Combined Effects of Cadmium and Azithromycin on Soil Nitrification Process

Abstract

Heavy metals and pharmaceuticals have polluted agricultural soils mainly through wastewater irrigation, fertilizers, and soil amendment with sewage sludge. This study aims to determine the synergetic toxic effect of Cd and the selected macrolide antibiotic, azithromycin (AZI), on ammonia-oxidizing bacteria (AOB) in soil, via analyzing nitrification inhibition. A short-term acute toxicity test was used to measure the formation of Nitrite (NO₂-N) to indicate the nitrification potential of the aerobic nitrosomonas bacteria in the germination period. Potential nitrification rates (PNRs) of five soil samples ranged between 3.782–17.642 mg NO₂-N/kg dm PNRs of soil samples positively correlated with organic matter content and neutral pH. PNRs of the tested soils were significantly affected by Cd and AZI contamination, with interactions exhibited for their simultaneous occurrence and soil pH. A significant difference (p < 0.05) was found when soil samples with pH 6.5–pH 8.5 contaminated with environmentally relevant concentrations of Cd (1 mg/kg–21 mg/kg) and AZI (1 mg/kg–9 mg/kg). 50% PNR inhibition after 11 mg/kg Cd and 5 mg/kg AZI contamination was determined for the soil sample at pH 8.5, with 3.782 mg NO₂-N/kg dm potential. From these outcomes, it was concluded that there was a risk of the soil nitrification process in case of simultaneous occurrence of Cd and AZI.

1. Introduction

The productivity of agricultural lands has become a critical issue in the world, where concerns about hunger are increasing. While there is a growing need for food, environmental pollutants threaten the sustainability of agricultural production. The main factors affecting soil pollution are industrial activities, such as mining, using fertilizers, pesticides, fungicides, soil conditioners, sewage sludge, irrigation with treated or insufficiently treated wastewater, etc. [1,2,3]. Cadmium (Cd) has accumulated in agricultural areas around Konya, Turkey, as a result of long-term wastewater irrigation and the use of phosphorus fertilizers. Additionally, the macrolide antibiotics azithromycin, clarithromycin, and erythromycin were found to be the most abundant compounds in digested sewage sludge used for soil amendment in the same region. Excessive heavy metal accumulation in the soil can cause the degradation of soil ecosystems and threaten soil fertility. Unfortunately, there is little information about the synergetic toxic effects of pharmaceuticals in sewage sludge and heavy metals in the soil after soil amendment.

Cd is among the most toxic heavy metals, which has no known essential function [4]. It has teratogenic and mutagenic effects on organisms, inhibits soil bacterial activity, and creates stress for plant growth [5,6]. Cd contamination deteriorates soil quality and threatens food safety and human health. While Cd pollution is mainly caused by industrial activities, phosphorus fertilizers, sewage sludge applications, and wastewater irrigation contaminate agricultural soils with Cd [7]. Cd has a long half-life in soil, estimated as 15–1100 years [8], and can bio-magnify in the soil-plant system, which is controlled by the soil type and plant species [9,10].

Macrolide antibiotics are among the most abundant pharmaceuticals extensively used for human and animal health [11]. Antibiotics and their metabolites have been released into the environment through wastewater and sewage sludge [12]. Antibiotics are excreted in manure generally in their main forms due to incomplete metabolism. While nearly 70% of these compounds in wastewater are accumulated in sewage sludge, 50% of sewage sludge has been used for soil amendment in European Member States, the United States, and China [13,14,15].

The amendment of soil with digested sewage sludge has become increasingly common, to benefit from its organic matter and nutrient content. However, sewage sludge also contains heavy metals and many other compounds, such as pharmaceuticals [16]. Cd and pharmaceuticals are often unavoidable contents of sewage sludge. Baize [17] reported that the long-term application of Cd-containing sewage sludge on soil in France resulted in 160 mg/kg dm mean Cd concentrations in the 1970s, which was reduced to 5.8 mg/kg dm in the 2000s after regulations. Topp et al. [18] determined macrolide antibiotics, including AZI, in the soil in the concentration range of 0.1 or 10 mg/kg dm after consecutive application of sewage sludge for five years. Cd and macrolide antibiotics pollution in soil and sewage sludge reported for various countries are given in

Table 1. Cd and macrolide antibiotics pollution in soil and sewage.

