**7. Antibiotics and hormones**

Sewage sludge (SS) obtained from waste water treatments plants contains organic matter and nutrients that, when properly treated, can be a valuable and safe resource for agriculture production systems [97, 99, 100]. Kentucky State University has been a pioneer in the use of municipal SS for land farming. Extensive work has been conducted by Antonious for more than 20 years. Composted municipal SS was proven to be a valuable fertilizer for many vegetable crops, including potatoes, peppers, broccoli, squashes, tomatoes, eggplants, onions, melons, cabbages, kales, and collards. After monitoring the bioaccumulation of heavy metals in plants grown in native soil mixed with municipal SS, data revealed that most of the heavy metals concentrations in edible plants were below their permissible limits and the plants were safe for human consumption. One of the outputs of this work was the transfer of this technol‐ ogy to the organic growers. Currently, municipal SS is commercially available as an organic base fertilizer known as Louisville Green (www.louisvillegreen.com). The intensive research carried out at KSU revealed the safety of SS as nutrient‐rich materials resulting from the treatment of SS at the Metropolitan Water Plant Facility in Louisville, KY, (**Figure 1**).

**Heavy metal Treatment μg g-1**

170 Organic Fertilizers - From Basic Concepts to Applied Outcomes

Each concentration in the table is an average of three replicates ± standard error.

**6.1. Quantification of trace elements (Ni, Pb, Cd) in soil**

inductively coupled plasma (ICP) spectrometer [7, 14, 96, 97].

**6.2. Quantification of trace elements (Ni, Pb, Cd) in edible plants at harvest**

fruits of plants grown under four soil management practices.

environmental impact of contaminated soils.

**7. Antibiotics and hormones**

YW = yard waste; SS = sewage sludge; CM = chicken manure; and NM = no‐mulch bare soil.

 **fresh fruit BAF**

SS 0.014 ± 0.007 1.007 NM 0.012 ± 0.004 0.731 CM 0.012 ± 0.006 0.876

SS 0.037 ± 0.001 0.808 NM 0.012 ± 0.005 0.222 CM 0.035 ± 0.012 0.765

**Pb** YW 0.016 ± 0.009 0.316

**Table 2.** Concentrations and bioaccumulation factor (BAF) of seven heavy metals in pepper (*Capsicum annuum* L.)

To monitor the concentration of trace elements in soil following the incorporation of soil amendments, samples collected from field plots should be oven‐dried at 105°C and ground manually with a ceramic mortar and pestle to pass a 1 mm sieve. Ten ml of concentrated nitric acid (HNO3) is added to each 1‐g of sieved dry powder, and the mixture is allowed to stand overnight and then heated for 4 h at 125°C on a hot plate. This mixture should be diluted with double‐distilled water (50 mL) and filtered through filter paper No.1 before quantification of heavy metals. Mehlich‐3 extractable Cd, Ni, and Pb can be determined in soil extracts using

For the determination of trace elements in plant tissues, fruit samples of the growing plants of comparable size are collected at random, washed with deionized water, and dried in an oven at 65°C for 48 h. The dried samples should be grounded manually with ceramic mortar and pestle to pass through 1 mm sieve. Bioavailability of heavy metals can be defined as the total metals available in the soil, whereas the bioaccumulation factor (BAF) is defined as the ratio of the metal in the plant divided by total metal in the soil [98]. As described earlier, BAF values below 1 are desirable and present levels that do not pose human health hazards, while BAF values >1 would be less favorable. Assessing the bioavailability and speciation of trace elements in native soil and soil mixed with organic amendments is crucial to determining the

Sewage sludge (SS) obtained from waste water treatments plants contains organic matter and nutrients that, when properly treated, can be a valuable and safe resource for agriculture

There is an emerging concern regarding the impact of endocrine disrupting compounds (EDCs) in reclaimed water and biosolids [101]. EDCs are exogenous agents **(**agents from outside the organism or system) that have the potential to interfere with the production, release, transport, metabolism, binding, or elimination of the natural hormones responsible for the body regulation of developmental processes [102]. EDCs could be one or more of the following chemicals: pesticides, plasticizers, natural chemicals found in plants (phytoestro‐ gens), pharmaceutical products, or hormones that are excreted in animal or human waste. Natural and synthetic estrogens are some of the most potent EDCs found in municipal wastewater. EDCs have been attributed as a cause of reproductive disturbance in humans and wildlife. Human exposure to these chemicals in the environment is a critical concern with unknown long‐term impacts [103].

