Heavy Metal Accumulation in Soil and Water in Pilot Scale Rice Field Treated with Sewage Sludge

Widespread use of chemical fertilizers in agricultural activities poses a high risk of multi-micro metal contamination in soils and potentially causes health issues through consumption of contaminated foods. Bio-organic fertilizers from sewage sludge have been regarded as a suitable substitute for chemical fertilizer for rice farming. In this study, we investigated accumulation of heavy metals (Cu and Zn) in soil, water and rice plant in three pilot-scale rice paddy fields treated with different fertilization schemes. The control field was treated with conventional chemical fertilizers while the soil of two treatment fields was mixed with biological sewage sludge obtained from a local wastewater treatment system in Vietnam at different ratios (1% and 3%). Initial results showed that heavy metals accumulated in the soil, water, and rice plant at varying levels and most of the Cu and Zn contents found in soils, water and rice products exceeded permissible Vietnamese standards (QCVN 03: 2008) and US EPA 503. Notably, the rice field whose soil was treated with sludge at 3% ratio showed a significantly lower accumulation of heavy metals in soil, water and in rice plant. However, treatment of sludge at this level seemed to cause higher heavy metal retention in soil after one harvest. Semi-quantitative risk analysis revealed that the risk of metal contamination in soil and water of the control field ranged from medium (RQ index between 0.1 and 1) to high risk (RQ index higher than 1) and that fertilization methods would also affect the level of risk to the environment.


Introduction
Rapid industrialization and urbanization have released a wide array of pollutants into soils, severely affecting agricultural activities and food production chains [1]. Among contaminants, heavy metals are of increasing concern because their accumulation through complex pathways may disseminate into soil, therefore impairing soil and water quality. As a result, humans could be exposed to heavy metals through consumption of agricultural products grown on those contaminated lands [2][3][4]. This raises the need for close monitoring of soil metal accumulation in maintaining food safety and ensuring ecological sustainability [5].
The use of fertilizers is essential for providing adequate nutrients and ensuring successful harvests. However, continued use of fertilizers and metal-containing pesticides and fungicides might contribute to elevated levels of metals in soils [6]. Additionally, chemical composition and usage of fertilizers could be drastically varied depending on the source of raw material and the nature of the soil. The accumulation of metals such as Cu, Zn and Cd has been found to be attributable to heavy agrochemical soil treatment [6][7][8][9]. Recently, the agricultural practice of farmers has shifted to the use of more sustainable and environmentally friendly sources, such as sludge produced from Treatment Works Treating Domestic Sewage (TWTDS). The treatment of sewage or domestic wastewater in TWTDS generates a wide range of biosolids, which include solid, semisolid and liquid residues. As a result, in order to be qualified for beneficial reuse in agriculture, biosolids should be subjected to further treatment in accordance with USEPA standards. One of the main uses of biosolids is to provide nutrients to agricultural crops and to sustain land quality thanks to soil amendment properties of these residuals. However, biosolids often contain a wide range of microorganisms (bacteria, viruses and other pathogens), metals (e.g., cadmium and lead), toxic organic chemicals (e.g., PCBs) and important nutrients (e.g., nitrogen and phosphorus). Therefore, biosolids are recommended to be subjected to further regulations to ensure safe and effective use in agricultural applications.
This study based on US EPA 503 [10] and Vietnamese standards to evaluate and control the accumulation of heavy metals in soil, water and rice products cultivated in fields treated with sludge as a biofertilizer. This practice, if successfully adopted, could change the traditional farming practices of the farmer from the conventional spraying of chemical fertilizers to the mixing of bio-fertilizers into the soil before cultivation, thereby contributing to reduced use of dispersal fertilizer. To be specific, we evaluated the levels of heavy metal accumulation in the soil, water and agricultural products in two consecutive rice crops using sludge as biofertilizer. The results are expected to contribute to the development of a reasonable scheme of fertilizer use, thereby contributing to the balance of the ratio between biological fertilizers and chemical fertilizers.