Soil	Reference	Sewage Sludge	Reference
Cd (mg/kg dm)
8.23–11.6 (range)	[20]	nd–24.46 (range)	[21]
9.45 (mean)	[22]	7.18 (mean)	[23]
0.1–4.2 (range)	[24]	49.26 (mean)	[25]
0.3–6.4 (range)	[26]	0.7–1.3 (range)	[27]
1.3–8.5 (range)	[28]	1.5–4.5 (range)	[29]
Macrolide Antibiotics (mg/kg dm)
-	-	nd–1.496 (total range)	[1]
-	-	5.315–8.466 (total range)	[30]
-	-	0.081–0.850 (AZI range)	[31]
-	-	0.200–0.666 (AZI range)	[32]
-	-	0.299 (AZI)	[33]

Note: nd: not determined.

. Recent research about soil antibiotic pollution focused on the formation of antibiotic-resistance genes in soil [19].

The working group of the authors of this article conducted a research study to define the effects of the long-term irrigation of agricultural areas in Konya, Turkey, with untreated municipal wastewater [20]. Significant pollution with Cd (8.23–11.6 mg/kg dm) was determined in the wastewater-irrigated soils of the sampling site. Soil Cd contamination was well over the Maximum Admissible Concentrations for Trace Elements in Agricultural Soils for the application of Sewage Sludge Regulation in Turkey, which was given as 1 mg/kg dm (6 ≤ pH < 7) or 1.5 mg/kg dm pH ≥ 7 (2010). Digested sewage sludge from an urban wastewater treatment plant (WWTP) has been used for soil amendment in the same area. A high-risk quotient (RQ > 1) was estimated for AZI, determined to be nearly 1.5 mg/kg dm in sewage sludge [1]. Questions have been raised about the toxic effect of the simultaneous occurrence of Cd and AZI on soil microbial activity in the region.

Ammonium (${\mathrm{NH}}_4^{+}$) oxidation bacteria (AOB), Nitrosomonas and Nitrobacter, and ammonium oxidation archea (AOA) in the soil ensure the conversion of ${\mathrm{NH}}_4^{+}$ content of the soil to useful nitrate (${\mathrm{NO}}_3^{-}$) form. The nitrification process transforms ${\mathrm{NH}}_4^{+}$) to ${\mathrm{NO}}_3^{-}$ (Equation (1) in two steps (R1 and R2). Nitrosomonas bacteria is responsible for the first step (R1), while Nitrobacter completes the transformation (R2). The first step (R1) conversion of ${\mathrm{NH}}_4^{+}$) to ${\mathrm{NO}}_2^{-}$ is assumed to be the limiting step of the nitrification process because of the sensitivity of Nitrosomonas when compared with Nitrobacter.

$$\mathrm{N}{\mathrm{H}}_4^{+}\stackrel{R1}{\to }\mathrm{N}{\mathrm{O}}_2^{-}\stackrel{R2}{\to }\mathrm{N}{\mathrm{O}}_3^{-}$$

(1)