The group of molecules identified as endocrine disruptors is highly heterogeneous and can be classified into several categories, such as hormones (natural and synthetic estrogen or steroids), pharmaceutical and personal care products, industrial chemicals, pesticides, combustion byproducts, and surfactants [104]. There is an increasing evidence that EDCs poses a health risk in humans and animals. EDCs have been associated with adverse effects on reproduction, breast development and cancer, prostate cancer, neuroendocrinology, thyroid, metabolism and obesity, and cardiovascular endocrinology [102]. The primary source of EDCs is municipal SS. Other sources include industrial manufacturing processes and agricultural waste [105]. Wastewater treatment plants are generally not designed for the removal of trace organic compounds (i.e., detected concentration occurs at nanograms per liter) such as pharmaceuti‐ cals and potential EDCs [105]. The treatment efficiency of most pharmaceuticals and personal care products was as low as 35% [104]. Variations in wastewater treatment processes and operational conditions are generally regarded as the reason for fluctuations in removal efficiencies and effluent concentrations [101]. EDCs removal methods fall into three categories: physical removal, biodegradation, and chemical advanced oxidation. Biodegradation is the primary removal mechanisms for EDCs in activated sludge systems, which are commonly used biological treatment techniques for municipal wastewater treatment. About 90% of natural steroids are degraded in the activated sludge system [103].

Other technologies have been studied to remove the remaining EDCs. These technologies include chemical removal, activated carbon, chlorination, ozonation, ultraviolet (UV) irradia‐ tion, and membrane separation [101]. Treatment plants are seeking viable alternatives to alleviate concerns over cost, energy consumption, and brine disposal [105]. The use of ozone is a unique option because it is a highly effective oxidant for removing the majority of trace organic contaminants, particularly the steroid hormones, and because it has potential for reduced energy and chemical requirements [106]. Despite the effectiveness of ozone, there are fewer than 10 recycled water facilities (RWF) in the US that currently use ozone [105]. Some plants use UV treatment alone which has not shown to be an economically reasonable option for removing estrogens from wastewater. The application of advanced oxidation processes such as photo‐oxidation which combines UV irradiation with ozone has achieved a high removal efficiency of EDCs [102]. EDCs are ubiquitous compounds with small molecular mass (<1000 Daltons), present in the range of ng L-1. These compounds are biologically active, and their trace level concentrations make the detection and analysis procedures very challenging [104]. Highly sensitive measurements are necessary, including chemical monitoring, such as liquid chromatography‐tandem mass spectrometry, gas chromatography‐tandem mass spectrometry, high‐performance liquid chromatography, and sensitive bioassay [103]. Determining the presence of EDCs in reclaimed water and municipal SS is important, because these contaminants may be introduced into the food chain through bioaccumulation. The use of SS and reclaimed water in agriculture is expected to improve rural communities and provides food crops that are consumer safe with little or no contaminants. Eleven EDCs (atrazine, bisphenol A, linuron, 4‐nonylphenol, butylbenzyl phthalate, diethylhexyl phthalate, 17β‐estradiol, 17α‐ethynylestradiol, estrone, octylphenol, and triclosan) should be monitored prior to land application. In addition, contaminant metals (arsenic, cadmium, lead, mercury, nickel, and zinc) should be also monitored. Data of this type of research are critical for understanding the behavior and potential accumulation of EDCs and heavy metals within the particular food, when reclaimed water and biosolids [107] are used for growing edible plants.