Field Experiment
The field experiments were conducted at Chau Thanh district, Tay Ninh province, Vietnam, one of the regions with the largest rice production in Vietnam. The climate of the region was warm and humid. The experimental paddy fields were located near Vam Co Dong River and belong to a typical farmer who had over 30 years of experience in rice farming. The soil was acrisol, which was largely comprised of fine-and medium-sized sand particles (40-55%) and clay particles (21-27%). The experiment was evaluated in two crops (summer-autumn and autumn-winter) in 2014. The effective yield was assessed after the autumn-winter crop.
The field was divided into three square fields with each field having the area of 30 × 30 m. Each sub field represented one out of three possible control and treatments. In the first sub field (Treatment 1), 50 kg of treated sludge [11], obtained from Binh Hung wastewater treatment plant [12], were mixed in the surface soil of the field at the beginning of the rice cultivation period. Treatment 2 was prepared similarly but with the weight of the sludge increased to 150 kg. The soil in the control field was not added with sludge. Time rice fertilization was performed as shown in Figure 1.
ChemEngineering 2021, 5, x FOR PEER REVIEW 2 of 8 and environmentally friendly sources, such as sludge produced from Treatment Works Treating Domestic Sewage (TWTDS). The treatment of sewage or domestic wastewater in TWTDS generates a wide range of biosolids, which include solid, semisolid and liquid residues. As a result, in order to be qualified for beneficial reuse in agriculture, biosolids should be subjected to further treatment in accordance with USEPA standards. One of the main uses of biosolids is to provide nutrients to agricultural crops and to sustain land quality thanks to soil amendment properties of these residuals. However, biosolids often contain a wide range of microorganisms (bacteria, viruses and other pathogens), metals (e.g., cadmium and lead), toxic organic chemicals (e.g., PCBs) and important nutrients (e.g., nitrogen and phosphorus). Therefore, biosolids are recommended to be subjected to further regulations to ensure safe and effective use in agricultural applications. This study based on US EPA 503 [10] and Vietnamese standards to evaluate and control the accumulation of heavy metals in soil, water and rice products cultivated in fields treated with sludge as a biofertilizer. This practice, if successfully adopted, could change the traditional farming practices of the farmer from the conventional spraying of chemical fertilizers to the mixing of bio-fertilizers into the soil before cultivation, thereby contributing to reduced use of dispersal fertilizer. To be specific, we evaluated the levels of heavy metal accumulation in the soil, water and agricultural products in two consecutive rice crops using sludge as biofertilizer. The results are expected to contribute to the development of a reasonable scheme of fertilizer use, thereby contributing to the balance of the ratio between biological fertilizers and chemical fertilizers.

Field Experiment
The field experiments were conducted at Chau Thanh district, Tay Ninh province, Vietnam, one of the regions with the largest rice production in Vietnam. The climate of the region was warm and humid. The experimental paddy fields were located near Vam Co Dong River and belong to a typical farmer who had over 30 years of experience in rice farming. The soil was acrisol, which was largely comprised of fine-and medium-sized sand particles (40-55%) and clay particles (21-27%). The experiment was evaluated in two crops (summer-autumn and autumn-winter) in 2014. The effective yield was assessed after the autumn-winter crop.
The field was divided into three square fields with each field having the area of 30 × 30 m. Each sub field represented one out of three possible control and treatments. In the first sub field (Treatment 1), 50 kg of treated sludge [11], obtained from Binh Hung wastewater treatment plant [12], were mixed in the surface soil of the field at the beginning of the rice cultivation period. Treatment 2 was prepared similarly but with the weight of the sludge increased to 150 kg. The soil in the control field was not added with sludge. Time rice fertilization was performed as shown in Figure 1.

Materials
The biological fertilizer used in the study was derived from sewage sludge after the centrifuge dewatering process of the Binh Hung wastewater treatment plant. The sludge was mixed in a 2:0.5 ratio with rice husk and then incubated anaerobically under mesophilic conditions (temperature of 30-35 • C) for about 60 days. The doses of biological fertilizer used in experiments were estimated, corresponding to the ratio of fertilizer and soil weight of 1% and 3%, respectively for Treatments 1 and 2. Chemical fertilizers were used in the study were NPK, and foliar fertilizer (Canximax brand) from local fertilizer shops.