As a result of the inhibition of AOB by environmental pollutants, ${\mathrm{NH}}_4^{+}$ containing nitrogenous fertilizers and organic nitrogen in the soil cannot be converted to ${\mathrm{NO}}_2^{-}$ and ${\mathrm{NO}}_3^{-}$ forms. Since ${\mathrm{NO}}_3^{-}$ is the essential form of nitrogen for plants, conversion of ${\mathrm{NH}}_4^{+}$ to ${\mathrm{NO}}_3^{-}$ is critical for agricultural activities [34]. Studies in the literature have focused on the independent toxic effects of heavy metals on AOB; however, the combined toxic effect of heavy metals with various environmental pollutants on the soil nitrification process is relatively less reported. Bai et al. [35] experimented with the effect of Cd addition on nitrification processes in soil samples taken from a constructed wetland in China. Incubation experiments were conducted for 15 mg/kg Cd and 100 mg/kg Cd. While no inhibition effect was found for a low level of Cd, 100 mg/kg Cd reduced nitrogen mineralization rate from 0.40 mg/kg dm to 0.21 mg/kg dm when compared to uncontaminated control soil. Smolders et al. [36] determined the PNRs of soil samples spiked with 0–200 mg/kg Cd. The PNRs of soil samples were reduced to 50–80% at the highest Cd contamination level. Rosendahl et al. [37] researched the persistence of difloxacin antibiotics in soil and its effect on soil nitrification potential. It was determined that even though difloxacin had a long half-life (>217 d), its effect on nitrogen turnover was limited. It was concluded that difloxacin formed non-extractible residues in soil, resulting in small bio-accessibility.

The effects of heavy metals on microbial populations in agricultural soils have been evaluated by different methods. The studies carried out were laboratory-scale studies, in which short-term acute effects were determined, or field studies, in which long-term chronic effects were determined [8]. In tests using long incubation times, few resistant microorganisms may cause the toxic effect to be undetectable. To activate the formation of microorganisms in the soil, test methods that add a large amount of external substrate do not give accurate results since they do not reflect the natural ecosystem. The most commonly used soil microbial toxicity test methods were tests using a single species, tests in which biomass or carbon and nitrogen transformations were measured, enzymatic tests, and tests in which changes in microbial diversity were determined [38,39]. Sauve et al. [40] determined the nitrification potential of soil samples contaminated with Pb or Cu using a 4 h incubation test. A positive correlation was determined for organic matter (OM) and the nitrification potential of soil samples, which was attributed to the existence of organic microsites favorable to heavy metal contamination over time. The nitrification potential of the soil samples reduced to low pH values below 6 and high pH values above 7.5, which was attributed to ${\mathrm{NH}}_4^{+}$ toxicity for the latter case.

The toxic effects of many environmental pollutants have been determined using aquatic and terrestrial organisms, and studies on the toxic effects of environmental pollutants on soil organisms are very limited. Previous researchers separately demonstrated the toxic effects of heavy metals and pharmaceuticals on soil microbial activity [35,41]. Only a recent publication questioned the simultaneous toxic effect of Cu, Zn, and oxytetracycline (OTC), reported by Tang et al. [42]. While OTC did not adversely affect soil respiration, the combined occurrence of OTC with Cu and Zn reduced the abundance of bacteria and fungi in the soil.

In this study, we aimed to determine the combined toxic effects of Cd and selected antibiotics, AZI on AOB, through testing soil PNR. Cd contamination of the soil and AZI content of the sewage sludge used for soil amendment in the selected study area have been demonstrated in previous research studies [1,20]. Therefore, it was aimed to determine the toxic effect of the simultaneous occurrence of these two contaminants on soil nitrification potential to evaluate the risk of soil amendment with digested sewage sludge for sustainable soil fertility.

2. Materials and Methods

2.1. Soil Sampling and Experimental Design

Soil samples were collected from agricultural lands in Konya City, located in the Central Anatolia region in Turkey (37°52′ N, 32°35′ E). The city is considered to be a granary area of the country, with a high production of wheat. The region has a temperate and arid climate, with a mean annual precipitation of 323 mm. Municipal wastewater of Konya City has been treated with conventional WWTP since 2010, removing carbon and partial nitrogen. The plant’s effluent is discharged into the main drainage channel, converging in Salt Lake downstream. Approximately 140 tons/day of treated sludge is produced in the plant and is periodically used for soil amendment. Soil samples were taken from the soil surface at a depth of 0–20 cm by digging a hole with the help of a shovel. The samples were sieved through a 200 µm sieve before analysis. The samples were covered and stored at 4 °C until analysis. Soil samples were analyzed within a month.