#### **8. Microorganisms and soil enzymes**

Government agencies, research scientists, farmers, and all citizens are looking for a healthy environment. Soil microorganisms are present in low amounts; however, they have a signifi‐ cant role in nutrient recycling to maintain the NPK, the main plant nutrients at the required level. Microorganism's biomass is often related to crop type, soil type, and landscape [108, 109]. Soil fungi usually constitute 75–95% of the soil microbial biomass and when we include bacteria, they are responsible for about 90% of the total energy flux of organic matter miner‐ alization in soil [110]. Among the main groups of microorganisms living with plant roots are saprotrophic fungi and mycorrhiza. Urease, invertase, phosphatase, cellulose, dehydrogenase, and amylase are among enzymes secreted by soil microorganisms (bacteria, fungi, protozoa, algae). These enzymes are responsible for degrading complex forms of organic matter and xenobiotics in soil and water ecosystems. Polysaccharides (sticky substances) are also pro‐ duced by soil microorganisms. These sticky substances play a significant role in sticking and adhering soil particles together and help the soil to resist erosion that can reduce agricultural productivity [111].

Other technologies have been studied to remove the remaining EDCs. These technologies include chemical removal, activated carbon, chlorination, ozonation, ultraviolet (UV) irradia‐ tion, and membrane separation [101]. Treatment plants are seeking viable alternatives to alleviate concerns over cost, energy consumption, and brine disposal [105]. The use of ozone is a unique option because it is a highly effective oxidant for removing the majority of trace organic contaminants, particularly the steroid hormones, and because it has potential for reduced energy and chemical requirements [106]. Despite the effectiveness of ozone, there are fewer than 10 recycled water facilities (RWF) in the US that currently use ozone [105]. Some plants use UV treatment alone which has not shown to be an economically reasonable option for removing estrogens from wastewater. The application of advanced oxidation processes such as photo‐oxidation which combines UV irradiation with ozone has achieved a high removal efficiency of EDCs [102]. EDCs are ubiquitous compounds with small molecular mass (<1000 Daltons), present in the range of ng L-1. These compounds are biologically active, and their trace level concentrations make the detection and analysis procedures very challenging [104]. Highly sensitive measurements are necessary, including chemical monitoring, such as liquid chromatography‐tandem mass spectrometry, gas chromatography‐tandem mass spectrometry, high‐performance liquid chromatography, and sensitive bioassay [103]. Determining the presence of EDCs in reclaimed water and municipal SS is important, because these contaminants may be introduced into the food chain through bioaccumulation. The use of SS and reclaimed water in agriculture is expected to improve rural communities and provides food crops that are consumer safe with little or no contaminants. Eleven EDCs (atrazine, bisphenol A, linuron, 4‐nonylphenol, butylbenzyl phthalate, diethylhexyl phthalate, 17β‐estradiol, 17α‐ethynylestradiol, estrone, octylphenol, and triclosan) should be monitored prior to land application. In addition, contaminant metals (arsenic, cadmium, lead, mercury, nickel, and zinc) should be also monitored. Data of this type of research are critical for understanding the behavior and potential accumulation of EDCs and heavy metals within the particular food, when reclaimed water and biosolids [107] are used for growing edible plants.

Government agencies, research scientists, farmers, and all citizens are looking for a healthy environment. Soil microorganisms are present in low amounts; however, they have a signifi‐ cant role in nutrient recycling to maintain the NPK, the main plant nutrients at the required level. Microorganism's biomass is often related to crop type, soil type, and landscape [108, 109]. Soil fungi usually constitute 75–95% of the soil microbial biomass and when we include bacteria, they are responsible for about 90% of the total energy flux of organic matter miner‐ alization in soil [110]. Among the main groups of microorganisms living with plant roots are saprotrophic fungi and mycorrhiza. Urease, invertase, phosphatase, cellulose, dehydrogenase, and amylase are among enzymes secreted by soil microorganisms (bacteria, fungi, protozoa, algae). These enzymes are responsible for degrading complex forms of organic matter and xenobiotics in soil and water ecosystems. Polysaccharides (sticky substances) are also pro‐ duced by soil microorganisms. These sticky substances play a significant role in sticking and

**8. Microorganisms and soil enzymes**

172 Organic Fertilizers - From Basic Concepts to Applied Outcomes

**Figure 5.** Kale and collard plants grown in soil amended with sewage sludge applied at 15 t acre-1 at Kentucky State University Harold Benson Research and Demonstration Farm (Franklin County, KY, USA).

