COMBINED EFFECT OF LOW-MOLECULAR-WEIGHT ORGANIC ACIDS AND CREOSOTE ON PHOSPHATASE ACTIVITIES IN SANDY

1234 Abstract. This paper assesses the impact of creosote and low-molecular-weight organic acids (LMWOAs) on the activity of acid phosphomonoesterase, alkaline phosphomonoesterase, phosphotriesterase, and inorganic pyrophosphatase in soil. The experiment was carried out on loamy sand samples with organic carbon content of 8.71 g·kg-1, with the following variable factors: dosages of creosote: 0, 0.5%, and 2.5%; type of LMWOAs: oxalic acid, tartaric acid, and citric acid in the amount of 50 mmol·kg-1 of soil; days of experiment: 1, 7, 14, 28, 56, 112. Obtained results showed that contamination with creosote caused decrease in the activity of soil phosphatases. The observed effect did not always increase with increase in the dosage of the pollutant. Among the assayed phosphatases, the biggest changes were noted in the activity of phosphomonoesterases. Application of LMWOAs to contaminated soil mainly effected the inhibition of phospha-


INTRODUCTION
Creosote is a wood-preserving product obtained by fractional distillation of crude coal tar and produced by high-temperature carbonization of bituminous coal (Gallego et al. 2008).It is composed of approximately 85% polycyclic aromatic hydrocarbons (PAH), 10% phenolic compounds, and 5% N-, S-, and O-heterocyclics (Simarro et al. 2013).Following the Regulation of the European Parliament and of the Council no.528/2012 of 22 May 2012, beginning from 1 May 2013, creosote, used for impregnation of railway track ties, has been considered a non-threshold carcinogen and it has been classified as a 1B carcinogen, and some of the PAHs have been considered as persistent, bioaccumulative, and toxic (Kukulska-Zając et al. 2014).
According to the estimations of the World Health Organization, at the beginning of the 21 st century, the annual production of creosote in the EU Member States was from 60 to 100 thousand tons (Ikarashi et al. 2005).Thus, large amounts of the substance may penetrate into the soil.Polycyclic aromatic hydrocarbons as well as other creosote components may cause negative impacts on the biochemical processes of the soil, including transformations of the phosphorus compounds.The amount of organic phosphorus in the upper soil layers remains in a wide range: from 20% to over 80% of the total phosphorus content (Turner and Haygarth 2005).The hydrolysis of esters and orthophosphoric acid anhydrides is catalyzed by a large group of enzymes, referred to as phosphatases (Nannipieri et al. 2011).The most commonly studied phosphatases include phosphomonoesterases, which catalyze the hydrolysis of organic phosphoric monoesters (Wang et al. 2011).Apart from these enzymes, soil contains phosphodiesterases and phosphotriesterases, which catalyze the hydrolysis of phosphate diesters and triesters, respectively, as well as an inorganic pyrophosphatase that catalyzes the decomposition of pyrophosphate to orthophosphates (Reitzel and Turner 2014).
Low-molecular-weight organic acids (LMWOAs) occur widely in soils and primarily originate from root exudation (Gao et al. 2003, White et al. 2003, Zhao et al. 2006, Lu et al. 2007).The most common LMWOAs identified in soils include oxalic, succinic, tartaric, fumaric, malic, and citric acids (Kpomblekou-A and Tabatabai 2003).LMWOAs have been known to disrupt the sequestering of soil matrix, and thereby enhancing the desorption of organic pollutants in soil (White et al. 2003).Consequently, based on the theory, it has been assumed that LMWOAs influence the PAH availability in the soil environ-ment.However, to date, little research has been conducted in this area, and there is limited information on the availability and sorption-desorption behavior of PAHs from natural soils by organic acids.
The aim of the study was to assess the effect of creosote and three low-molecular-weight organic acids, namely oxalic, tartaric, and citric acids on the activity of phosphatases (acid phosphomonoesterase, alkaline phosphomonoesterase, phosphotriesterase, and inorganic pyrophosphatase) in loamy sand.