Soil samples were taken from five districts in Konya City (S1, S2, S3, S4, S5). Sampling areas were selected to have different soil physicochemical properties. Three samples were taken from the same district to find the least Cd-contaminated soil samples. Additionally, three soil samples were taken from each sampling point and homogenized before experiments. The least contaminated soil samples taken from the study area were used in controlled laboratory experiments. Soil samples were experimented on with their original pH with no modification. Environmentally relevant and excessive concentrations of Cd and AZI were spiked to soil samples with different pH levels to determine nitrification inhibition compared with control soil (CS) that was not exposed to contaminants.

A total of 17 experiment sets were performed to determine PNR inhibition. Environmentally relevant concentrations of Cd (1 mg/kg–21 mg/kg) and AZI (1 mg/kg–9 mg/kg) were experimented on. The second-degree polynomial model predicted the relationship between the experimental variables and the response (Y). For this, five levels for each variable were experimented on. The central level was coded as 0 (mid), low and high levels were coded as −1 and +1, respectively, while low and high control levels were coded as −2 and +2, respectively. The experimental range for the coded independent variables is given in

Table 2. Coded experimental range and variables for experimental design.

Factor	Variable	Level
		−2 (−α)	−1 (Low)	0 (Mid)	+1 (High)	+2 (+α)
X1	Cd (mg/kg)	1	6	11	16	21
X2	AZI (mg/kg)	1	3	5	7	9
X3	pH	6.5	7.0	7.5	8.0	8.5

. While the mid-level pollution was selected as an environmentally relevant concentration, the high level was selected for excessive contamination. To avoid systematic error, experiments were performed in a randomized order. Each experiment was performed with three replicates, and the results were given in the form of mean. Design-Expert^® package, version 11.0.3 (Stat-Ease Inc., Minneapolis, MN, USA) software was used for the statistical analysis of obtained data. The analysis of variance (ANOVA) was used to analyze the experimental results obtained for Cd and AZI-contaminated soil samples in various pH levels and validate the significance of the quadratic model. p values less than 0.05 were considered to be significant.

2.2. Acute Ammonium Oxidation Toxicity Test

The acute ammonium oxidation toxicity test helps to determine the presence and vital activity of nitrifying bacteria (nitrosomonas) in the soil [43]. It differs from other toxicity tests as it is a test performed without the addition of an external organism. The soil must be fresh for the test to be performed (maximum of 3 months at 4 °C). The amount of NO₂, an intermediate in the nitrification process, was measured spectrometrically in accordance with the sulfanilamide colorimetric method (SM 4500 NO₂-B). Conversion of ${\mathrm{NO}}_2^{-}$ to ${\mathrm{NO}}_3^{-}$ was inhibited using 2 mL KCl (1 mol/L) for 2 mL aliquot samples taken from the incubation solution. Nitrification was promoted by adding the optimum dose of NaClO₃ (0.5 mol/L), 28 mL/L KH₂PO₄ (0.2 mol/L), 72 mL/L K₂HPO₄ (0.2 mol/L), and 0.198 g/L (NH₄)₂SO₄ to the incubation solution as substrate. Higher amounts of KH₂PO₄, K₂HPO₄, and (NH₄)₂SO₄ increase the nitrification potential in soil; however, excess NaClO₃ inhibits the nitrification process. Since large amounts of external substrate do not reflect the natural ecosystem, only limited amounts of KH₂PO_4, K₂HPO_4, and (NH₄)₂SO₄ were used to stimulate nitrification. The optimum dose of NaClO₃ was investigated for 10 mL/L, 20 mL/L, and 30 mL/L to determine the most suitable incubation solution [40]. The optimum dose was determined for 20 mL/L NaClO₃ for all samples.

