dose III), were lower at day one, and decreased further at day 12. At that term F content after application of the highest F dose was comparable with that of soil fortified with 570 mg F (dose II) and was about 595 mg F kg^-1 d. m. soil. At day 48 the amount of fluorine soluble in 2 M HClO₄ was similar to that found at day 12. Statistical analysis proved that amounts of fluorine soluble in 0.01 M CaCl₂ and 2 M HClO₄ in tested soils differs significantly (LSD_0.05). The highest levels of fluorine soluble in 0.01 M CaCl₂ were found in the lightest soil (soil 1), in that soil the content of fluorine soluble in 2 M HClO₄ was low at the begin of the experiment.

Introduction of NaF dissolved in water caused changes of soil pH toward alkaline reaction. With the increase of NaF doses the soil pH also increased, soil 2 and 3 differed only slightly, and soil 1 showed a lower pH. At day 1, pH of soil 1 increased from 6.5 to 7.9 and at day 48 (last day) from 6.8 to 7.6. The pH of soil 2 increased at day one from 7.1 to 8.5, from 7.7 to 8.5 at day 48, and in soil 3 at day one from 7.3 to 7.9, at day 48 from 7.7 to 8.7. The pH in KCl changed in a similar manner, though it was lower (Tab. 3).

Fig. 1 to 4 shows the inhibition of enzyme activities caused by the addition of fluoride. The applied sodium fluoride caused a decrease phosphatases activity in all three soils tested (Fig. 1 and 2). The strongest decrease of the activity of both acid and alkaline phosphatase was seen in soil 1 – poor in organic matter and colloids. The inhibition varied greatly with the doses applied. Sodium fluoride caused an inhibition of alkaline phosphatase by ca 57% greater than that of the acid phosphatase (ca 40%) at the same day of experiment i.e. day 48. The activity of acid phosphatase was less decreased in soil 2 (the maximal at day 24, of 27%, caused by the third NaF dose), the least effect in soil 3 – the richest in organic carbon, in that soil the highest inhibition reached 23% at day 12 and the highest NaF dose. Similarly behaved the alkaline phosphatase, though to a greater extent (Fig. 2). With the increase of organic matter content in soil, the inhibition of the phosphatases activity decreased, and lesser amounts of 0.01 M CaCl₂ soluble fluorine forms were found. In soil 2 and 3, at the first day and the lowest NaF dose, inhibition of the phosphatases was low and the activity of these enzymes resembled that in control soil, but with the inhibition increased.

Inhibition of β-glukosidase in all three tested soils increased when the concentration of NaF added increased (Fig.3). A more than 50% inhibition occurred in soil 1 and 2 as effect of the highest dose of fluoride, and was recorded at day 24. The highest dose of fluoride decreased the activity of β-glukosidase in all three soils. The activity of – β-glukosidase in soil 2 and 3 was slightly inhibited by the first and second NaF concentrations – string at day 28. The extent of that inhibition depended also on the soil kind.

The activity of dehydrogenases decreased with the increase of NaF doses applied, and also with the time of its influence. At day 48 the enzyme activity was only 50% (in soil 2 and 3). At some times the fluorine stimulated the activity of soil dehydrogenases, but the overall effect had been inhibition (Fig. 4).

Inhibition of enzyme activity was positively correlated with the amount of fluorine soluble in 0.01 M CaCl₂ and 2 M HClO₄ (tab.4). In most cases the correlation coefficient was above 0.9. The highest degree of correlation showed b-glukosidase. In soil 1 and 3 at all sampling days there was a correlation between fluorine forms soluble in 0.01 M CaCl₂ and rate inhibition. The least number of significantly correlation coefficients was found for alkaline phosphatase, though these coefficients were rather high. The correlation coefficients decreased in the case of acid and alkaline phosphatases and therefore were no significance correlations with increasing time of influence both forms of F (soluble in 0.01 M CaCl₂ and 2 M HClO₄) in the soil.

