Studies exploring the phytotoxicity of individual pharmaceuticals or classes of pharmaceuticals are useful to highlight high-risk compounds and/or the potential mechanism of toxicity. CECs are, however, often introduced into the environment in complex mixtures and these mixtures can affect the uptake and translocation of individual compounds . Some studies report positive effects on plants exposed to CEC mixtures under environmentally relevant conditions. For instance, TWW irrigation increased tomato and lettuce yield compared to freshwater irrigation . Exposure of lettuce seedlings to a mixture of 11 CECs significantly altered plant metabolic pathways, including the citric acid cycle and pentose phosphate pathway, and decreased chlorophyll content in a dose-dependent manner . Also, exposure to 18 CECs at concentrations ranging from 5 to 50 µg L,-1 induced oxidative stress in cucumber seedlings and caused upregulation of enzymes associated with detoxification reactions . Literature on the toxicity of a number significant CECs to terrestrial plants is still very limited, and many of the studies have utilized concentrations that are orders of magnitude higher than those seen in the environment. Studies on the toxicity of mixtures in terrestrial plants are also limited, but warrant attention as several studies have indicated that mixtures can induce effects not observed from individual compounds . The ability of plants to detoxify these compounds through metabolism also merit further research. Overall, more research is needed on the toxicity of a wider range of CECs in plants under environmentally relevant conditions to more accurately assess the impacts of CECs in the agro-environment. The potential for exposure to, and toxicity of, CECs has been investigated in several aquatic invertebrate species. Toxicity end-points such as endocrine disruption, changes in growth, time to development, and mortality rates have been considered in these studies . Studies addressing the effects of CECs on terrestrial invertebrates are, however, few. Of the published studies on terrestrial invertebrates, the earthworm Eisenia fetida has been examined mainly due to their increased susceptibility andecological importance . Literature pertaining to toxicities of various classes of CECs to terrestrial invertebrates is discussed below. Like in terrestrial plants, antibiotics can also induce toxicity in terrestrial invertebrates. Exposure to environmentally relevant concentrations of antibiotics caused mortality to earthworms and/or induced oxidative stress and genotoxicity in E. fetida. For instance, high concentrations of tetracycline and chlortetracycline inhibited antioxidant enzymes superoxide dismutase and catalase while these enzymes were stimulated at lower doses , and DNA damage was induced along a dose-dependent curve . Also, chlortetracycline can reduced juvenile earthworm and cocoon counts in E. fetida .

Environmentally relevant concentrations of three antibiotics, lincomycin, ciprofloxacin, and oxytetracycline increased mortality and development time in cabbage loopers when reared on an artificial diet and treated tomato plants . Further, the three antibiotics altered the microbiome inside cabbage loopers and mosquitos but did not impact development time of mosquitoes . However, antibiotic exposure did not induce toxicity in aphids reared on bell peppers . Antibiotic toxicity in terrestrial invertebrates, therefore, appears to depends upon the specific antibiotics, concentrations, bioavailability, invertebrate species, and environmental conditions.Exposure to NSAIDs caused acute and sub-acute adverse effects in terrestrial invertebrates, including earthworms . Pino et al. assessed lethality of E. fetida cultivated in artificial soil as a result of exposure to 18 pharmaceuticals. Ibuprofen had the lowest LC50 at 64.8 mg kg-1 followed by diclofenac at 90.5 mg kg-1 . Exposure to diclofenac resulted in a dose-dependent decrease in survival and reproduction of Folsomia candida in spiked soils . However, it should be noted that these LC50 values were much higher than what may be expected in the real environment. At sub-acute concentrations , diclofenac induced significant genotoxicity in Folsomia candida, including induction of the up-regulation of transcriptional processes and genes associated with the immune response . Acetaminophen increased E. fetida mortality along both a dose-dependent curve and over time [7-28 d ]. In the mosquito species Culex quinquefasciatus, exposure to water contaminated with an environmentally relevant concentration of acetaminophen resulted in increased susceptibility to Bacillus thuringien israelensis and increased larval development time . Acetaminophen at environmentally relevant concentrations also significantly increased days to adulthood in cabbage loopers reared on an artificial diet. However, a similar effect was not observed when cabbage loopers were reared on acetaminophen-treated tomato plants . Similarly, the development time for aphids reared on acetaminophen treated bell pepper was not affected by acetaminophen . Therefore, like for other CECs, effeccts of NSAIDs on terrestrial invertebrates are species, compound, and environment specific.