As the test principle, considering the soil water content, 100 mL of incubation solution was added to 25 g of soil sample. The flasks containing the control soil without contaminant and samples spiked with Cd and AZI were covered with aluminum foil with an open top. Spike concentrations of Cd and AZI were adjusted according to the experimental design given in Table 2. It was placed in a shaking incubator at 26 °C, 120 rpm in aerobic conditions. The linearity of ${\mathrm{NO}}_2^{-}$ formation, which was an indicator of the formation of AOB, was evaluated by taking samples at the end of each hour. 6 samples were taken in 6 h within the germination period of nitrifying bacteria. The samples were centrifuged (3000 cycles/2 min) before ${\mathrm{NO}}_2^{-}$ analysis. The PNR inhibition percent of Cd and AZI spiked soil samples was determined by comparing the PNR of control soil samples at the end of the incubation period.

2.3. Analytical Methods for the Determination of Soil’s Physicochemical Properties and Heavy Metals

Dry matter (%) content of soil samples was determined in accordance with the ISO 11,465 method [44]. The pH value was determined for 1:5 (v/v) suspension formed by a 5 g soil sample dried in the laboratory and 25 mL distilled water, using a pH-meter (Hach^®, Loveland, CO, USA) in accordance with ISO 10390 [45]. The dry soil sample was weighed and placed in the shaker bottle with 100 mL water at 20 + 1 °C, filtered after shaking for 30 min, and kept at 25 °C for an Electrical Conductivity (EC) measurement using the electrometric method in accordance with ISO 11265 [46]. Clay/silt/sand compositions of soil samples were determined with the Bouyoucos Hydrometer method using Atterberg cylinders (ASTM D 422–63) [47]. Cation exchange capacity (CEC) was determined by the ammonium acetate saturating and distillation procedure using a Kjeltec 8100 (FOSS Analytical, Hilleroad, Denmark) distillater. Soil organic matter (OM) was determined according to the Walkley-Black method [48], which depends on oxidizing carbon with potassium dichromate and back titration of the excess dichromate with ferrous sulphate. The total Nitrogen (TN) content of soil samples was determined according to the modified Kjeldahl Method (ISO 11261) [49]. Microwave (CEM, MarsXpress, Charlotte, NC, USA) assisted acid digestion (9.5 mL, 65% HNO₃/0.5 mL, 37% HCl for 0.5 g soil sample) was performed for extraction of target heavy metals in soil samples, prior to Inductively Coupled Plasma—Optical Emission Spectrometry (ICP-OES 5300, Perkin Elmer, Waltham, MA, USA) analysis in accordance with the EPA 6010 D standard method [50]. The physicochemical properties of 5 soil samples used for the toxicity test are given in

Table 3. Physicochemical properties of soil samples.

Sampling Site	pH	EC mS/cm	Soil Texture	OM%	CaCO3%	P2O5 kg/da	K2O kg/da	Na mg/kg	Ca mg/kg	TN%	Cd mg/kg
S1	8.5	0.57	Clay	0.82	17.06	0.06	36	312	3364	0.140	1.2
S2	6.5	0.35	Clay Loam	1.53	1.56	5.21	33	50	881	0.153	1.5
S3	8.0	0.50	Loam	1.45	63.92	4.98	96	54	3015	0.149	0.8
S4	7.5	0.67	Clay	1.67	11.46	0.92	143.4	51	3.14	0.145	1.4
S5	7.0	1.14	Clay	4.36	20.78	25.88	65.4	119	2109	0.104	0.8

. Samples are given using the same letter with consecutive numbers (S1, S2, S3, S4, S5).

3. Results

3.1. Nitrification Potential of Soil Samples

The PNR of soil samples obtained for optimum experimental conditions is given in Figure 1. S1 (3.782 mg NO₂-N/kg dm) and S2 (4.52 mg NO₂-N/kg dm) were determined to have lower nitrification potential when compared with S3 (15.515 mg NO₂-N/kg dm), S4 (15.383 mg NO₂-N/kg dm), and S5 (17.642 mg NO₂-N/kg dm).

One of the most important physicochemical properties affecting the nitrification potential of the soil was the OM content. The (OM) content of S1 was 0.82%, while the (OM) content of S5 was 4.36%. The increase in (OM) content increased the PNR at the end of the 6 h incubation period. A positive correlation was determined between (OM) and PNR, with a 0.6 R² value (Figure 2). The low amount of (OM) in the soil adversely affected the biological activity and physical and chemical soil fertility of the bacteria. This situation inhibited the effective working of bacteria. It caused N losses to the atmosphere with its low N buffering and filtering capacity in the soil.