**Figures 5** and **6** revealed that kale, collard, and pepper were successfully grown in SS and CM amended soils that contained greater soil urease and invertase activities. On the contrary yard waste (**Figure 4**) used at KSU Research Farm revealed that, its application in spring broccoli did not alter soil urease or invertase activities to any appreciable extent (data not shown).

SS increased soil urease and invertase activity (**Figure 7**). Urease is an enzyme that depends on Ni for its activity [112]. Accordingly, Ni in SS might be the cause of elevated urease activity. This increase could be due to the presence of urea, the substrate of the enzyme urease. SS obtained from municipal plants contains great amounts of enzymatic substrates [113]. SS used at KSU for growing many vegetable crops including broccoli contained 1.2 μg Ni g-1 dry soil. However, broccoli plants showed normal growth in the field without any apparent symptoms of Ni toxicity or deficiency. Results indicated that the addition of SS to native soils has increased total crop marketable yield compared to no‐mulch native soils (data not shown). Indicating that incorporation of organic materials, such as municipal SS, into soil promotes microbiolog‐ ical activity. Microbial activity and soil fertility are closely related because it is through the biomass that the soil mineralization of the important organic elements (C, P, and N) occurs. Accordingly, soil biological monitoring is a potential and sensitive indicator of soil ecological stress for early restoration. **Figure 7** revealed that soil invertase and urease activities were increased by 89 and 47%, respectively, in SS compared to native soil.

**Figure 6.** Hot pepper plants grown in soil amended with chicken manure applied at 15 t acre-1 at Kentucky State Uni‐ versity Harold Benson Research and Demonstration Farm (Franklin County, KY, USA).

**Figure 7.** Invertase activity expressed as mg glucose released g-1 dry soil and urease activity expressed as mg NH4–N released g-1 dry soil h-1.

Tabatabi and Bremner [114] used a simple method for the quantification of urease activity in soil. In their method, they used a few grams of soil (about 5 g) and added 10 mL of 0.1 M phosphate buffer in a volumetric flask kept in water bath at 30°C for 1 h to allow the soil temperature to equilibrate. The liberated NH4 <sup>+</sup> ions were determined by the selective electrode method [115]. For standardization, a series of standard solutions of NH4 Cl covering the concentrations of 0.1–100 μg NH4–N mL-1 of water was prepared. In this method, urease activity was expressed as mg NH4–N released g-1 dried soil during the 1 h incubation at 30°C [17]. For invertase quantification in soil, the method described by Balasubramanian et al. [116] was used. For standardization, a calibration curve was obtained using analytical grade glucose in the range of 10–50 μg mL-1 glucose standards.

Addition of SS, as a source of organic matter, to agricultural soil reduced the C/N ratio (**Table  3**). Kizilkaya and Bayrakli [117] added nitrogen as ammonium sulfate, (NH4)2 SO4, to reduce the C/N ratio in soil from 9:1 to 6:1 and 3:1. This resulted in a rapid increase in soil enzymatic activities. Investigators [118, 119] reported that reducing the C/N ratio is indicative of a high OM decomposition rate. On the contrary, OM with high level of C/N ratios has relatively low rates of decomposition and causes low rates of N‐mineralization [120] in soil.


Statistical comparisons were done between three soil management practices (each replicated six times) for each soil parameter. Values in each row accompanied by the same letter are not significantly different (P > 0.05) using Duncan's multiple range test.

**Table 3.** Soil properties in the rhizosphere of broccoli plants grown in native soil amended with sewage sludge and soil amended with yard waste at KSU Harold Benson Research and Demonstration Farm, (Franklin County, Kentucky, USA).