Methods of Analysis and Sampling
Twelve soil samples, six water samples in the field and six agricultural product samples (leaf, trunks and grains of the rice plant) were taken at the end of the summer-autumn and autumn-winter crops collection in three experimental models. Samples were taken according to the method ISO 5297:1995 (soil) at two depths (0-15 cm and 15-30 cm) and ISO 5992:1995 (water) in a paddy field. Heavy metals were extracted from soil samples by the method TCVN 6649:2000 (ISO 11466:1995, Soil quality-Extraction of trace elements in aqua regia). The content of heavy metals was analyzed by TCVN 6193-1996 (ISO 8288-1986) with the method of flame atomic absorption AAS machine and ICP-MS machine. Specific methods for each heavy metal element such as Pb, Cu, Zn, Cd and Ni are EPA 3050 SMEWW 3120, 2012, EFA SW 846, method 3050B and TCVN 6647:2007, respectively.

Risk Assessment
Risks impact on the environment and human health will be calculated according to the semi-quantitative method [13,14]. The semi-quantitative risk was calculated using the formula RQ = MEC/PNEC, MEC (measured environmental concentration) and PNEC (worst-case predicted no effect concentration). The level of risk depends on the outcome RQ index calculation in which RQ levels ranging from 0.01 to 0.1 is classified as low risk, from 0.1-1 as average risk and higher than 1 as high risk [15].
The results of RQ calculation are calculated through formulas and graphical representations by Excel 2016.

Accumulation of Heavy Metals in Sludge
The results of heavy metals concentrations in the experimental chemical and biofertilizers are shown in Table 1 and compared to TCVN 562:2002 MARD standard (Vietnamese Ministry of Agriculture and Rural Development-Industry standards on microbial organic fertilizers from domestic waste). Both used fertilizers contain a sufficient amount of Pb, Cd and Ni. Bio-fertilizer contains Pb (67.96 mg/kg), Cd (1.515 mg/kg) and Ni (27.45 mg/kg). These heavy metal concentrations are much lower (5-50 times) than allowable standards based on the National Technical Regulation on Hazardous Thresholds for Sludges (QCVN 50:2013/BTNMT) with thresholds being Pb < 300 mg/kg, Cd < 10 mg/kg and Ni < 1400 mg/kg. For reference, pollutant limits of US EPA 503 (Ceiling Concentration Limits for All Biosolids Applied to Land) of Cd, Lead, Ni, Zn, and Cu are 85, 840, 420, 7500 and 4300 mg/kg, respectively. Although these metals are highly toxic and have the potential to cause cancer, their accumulated concentration in fertilizers is shown to be low and could be further reduced after applying into the field at low dosage. On the other hand, the contents of Cu and Zn present in the biofertilizer have exceeded the permissible TCVN standard. Therefore, in subsequent analyses, we focused on quantifying the accumulation of Cu and Zn in the soil and water. Most of the previous studies indicate that crop production benefits from the use of soil sludge, but the presence of toxic metals such as Cd, Ni, Pb, and Zn can accumulate in plant tissues and can contaminate the food chain [16][17][18].
The results on the accumulation of heavy metals in the fertilizer materials of urban sewage sludge, predominated by Cu (105-1058 mg/kg) and Zn (134-1268 mg/kg), are also consistent with the results of some studies such as Saha (2017) and Smith (2009) [19,20]. Those studies also showed that due to the characteristic of most urban wastewater, highly toxic heavy metals such as Cd, Pb, Ni, Cr exist but with negligible concentrations, hardly exceeding the allowable limit standard US EPA 503 as information in Figure 2. Thus, concerns for municipal sludge when applied to agriculture should focus mainly on Zn and Cu metals.  Most of the previous studies indicate that crop production benefits from the use of soil sludge, but the presence of toxic metals such as Cd, Ni, Pb, and Zn can accumulate in plant tissues and can contaminate the food chain [16][17][18].
The results on the accumulation of heavy metals in the fertilizer materials of urban sewage sludge, predominated by Cu (105-1058 mg/kg) and Zn (134-1268 mg/kg), are also consistent with the results of some studies such as Saha (2017) and Smith (2009) [19,20]. Those studies also showed that due to the characteristic of most urban wastewater, highly toxic heavy metals such as Cd, Pb, Ni, Cr exist but with negligible concentrations, hardly exceeding the allowable limit standard US EPA 503 as information in Figure 2. Thus, concerns for municipal sludge when applied to agriculture should focus mainly on Zn and Cu metals.