Another important physicochemical property of the soil samples was the pH values. The inhibition of nitrification at high pH was due to ${\mathrm{NH}}_4^{+}$ toxicity or the increased solubility of metals. With the increase in the solubility of metals, the pH balance changed, and when the pH value rose above 8, nitrification was inhibited. At the end of the 6-h incubation period in this study, the PNR of S1 with pH 8.3 was 3.782 mg NO₂-N/kg dm, while the PNR of S5 with pH 6.87 reached 17.642 mg NO₂-N/kg dm value.

Poor correlations were determined for soil EC, CaCO₃, P₂O₅, Na, Ca, and TN values and soil PNR, with 0.42, 0.22, 0.28, 0.26, 0.02, and 0.24 R² values, respectively (Figure 2). A meaningful correlation was determined for soil K₂O and soil PNR, with a 0.52 R² value. K is one of the macronutrients in the soil, effective on soil microbial activity. In this study, the soil sample with 33 kg/da K₂O had 4.52 mg NO₂-N/kg dm PNR, while it was 15.38 NO₂-N/kg dm for the soil with 143.4 kg/da K₂O.

3.2. Model Fitting and Statistical Analysis

A total of 17 experiments, with three replicates, in a randomized order, were performed to investigate the PNR inhibition effect of initial Cd and AZI contamination on soil samples with various pH values. The experimental design matrix is given in

Table 4. Design matrix and observed responses.

Run	Codified Variables			No Codified Variables			Response for PNR Inhibition (%)
	X1 Cd (mg/kg)	X2 AZI (mg/kg)	X3 pH	X1 Cd (mg/kg)	X2 AZI (mg/kg)	X3 pH
1	−2	0	0	1	5	7.5	3
2	0	2	0	11	9	7.5	29
3	−1	−1	−1	6	3	7.0	10
4	1	1	−1	16	7	7	31
5	0	0	−2	11	5	6.5	30
6	0	0	0	11	5	7.5	21.5
7	2	0	0	21	5	7.5	35
8	0	0	0	11	5	7.5	22
9	0	0	2	11	5	8.5	50
10	1	−1	−1	16	3	7	22
11	1	−1	1	16	3	8	32
12	−1	−1	1	6	3	8	17
13	−1	1	−1	6	7	7	14
14	0	−2	0	11	1	7.5	13
15	0	0	0	11	5	7.5	21
16	1	1	1	16	7	8	46
17	−1	1	1	6	7	8	21

, along with the observed responses for PNR inhibition. While the minimum PNR inhibition of 3% was determined for 1 mg/kg Cd and 5 mg/kg AZI for the soil at pH 7.5, the highest PNR inhibition of 50% was determined for 11 mg/kg Cd, and 5 mg/kg AZI, for the soil at pH 8.5. Even though Cd concentration was nearly doubled in the 7th experimental run, PNR inhibition was 35% for the soil at pH 7.5, revealing the importance of soil pH.

The data for Cd and AZI contamination of soil samples at various concentration levels and pH were fitted to the quadratic model. The statistical significance of the data was tested by ANOVA, as given in

Table 5. ANOVA results for the quadratic model for Cd and AZI at different soil pH.

Source	Sum of Squares	F-Value	p-Value
A-Cd	1105.56	638.55	<0.0001
B-AZI	248.06	143.28	<0.0001
C-pH	390.06	225.29	<0.0001
AB	28.12	16.24	0.003
AC	15.12	8.74	0.0161
A2	8.50	4.91	0.0540
C2	494.27	285.48	<0.0001
Residual	15.58
Lack of Fit	15.08	8.62	0.1079
Pure Error	0.5000
Cor Total	2388.94

. The low p values (p < 0.05) indicated the high significance of inhibition data. The high value of the squared correlation coefficient (R²) of 0.9935 indicated that variation could be explained by the model. The predicted R² of 0.9699 is in reasonable agreement with the adjusted R² of 0.9884, where the difference is less than 0.2. The high R² and adjusted R² explained the significance of PNR inhibition data obtained for various concentration levels of Cd and AZI at different pH levels of the soil. A low coefficient of variation, calculated as 5.36%, ensures the high precision and reliability of the experiments. The model is satisfactory since predicted versus experimental data have a high correlation, as given in Figure 3.