MATERIALS AND METHODS
The testing was performed on soil samples taken from the topsoil at the Agricultural Experimental Station in Lipnik (53°24'N, 14°28'E), located in the West Pomeranian Voivodeship, Poland.According to the classification of the United States Department of Agriculture, it is a soil with granulometric composition of loamy sand.The content of particular fractions, expressed in g•kg -1 , was as follows: sand (0.05-2 mm) -748.6;silt (0.002-0.05 mm) -231.3; and clay (<0.002 mm) -20.1.The soil contained, in g•kg -1 : C org -8.71 and N tot -0.97.Its hydrolytic acidity was 9.9 mmol(+)•kg -1 , and the pH value in 1 mol KCl•dm -3 was 6.4.The soil was air-dried and sieved through a 2-mm mesh.
The experiments were carried out in triplicate under laboratory conditions, with the following variable factors: (a) creosote dosages: 0, 0.5%, and 2.5%; (b) type of LMWOAs: oxalic acid, tartaric acid, and citric acid; (c) days of experiment: 1, 7, 14, 28, 56, and 112.The content of LMWOAs added to soil was 50 mmol•kg -1 .The 1-kg soil samples were adjusted to 60% maximum water holding capacity, and they were incubated in tightly closed glass containers at a temperature of 20°C.
Based on the mean activity of assayed soil phosphatases, indices on effects of creosote and LMWOAs were calculated using the following formulas (Kaczyńska et al. 2015): Where: IF C -index of the creosote effect; IF LMWOAs -index of low-molecular-weight organic acids effect; A 0 -activity of phosphatases in non-contaminated soil with the creosote; A C -activity of phosphatases in soil contaminated with the creosote; A LMWOAs -activity of phosphatases in the soil treated with low-molecular-weight organic acids.
If IF=1, there is no influence of the tested factor on oxidoreductases.If IF<1, there is inhibition of the oxidoreductases activity by the tested factor and if IF>1, there is stimulation of the oxidoreductases activity by the tested factor (Stręk and Telesiński 2016a).
The results of the studies were determined statistically, using a statistical software package Statistica v. 13.1 (Statsoft, Inc.).Based on the analysis of the effect measure η 2 by variance analysis -ANOVA, the percentage shares of all variable factors affecting the activity of phosphatases were defined.Homogeneous groups were calculated using the Tukey's test with p<0.05.

RESULTS AND DISCUSSION
The activities of phosphatases in non-contaminated soil with creosote were 111.24-288.81mg p-NP•kg -1 dm•h -1 , 15.83-35.51mg p-NP•kg -1 dm•h -1 , 18.25-22.55mg p-NP•kg -1 dm•h -1 200.78-259.44mg p-PO 4 3-•kg -1 dm•h -1 for Pac, Pal, PT, and IPP, respectively (Table 1).Application of all LMWOAs caused a significant decrease in Pac and Pal in the whole experiment.The highest inhibition was observed for Pac and Pal activity in soil treated with oxalic acid on day 1 (96.54%,compared to control) and on day 7 (85.08%,compared to control), respectively.IPP activity was significantly decreased during all experiment in soil with the addition of oxalic acid and tartaric acid, whereas application of citric acid inhibited IPP activity only from day 1 to day 28.On almost all test days, the activity of PT in soil treated with LMWOAs was significantly lower than or similar to control.Moreover, on day 1, increase in PT activity was reported in soil with the addition of tartaric acid.Renella et al. (2007) have shown that the activity of phosphatase in the rhizosphere soil layer was generally stimulated by LMWOAs.The alkaline phosphomonoesterase activity was significantly stimulated by citric acid in both clay and sandy soils, whereas the acid phosphomonoesterase activity was significantly stimulated by citric acid in the clay soil and by citric acid and oxalic acid in the sandy soil.Phosphodiesterase activity was significantly increased by citric acid in the sandy and clay soil.Furthermore, Huang et al. (2003) reported that LMWOAs may have an effect on the sorption/ desorption activity of acid phosphomonoesterase in soil.
In the whole experiment, creosote at both doses caused a decrease in alkaline phosphomonoesterase activity in soil.Generally, creosote dosage of 2.5% induced higher inhibitory effect on Pac activity than the dosage of 0.5%.Activity of Pal, PT, and IPP in soil containing creosote at the dosage of 0.5% was mainly lower than the control.However, soil contamination with creosote at the dosage of 2.5% caused also an increase in activity of Pal, PT and IPP.This stimulation was the highest for Pal on day 14 (Table 2).
Summarizing the evaluation of the influence of creosote on soil phosphatases, one may ascertain that creosote at the dosage of 0.5% inhibited the activity of Pac, Pal, PT, and IPP (Fig. 1).The mean index of the effect of creosote (IF C ) on the activity of Pac and PT decreased, together with an increase in the dosage of creosote.Inhibition of the activity of different phosphatases in soil contaminated with polyaromatic hydrocarbons (PAHs) and other petroleum products was reported by many researchers (Bielińska et  The effect of LMWOAs on the activity of phosphatases in soil contaminated with creosote proved to be diversified, and depended on the enzyme, incubation time, creosote dosage and the form of LMWOAs.In soil with creosote at the dosage of 0.5%, on the most days of experiment, application of all LMWOAs affected Pac, Pal, and IPP negatively, but PT -favorably (Table 3).Furthermore, on days 56 and 112, the activity of IPP was stimulated.However, in soil contaminated with creosote at the dosage of 2.5% after treatment with LMWOAs, a decrease in the activities of Pac and IPP and an increase in PT activities were mainly observed.For Pal, stimulation of the activity was reported on days 1, 56, and 112, whereas at other measurement dates, application of LMWOAs caused inhibition (Table 4).The same letter means a homogenous group in the columns for an enzyme (p<0.05);abbreviations as in Table 1 From the index of LMWOAs effect (IF LMWOAs ) in soil contaminated with creosote, mean values of below 1 were observed in the activity of Pac for both 0.5% and 2.5% creosote dosages, for Pal at creosote dosage of 0.5%, and for IPP at creosote dosage of 2.5%.Moreover, in soil with creosote at the dosage of 0.5%, mean IF LMWOAs was lower than 1 after the application of citric acid.However, mean values of IF LMWOAs above 1 were reported for PT in soil with both creosote dosages treated with all LMWOAs, for Pal in soil with creosote at the dosage of 2.5% treated with all LMWOAs, and for IPP in soil with creosote at the dosage of 0.5% treated with oxalic acid and tartaric acid (Fig. 2).The information about the impact of LMWOAs on the enzyme activity in soil contaminated with petroleum hydrocarbons is limited.However, some studies have shown that LMWOAs could accelerate the degradation and desorption of polycyclic aromatic hydrocarbons (PAHs) in soils (LeFevre et al. 2013, Ling The data presented in Table 5 indicates, unequivocally, that the activity of phosphatases varied over time.Also, it depended on the creosote dosage.The share of this factor in the formation of dehydrogenase activity ranged from 10.81% (Pal) to 34.49% (IPP).The application of the LMWOAs affected all phosphatases, significantly.The share of this factor in the initiation of the activity ranged from 6.67% (PT) to 78.89% (Pac).Also, the incubation time of the soil affected these enzymes, significantly.This independent variable determined the activity of phosphatases in the range from 5.68% (Pac) to 60.18% (PT).