Heavy Metal Residues after Two Harvests
Zn content measured in soils collected at different depths in three experimental rice fields after the first and the second rice harvest is shown in Figure 3. After the first harvest, the topsoil in the control field accumulated around 239.288 mg/kg of Zn, which was the highest and far exceed the QCVN 03-standard (Vietnamese National technical regulation on the allowable limits of heavy metals in the soils), and Treatment 2 showed the lowest topsoil Zn content. For deep soil, Zn accumulation in the field with Treatment 2 was slightly higher than the QCVN standard. Zn accumulation in water measured in the control and the Treatment 1 field was generally higher than that of Treatment 2. This could be due to the more intensive use of chemical fertilizers during rice cultivation in this Treatment. After the second harvest, Zn accumulation increased in the topsoil in the control and Treatment 1 but then decreased in the Treatment 2. In contrast, the deep soil, collected at a depth from 15 to 30 cm, showed significantly higher Zn accumulation than the topsoil due to the deposit of fertilizers.

Heavy Metal Residues after Two Harvests
Zn content measured in soils collected at different depths in three experimental rice fields after the first and the second rice harvest is shown in Figure 3. After the first harvest, the topsoil in the control field accumulated around 239.288 mg/kg of Zn, which was the highest and far exceed the QCVN 03-standard (Vietnamese National technical regulation on the allowable limits of heavy metals in the soils), and Treatment 2 showed the lowest topsoil Zn content. For deep soil, Zn accumulation in the field with Treatment 2 was slightly higher than the QCVN standard. Zn accumulation in water measured in the control and the Treatment 1 field was generally higher than that of Treatment 2. This could be due to the more intensive use of chemical fertilizers during rice cultivation in this Treatment. After the second harvest, Zn accumulation increased in the topsoil in the control and Treatment 1 but then decreased in the Treatment 2. In contrast, the deep soil, collected at a depth from 15 to 30 cm, showed significantly higher Zn accumulation than the topsoil due to the deposit of fertilizers. ChemEngineering 2021, 5, x FOR PEER REVIEW 5 of 8 Figure 3. Zn in soil, water in paddy field, farm produce over two rice crops. Figure 4 indicates that the control and Treatment 1 field show considerably higher Cu content in topsoil than that in Treatment 2. However, for soils collected at higher depth, Treatment 2 accumulated excessively high Cu, possibly because the metal content in the fertilizer was not washed away during irrigation and rainfall or due to accumulation from other unknown sources. The rice harvested in Treatment 2 seemed to have a lesser accumulation of Cu compared with that of other experimental fields. Compared to the control, Treatment 1 had reduced Cu accumulation in the soil and water, possibly due to reduced fertilization. In general, the highest heavy metal content was found in the topsoil of the control sample, followed by topsoil in Treatment 2 field and then in the Treatment 1. For deep soil, Treatment 2 showed the highest level of accumulation of heavy metals. Current results also revealed that later heavy metal contents in the soil after a later harvest season seemed to be higher than those in a prior harvest. This finding is corroborated by a previous study where high Cu, Pb and Zn contents were detected in permanent rice fields in Dong Thap province, Vietnam [21].