DISCUSSION

Results of this study indicate that the presence in soil of various forms of fluorine soluble in 0.01 M CaCl₂ and in 2 M HClO₄ inhibits the activity of soil enzymes: acid and alkaline phosphatase, β-glukosidase and dehydrogenase. The highest levels of fluorine soluble in 0.01 M CaCl₂ were found in light soil, and with the increase of organic matter and floatable parts in soil – the content of fluorides decreased. [24] have found that the fluorine content in soils had been between 20 and 1660 mg kg^-1, and depended mostly on the soil’s grain composition. Soils of large content of floatable parts contained mostly more fluorine than those of lesser content of such particles. The solubility of fluorine in 0.01 M CaCl₂ was minute, from 0-1%, and attained higher levels in layers of greater content of organic matter. [1] pointed out that the mobility of fluorine depends to a great extent on the pH. Retention of fluorine decreases with the increase of soil pH. In our study pH increased by ca 1 unit if the soil had been treated with 50 mmol NaF solution (dose III). Perhaps that change had also an impact on the content of 0.01 M CaCl₂ and 2 M HClO₄ – soluble fluorine in soil.

Many authors point to an inhibition of enzyme activity caused by fluorine. [3] which have assessed the role of residues of various chemicals in the environment, and their impact on the activity of eight enzymes, stressed the clearly inhibitive action of NaF towards urease, and also found that some salts had been in this aspect of much lesser importance, e.g. NaCl. Similar effects have reported also by [7]. [12] studied the inhibition of jack bean urease covalently immobilized on the chitosan membrane by Ni²⁺ and Fˉ ions. The stability of the complex EI (enzyme inhibitor) is characterized by it inhibition constant K_i. The increase of this value was for Fˉ 26.5 fold and for Ni²⁺ 17.9-fold. [14] were measured the biomass carbon (C_mic) and enzyme activities at 12 sites along a gradient of former emissions of pollutants resulting from phosphate production. They found high total concentrations of cadmium in the dust (up to 33 mg kg^-1 dw) and fluoride (5300 mg kg^-1 dw) in contaminated sites. Accumulation of partially decomposed plant matter, soil respiration and dehydrogenase activity paralleled the increase of dust deposits, whereas microbial biomass decreased along that gradient. These authors obtained a significant negative correlation between the C_mic and C_org ratio and the concentration of pollutants; in contrast, the C_mic and the dehydrogenase activity were positively correlated to C_mic ratio. They suggested that the low C_mic values and the enhanced activities in the contaminated soils are a response of microbial communities to environmental stress or ecosystem disturbances. In our study high doses of NaF inhibited the activity of dehydrogenases, thus confirmed the findings of [14]. [6] reported an increased (7-8-fold) concentration of fluorine (tolerable amount 4-5-fold) in soil near aluminum factory. She found no differences in the dimensions of bacterial and fungi biomass along the gradient of pollution, but there were changes in the taxonomic structure of soil mycoflora and in the level of their domination along the gradient pollution. The applied NaF caused remarkable inhibition of the activity of both phosphatases, also of b-glukosidase, and this process was connected with the NaF dose and kind of soil. These data confirm those reported [18]. They have found, that fluoride was an inhibitor of heme catalase, and could also bind and inhibit copper-based oxidases and urease. According to the authors mentioned above, fluoride can affect a variety of pyrophosphatases and other phosphatases, e.g. kinetoplasts enzymes of Leishmania which is particularly sensitive to NaF and could be inhibited by 0.2 mM fluoride.

CONCLUSIONS

In general, addition of NaF in three various doses (10, 30 and 50 mM) to soil of diverse content of organic matter and mechanical composition has caused the appearance in soil various amounts of fluorine compounds soluble in 0.01 M CaCl₂ and 2 M HClO₄. The levels of the fluorine compounds depended on the kind of soil (the greatest amounts of water soluble compounds had been found in the lightest soil), and also were related to the NaF dose applied and time of the experiment duration. The content of the fluorine forms was correlated with the activity of soil enzymes. The greatest inhibition pertained to the phosphatase: alkaline, acid, next b-gukosidase and dehydrogenases. As far the activity of dehydrogenases was concerned, a temporary increase had also been noticed. The inhibition of enzymes was highly correlated with the content of the various fluorine forms in soil, the highest significance of that correlation pertained to b-glukosidase.

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