3.3. Effect of Cd, AZI, and Soil pH on PNR Inhibition through Response Surface Methodology

The response surface contour plots of the second-order quadratic model, proposed by Box Behnkan, were used to interpret obtained experimental data. For this, one variable was kept at a fixed level, and the other two variables were kept in the low and high experimental ranges. The effect of soil Cd concentration (X1), AZI concentration (X2), and pH (X3) on PNR inhibition % are given in Figure 4. Figure 4a presents the PNR inhibition effect of Cd and AZI contamination obtained for soil samples at pH 7. Neutral soil pH reduced PNR inhibition to 30% for 16 mg/kg Cd and 7 mg/kg AZI contamination levels. Figure 4b presents the PNR inhibition effect of Cd and pH, obtained for a 6 mg/kg AZI concentration. There were higher inhibition effects in this case, showing an increasing effect of soil pH. Figure 4c presents the PNR inhibition effect of AZI and pH obtained for 15 mg/kg Cd. Results show that Cd contamination has a higher PNR inhibition effect than AZI.

4. Discussion

The toxic effect of the tested contaminants was significantly affected by the soil type. It was determined that soils with low PNR were more sensitive to Cd and AZI contamination. Higher OM content of S5 and neutral pH resulted in higher PNR. It must be remembered that the OM of the soil increases the accumulation of contaminants in the soil. Sorption–desorption reactions greatly affect the mobility of heavy metals and also control their bioavailability [51]. It was reported that over 60% of the Cd in soil might be bound to soil OM [52]. Heavy metals, in association with the solid phase of the soil, are immobile. Adsorption on soil particles reduces bioavailability, which also explains the findings of this study. Tang et al. [42] evaluated the acute toxic effect of OTC antibiotics with Zn and Cu on the microbial activity of soil by measuring N₂O. The findings of this study revealed that the soil type influenced the toxic effects of the contaminants.

The time-dependent toxic effect of Cd and AZI on the nitrification process was determined in this study. A diminished toxic effect was determined for all tested soil types and contamination levels. While the toxic effect varied between 15–89% for the first hour of the test, it reduced to 3–50% by the end of the incubation. This type of recovery was reported by other researchers. Rodríguez-González et al. [53] evaluated the potential toxic effect of clarithromycin on soil microbial activity. The toxic effect of clarithromycin on the soil bacterial community was found to be time-dependent. The toxic effect decreased between the 1st and 8th incubation days, and the bacterial community recovered after 42 days. Decreasing toxicity was attributed to the adsorption of clarithromycin on soil particles and degradation.

Persistence of antibiotics and heavy metals in soil results in continuing toxic effects. The long-term effect of the field applications, carried out annually, has been questioned. Rosendahl et al. [37] determined >217 d half-life for difloxacin in bulk soil. The half-life of enrofloxacin in soil was reported as 152 d [54]. Topp et al. [18] conducted a laboratory scale study to determine the dissipation kinetics of macrolide antibiotics. The half-life of AZI was nearly 13 days after the 10 mg/kg soil treatment concentration. No carry-over of macrolide antibiotics from year to year was determined. Attention has been drawn to the formation of antibiotic-resistant genes in the soil.