Risk Assessment of Heavy Metal Residues in the Environment
Certain heavy metals play an important role in the lives of organisms and are known as micronutrients. However, the borderline between heavy metal sufficiency and heavy metal excess is often very subtle and some heavy metals could be toxic to soil organisms [22]. According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific,  Figure 4 indicates that the control and Treatment 1 field show considerably higher Cu content in topsoil than that in Treatment 2. However, for soils collected at higher depth, Treatment 2 accumulated excessively high Cu, possibly because the metal content in the fertilizer was not washed away during irrigation and rainfall or due to accumulation from other unknown sources. The rice harvested in Treatment 2 seemed to have a lesser accumulation of Cu compared with that of other experimental fields. Compared to the control, Treatment 1 had reduced Cu accumulation in the soil and water, possibly due to reduced fertilization.
ChemEngineering 2021, 5, x FOR PEER REVIEW 5 of 8 Figure 3. Zn in soil, water in paddy field, farm produce over two rice crops. Figure 4 indicates that the control and Treatment 1 field show considerably higher Cu content in topsoil than that in Treatment 2. However, for soils collected at higher depth, Treatment 2 accumulated excessively high Cu, possibly because the metal content in the fertilizer was not washed away during irrigation and rainfall or due to accumulation from other unknown sources. The rice harvested in Treatment 2 seemed to have a lesser accumulation of Cu compared with that of other experimental fields. Compared to the control, Treatment 1 had reduced Cu accumulation in the soil and water, possibly due to reduced fertilization. In general, the highest heavy metal content was found in the topsoil of the control sample, followed by topsoil in Treatment 2 field and then in the Treatment 1. For deep soil, Treatment 2 showed the highest level of accumulation of heavy metals. Current results also revealed that later heavy metal contents in the soil after a later harvest season seemed to be higher than those in a prior harvest. This finding is corroborated by a previous study where high Cu, Pb and Zn contents were detected in permanent rice fields in Dong Thap province, Vietnam [21].

Risk Assessment of Heavy Metal Residues in the Environment
Certain heavy metals play an important role in the lives of organisms and are known as micronutrients. However, the borderline between heavy metal sufficiency and heavy metal excess is often very subtle and some heavy metals could be toxic to soil organisms [22]. According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific, In general, the highest heavy metal content was found in the topsoil of the control sample, followed by topsoil in Treatment 2 field and then in the Treatment 1. For deep soil, Treatment 2 showed the highest level of accumulation of heavy metals. Current results also revealed that later heavy metal contents in the soil after a later harvest season seemed to be higher than those in a prior harvest. This finding is corroborated by a previous study where high Cu, Pb and Zn contents were detected in permanent rice fields in Dong Thap province, Vietnam [21].