CONCLUSIONS
Soil contaminated with creosote oil resulted in the inhibition of the soil phosphatase activities.The observed effect was not always increased with the increase of the contamination dosage.The greatest changes among the determined enzymes, caused by creosote oil, occurred for the activity of phosphomonoesterases.The introduction of low-molecular-weight organic acids, both to non-contaminated soil and soil contaminated with creosote, resulted primarily in the reduction of phosphatase activity.The biggest visible effect was in the case of acid phosphomonoesterase.A comparison of the effect of different organic acids such as oxalic, tartaric, and citric allows concluding that citric acid was characterized with the lowest toxicity to soil phosphatases.

TABLE 2 .
INDICES OF THE CREOSOTE EFFECT (IF C ) ON THE ACTIVITY OF SOIL PHOSPHATASES

TABLE 3 .
INDICES OF THE LMWOAS EFFECT (IF LMWOAs ) ON THE ACTIVITY OF PHOSPHATASES IN SOIL CONTAMINATED WITH CREOSOTE AT THE DOSAGE OF 0.5%

TABLE 4 .
INDICES OF THE LMWOAS EFFECT (IF LMWOAs ) ON THE ACTIVITY OF PHOSPHATASES IN SOIL CONTAMINATED WITH CREOSOTE AT THE DOSAGE OF 2.5%The same letter means a homogenous group in the columns for an enzyme (p<0.05);abbreviations as in

Table 1
(Gao et al. 2015, Sun et al. 2016nd that rhizosphere of ryegrass potentially enhanced the dissipation or biotransformation of a range of PAHs including 5-and 6-ring PAHs.Ling et al. (2015)have reported that the availability of phenanthrene and pyrene in soils increased with the increasing concentration of LMWOAs.Further, several studies suggested that desorption of PAHs in soils could be significantly enhanced with the presence of LMWOAs, thus improving the bioavailability and biodegradation of the residual pollutants(Gao et al. 2015, Sun et al. 2016).

TABLE 5 .
PARTICIPATION OF VARIABLE FACTORS IN THE FORMATION OF PHOSPHATASE ACTIVITIES (%)