This study also shows a concentration-dependent toxic effect of Cd and AZI. While 1 mg/kg Cd and 5 mg/kg AZI caused only 3% PNR inhibition at pH 7.5, 21 mg/kg Cd and 5 mg/kg AZI caused 35% PNR inhibition at the same conditions. The inhibitory effect reached a critical 50% value at the end of the incubation when the pH was 8.5 even, Cd was 11 mg/kg, and AZI was 5 mg/kg. This finding reveals the importance of soil pH. Smolders et al. [36] determined that the nitrification potential increased as the soil pH value changed from acidic to neutral. While 0.4 mg N/kg day was formed in soil with pH 3.6, 2 mg N/kg day was formed in soil with pH 7. Sauve et al. [40] reported that the nitrification potential of the soil with pH 4.54 (OM: 2.91%) was 25 µmol/kg per day, while the nitrification potential of the soil with pH 7.84 (OM: 2.55%) was 303 µmol/kg day. As the pH rose above 8, nitrification was inhibited. Nitrification was found to be low in acidic soils below pH 6, as well as in soils with fficien soils above pH 8. It has been determined that the optimum pH range for nitrification is in the range of pH 6.5–7.5. A dose-response pattern was also obtained by Toth et al. [41]. The inhibitory effect of a commonly used veterinary antibiotic, sulfadimethoxine, on the nitrification potential of tested soil was reported for 0–200 µg/kg sulfadimethoxine concentrations. While the soil nitrification potential was nearly 0.50 mg NO₂-N/kg soil dm for the first day, it was reduced to nearly 0.20 mg NO₂-N/kg soil dm for 50 µg/kg sulfadimethoxine dose.

Soil structure affects N leaching as it determines the water movement in the soil. Fine-structured soils (clay) are less sensitive to N transport as they have lower water permeability than coarse-grained (sandy) soils. However, clay soils are more prone to nitrogen loss by denitrification. S1, S4, and S5 soil samples in this study have a clay soil texture, while S2 has a clay loam and S3 has a loam structure. Clay minerals affect the mobility and bioavailability of antibiotics and heavy metals in the soil [55]. Fang et al. [56] reported that the sorption of gemfibrozil was higher for sandy loam soil than for sandy soil, with 17.8 and 20.6 days half-lives, respectively.

Inorganic salts such as phosphorus, potassium, calcium, and magnesium are among the cell building materials of AOB. However, soil physicochemical properties did not show a clear effect on PNR, except for soil pH, OM, and K, in this study. Similar results were reported by Rodriquez-Gonzales et al. [53]. While soil pH and cation exchange capacity affected the toxic effect of clarithromycin on the soil bacterial community, the effects of soil nutrients were not clear.

The findings of this research study prove that there is a risk for the soil nitrification process in case of the simultaneous occurrence of Cd and AZI, selected to represent macrolide antibiotics in soil. Inhibition of PNRs of the tested soils has proved the toxic effects of Cd and AZI on AOB, which was influenced by the soil type. The PNR inhibitory effect was determined in the order of AZI < Cd < pH. Soil samples containing higher OM and neutral pH were determined to have higher PNRs. Soil samples, which had higher PNRs, were more resistant to the toxic effects of Cd and AZI. It was concluded that soils with poor PNR had higher risks of toxic effects.

5. Conclusions

Approximately 140 tons/day of treated sewage sludge with 1.5 mg/kg dm AZI content has been periodically used for soil amendment in Konya City. Farmers could apply various amounts of sewage sludge to their soils with no limitation. Since a significant Cd pollution (8.23–11.6 mg/kg dm) was determined in the region, a risk for soil PNR inhibition was clear according to the findings of this study. While the inhibition of PNR was relatively low for soils with neutral pH values, 50% PNR inhibition was determined for environmentally relevant concentrations of Cd and AZI for alkaline soil samples. This means that half of the nitrogen fertilizers applied to the soil could be wasted in alkaline soils. Besides, neither soil has sole Cd contamination, nor sewage sludge the only AZI content. The simultaneous occurrence of various heavy metals and pharmaceuticals has been ignored. Pharmaceuticals were not included in the regulations on applying sewage sludge to the soil, and the danger of synergistic effects with heavy metals has not been recognized. While the sewage sludge is applied to the soil to benefit from its nutrient content, it may adversely affect the nitrogen fertilizer utilization efficiency of the plants. Replicated annual applications may reduce soil fertility. This study aims to draw attention to this issue.