Risk Assessment of Heavy Metal Residues in the Environment
Certain heavy metals play an important role in the lives of organisms and are known as micronutrients. However, the borderline between heavy metal sufficiency and heavy metal excess is often very subtle and some heavy metals could be toxic to soil organisms [22]. According to Krivokapic, the toxicity of heavy metals against soil organisms could be ranked following the descending order: Hg > Cd > Cu > Zn > Pb [23]. Heavy metals also contribute to controlling pesticides and pathogenic microorganisms [24]. To be specific, high concentrations of Cu have been to be responsible for the reduction of the number of bacteria and nematodes [25], and high accumulation of Zn would reduce the number of arthropodae, especially mites, fungi and bugs such as brown planthopper [26]. Contamination of Cu in the soil might also lead to reduced microbial diversity and also to reduced soil microbial biomass from 44% to 36% compared with unpolluted soil [27]. In this analysis, the risk of Zn ( Figure 5) and Cu ( Figure 6) contamination in soil and water of paddy rice fields was calculated and the results are expressed as follows.
ChemEngineering 2021, 5, x FOR PEER REVIEW 6 of 8 high concentrations of Cu have been to be responsible for the reduction of the number of bacteria and nematodes [25], and high accumulation of Zn would reduce the number of arthropodae, especially mites, fungi and bugs such as brown planthopper [26]. Contamination of Cu in the soil might also lead to reduced microbial diversity and also to reduced soil microbial biomass from 44% to 36% compared with unpolluted soil [27]. In this analysis, the risk of Zn ( Figure 5) and Cu ( Figure 6) contamination in soil and water of paddy rice fields was calculated and the results are expressed as follows.  Semi-quantitative risk assessment of Zn content in soil indicated that the risk indexes were above-average risk in either or both water and soil assessments in Figure 5, especially with more heavy use of chemical fertilizers in the control field. Regarding Cu contamination risk ( Figure 6) in soil, the deep, continuously mixed soil in Treatment 2 field seemed to accumulate the highest Cu content, much higher than the risk standard, and Treatment 1 showed moderate risk with relatively lower Cu accumulation than the remaining samples. Regarding Cu accumulation in water, Treatment 2 seemed to fulfil the low-risk standard while the risk index of Treatment 1 seemed to fluctuate around the medium risk threshold. The Cu content in water in control was much higher than the medium risk threshold.
In general, the calculated risk figures suggested that Treatment 2 offered a relatively safe aquatic environment with very low Cu and Zn concentrations. This could be explained by the method of biosludge application of Treatment 2, which is to mix with soil before sowing, and the absence of chemical spraying during cultivation, suggesting that fertilization methods would also affect the level of risk to the environment. high concentrations of Cu have been to be responsible for the reduction of the number of bacteria and nematodes [25], and high accumulation of Zn would reduce the number of arthropodae, especially mites, fungi and bugs such as brown planthopper [26]. Contamination of Cu in the soil might also lead to reduced microbial diversity and also to reduced soil microbial biomass from 44% to 36% compared with unpolluted soil [27]. In this analysis, the risk of Zn ( Figure 5) and Cu ( Figure 6) contamination in soil and water of paddy rice fields was calculated and the results are expressed as follows.  Semi-quantitative risk assessment of Zn content in soil indicated that the risk indexes were above-average risk in either or both water and soil assessments in Figure 5, especially with more heavy use of chemical fertilizers in the control field. Regarding Cu contamination risk ( Figure 6) in soil, the deep, continuously mixed soil in Treatment 2 field seemed to accumulate the highest Cu content, much higher than the risk standard, and Treatment 1 showed moderate risk with relatively lower Cu accumulation than the remaining samples. Regarding Cu accumulation in water, Treatment 2 seemed to fulfil the low-risk standard while the risk index of Treatment 1 seemed to fluctuate around the medium risk threshold. The Cu content in water in control was much higher than the medium risk threshold.
In general, the calculated risk figures suggested that Treatment 2 offered a relatively safe aquatic environment with very low Cu and Zn concentrations. This could be explained by the method of biosludge application of Treatment 2, which is to mix with soil before sowing, and the absence of chemical spraying during cultivation, suggesting that fertilization methods would also affect the level of risk to the environment. Semi-quantitative risk assessment of Zn content in soil indicated that the risk indexes were above-average risk in either or both water and soil assessments in Figure 5, especially with more heavy use of chemical fertilizers in the control field. Regarding Cu contamination risk ( Figure 6) in soil, the deep, continuously mixed soil in Treatment 2 field seemed to accumulate the highest Cu content, much higher than the risk standard, and Treatment 1 showed moderate risk with relatively lower Cu accumulation than the remaining samples. Regarding Cu accumulation in water, Treatment 2 seemed to fulfil the low-risk standard while the risk index of Treatment 1 seemed to fluctuate around the medium risk threshold. The Cu content in water in control was much higher than the medium risk threshold.
In general, the calculated risk figures suggested that Treatment 2 offered a relatively safe aquatic environment with very low Cu and Zn concentrations. This could be explained by the method of biosludge application of Treatment 2, which is to mix with soil before sowing, and the absence of chemical spraying during cultivation, suggesting that fertilization methods would also affect the level of risk to the environment.

Conclusions
This study aimed to measure the accumulation of Cu and Zn in soil and water of rice paddy fields under treatment of bio-sludge in lieu of conventional fertilizers. The results revealed a considerably higher accumulation of Cu and Zn in deep soils of the field added with a high proportion of bio-sludge (Treatment 2), possibly due to the penetration and retention of the metal content from sludge at the lower soil layer. However, Treatment 2 seemed to result in significantly lower Cu and Zn concentrations measured in a water environment, suggesting that one-time bio-sludge addition into the soil at the beginning of the rice sowing might limit the dissemination of heavy metals into the water. The heavy metal residuals on the leaves and stalks of rice harvested in the field of Treatment 2 were also the lowest. Risk assessment results indicate a high risk of Cu contamination in deep soils of Treatment 2. Hence, to minimize risks and reduce diseases, it is recommended to combine two chemicals with biological fertilizers and adopt a reasonable fertilization method for limiting the concentration of heavy metals.