Many antimicrobials and preservatives, including the common environmental contaminants triclocarban, triclosan, and parabens are amongst the most frequently detected in TWW and bio-solids . Partitioning of these CECs into bio-solids suggests that soil-dwelling organisms are at greater risks of exposure as they preferentially consume organic matter rich soils and bio-solids . Triclocarban, triclosan, and methyl-triclosan have been detected in the tissues of earthworms collected from field sites that were amended with bio-solids 4 years prior to the worm collection . After 28-d exposure to triclosan at ≥50 mg kg-1 in soil E. fetida had significantly increased SOD and CAT activities and increased concentrations of malondialdehyde , a chemical indicative of lipid peroxidation and DNA damage in E. fetida . Lin et al. reported negative impacts of triclosan exposure on E. fetida reproduction including decreases in the number of cocoons and juveniles. Triclosan also decreased the biomass, shell diameter,blueberries in containers growing and food intake in a terrestrial snail at concentrations ≥ 40 mg kg-1 . Further, triclosan exposure increased CAT and SOD activities and MDA concentration in A. fulica in a dose-dependent manner . However, no adverse effects were observed in E. fetida cultivated in triclosan-amended bio-solids at environmentally relevant concentrations . Triclocarban is more persistent in the environment than triclosan and is known to bio-accumulate in earthworm tissues . However, information on its toxicity to terrestrial invertebrates remains limited. For example, in Synder et al. exposure to triclocarban at concentrations ≥ 77 mg kg-1 for 2-4 weeks resulted in a trend towards increased mortality; however, the variations in data were too high to discern any statistically significant trend. Exposure ≥ 400 mg kg-1 to methyl paraben in soil resulted in increased abnormalities in earthworms where a normal survival-EC50 value of 397 mg kg-1 was estimated . An acute exposure to methyl paraben in soil at ≥ 60 mg kg-1 increased F. candida mortality and chronic exposure at concentrations ≥150 mg kg-1 decreased the reproductive rate . However, methyl paraben is often detected at concentrations ranging from 15.9 – 203.0 µg kg-1 in sewage sludge, levels that are well below the concentrations where toxicity was observed .The studies highlighted above suggest that CECs are ubiquitous in the environment and that exposure, even at environmentally relevant concentrations, these contaminants may be hazardous for terrestrial organisms. However, studies also suggest that these organisms can metabolize, transform and detoxify these CECs. The interplay between the toxicological effects of CEC exposure and an organism’s ability to take up and metabolize these contaminant is poorly understood and serves as significant knowledge gaps in understanding the fate and risks of CECs in terrestrial environments. These gaps must be addressed to gain better risk assessments of CECs during the use of bio-solids and treated wastewater in the agro-environment.

To address these gaps, we carried out a series of experiments utilizing plant cell cultures, hydroponic cultivations, earthworm incubations, high-resolution mass spectrometry, 14C-tracing, and enzyme assays to systematically evaluate the fate, metabolism, and biological effects of sulfamethoxazole, diazepam, naproxen, and methyl paraben and their major metabolites in terrestrial organisms under laboratory conditions. The four CECs were selected based on their detection in TWW and bio-solids, their range of physicochemical properties and uses, and the paucity of information about their fate and impacts in the literature . The study systems included Arabidopsis thaliana cells cultures, radishes, cucumbers, and E. fetida. Organisms were selected due to their extensive use in the literature, commercial availability, and worldwide agricultural relevance. Over the past two decades, pharmaceuticals and personal care products have emerged as contaminants of environmental concern due to their extensive use and continuous emission into the environment . PPCPs are released into the environment primarily through the disposal of treated wastewater and bio-solids from wastewater treatment plants . As climate change and population growth places an increasing stress on freshwater resources, especially in arid and semi-arid regions, communities have turned to utilizing municipal treated water for agricultural irrigation, which may result in soil contamination by PPCPs . Furthermore, the heavy use of some pharmaceuticals, particularly antibiotics, for disease control and growth promotion in intensive animal farming also contributes to contamination of agricultural fields when animal wastes are used for fertilization . The presence of PPCPs in irrigation water and soil can lead to contamination of food crops if plants can substantially accumulate these compounds. Various studies over the last decade have sought to quantify plant uptake of PPCPs, and in general, only low levels of PPCPs have been found in edible tissues . The majority of studies to date have only targeted the parent form of PPCPs for analysis. However, plants have a cascade of enzymes that may extensively transform xenobiotics such as PPCPs after uptake . Recently several published studies have explored the metabolism of pharmaceuticals in plants . Therefore, consideration of metabolism and biologically active metabolites is much needed for a better understanding of the fate and risks of PPCPs in the soil-plant system. Higher plants have many detoxification enzymes similar to those in animals. These enzymes function in plants as a ‘green liver’ . In general, metabolism of xenobiotics includes three phases. Phase I involves modification reactions such as oxidation, hydrolysis, and dealkylation reactions introducing reactive sites to the molecule. Phase II is characterized by conjugation with large polar bio-molecules, such as sugars and amino acids, to further increase the polarity of the xenobiotic. Phase III is typified by sequestration, resulting in the formation of bound residues . As shown for many xenobiotics in mammals and plants metabolites from phases I and II often retain biological activity , and therefore should not be discounted. In this study, sulfamethoxazole was selected as the compound of interest because of its prevalence in WWTP effluents and increasing concerns over the propagation of antibiotic resistance .