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, bio-availability, 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 ,gardening pots plastic 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, effects 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, 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 .Terrestrial invertebrates are more likely to be exposed to complex mixtures of CECs in the environments, including agricultural soils . However, studies addressing the effects of CEC mixtures on terrestrial invertebrates are scarce. E. fetida reared in bio solid-amended soils suffered a decrease in cocoon counts and a decrease in adult survival at 8 weeks with 4% bio solids amendment rates .
High, but environmentally relevant, rates of bio solids application also caused mortality in E. fetida and F. candida; even lower application rates hindered reproduction in both species . However, bio solids are extremely complex in composition with numerous other constituents, including metals and salts, and it is difficult to attribute their effects solely to CECs. Environmentally relevant concentrations of mixtures of antibiotics, hormones, acetaminophen, and caffeine were found to extend development time, increase mortality, and alter the microbiome of mosquitos and cabbage loopers . Collectively, the aforementioned studies suggest that CECs may have increased toxicity when present in mixtures, and adverse effects at environmentally relevant concentrations are likely, but need to be further confirmed.Currently, knowledge on the fate and toxicity of CECs in terrestrial invertebrates is still very preliminary. The limited studies conducted so far shown chemical-specific and species-specific effects of CECs in the environment. It is known that the bio-availability of these compounds and environmental conditions will play a large role in the overall accumulation and toxicity of the CECs in terrestrial invertebrates . However, information on bio-availability under differing environmental conditions is still lacking for most CECs. Similarly, the potential for trophic transfer warrants further investigation. Overall, more research involving a wider range of compounds, especially in the context of environmental relevance, is needed to more accurately assess the fate and toxicity of CEC in terrestrial invertebrates. 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,plastic pots with drainage holes 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 . Since its introduction in 1961 sulfamethoxazole has been widely prescribed due to its potency against both gram-positive and gram-negative bacteria . Currently, sulfamethoxazole has been detected from ng L-1 to µg L-1 in surface and effluent waters and µg kg-1 to mg kg-1 in soils and manure .Recent long-term studies of waste-water application under realistic field conditions have highlighted the potential for sulfamethoxazole to be taken up and translocated in crop plants, including to the fruit . The structures of sulfamethoxazole metabolites, including conjugates from Phase II metabolism, were identified using high-performance liquid chromatography coupled with time-of-flight high-resolution mass spectrometry and further quantified using ultra-high performance liquid chromatography in tandem with a triple quadrupole mass spectrometry . Furthermore, Phase III terminal products in the form of bound residues were quantified using 14C labeling. Arabidopsis thaliana cells were selected as the experimental organism due to their extensive use in the literature, commercial availability, and their membership in the commonly consumed Brassica family . Further, Arabidopsis thaliana plants are found worldwide under several common names and are consumed by a wide variety of animals as well as humans . Cucumber was selected in the hydroponic experiment due to the fact that it is often consumed raw, rapid growth, and amiability to soilless culture .Non-labeled sulfamethoxazole was purchased from MP Biomedicals . Sulfamethoxazole-d4 was purchased from C/D/N Isotopes and 14C-labeled sulfamethoxazole was obtained from American Radiolabeled Chemicals . Stock solutions of 14C-sulfamethoxazole and non-labeled sulfamethoxazole were prepared in methanol to reach a specific radioactivity of 1.2 × 103 dpm µL-1 and a chemical concentration of 1.0 mg mL-1 , respectively. HPLC grade acetonitrile and methanol were used for extraction along with ultra pure water. Mobile phases were prepared using Optima™ LC/MS grade methanol and deionized water. Standards were prepared in HPLC grade methanol and stored in the dark at -20 °C. All solvents used in this study were purchased from Fisher .PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at the Ohio State University . The cells were maintained in liquid suspension culture at 25 °C and rotated at 130 rpm in the dark according to the ARBC protocol . To explore metabolism of sulfamethoxazole in A. thaliana cells, 7 mL of cell culture was inoculated in 43 mL fresh culture and cultivated for 96 h at 25 °C and 130 rpm in the dark to produce the seed culture. A 30 µL aliquot of the non-labeled stock solution and 10 µL aliquot of 14C-sulfamethoxazole were spiked into 30 mL of A. thaliana cell culture, resulting in a nominal initial concentration of sulfamethoxazole of 1 µg mL-1 and a specific radioactivity of 1.2 × 103 dpm mL-1 . Simultaneously, control treatments were prepared by autoclaving cell suspensions before chemical spiking , flasks containing sulfamethoxazole without cells , and flasks containing live cells but no sulfamethoxazole . These control treatments were used to determine adsorption, abiotic degradation, and potential toxicity to cells.