The land application of TWW and bio-solids can introduce contaminants of emerging concern into terrestrial environments . Consequently, a range of CECs have been detected in agricultural soils . Literature pertaining to the effects of CECs in terrestrial ecosystems is, however, limited . The majority of previous research has been concerned with the fate and effects of CECs in plants , and only a few studies have considered CECs in terrestrial invertebrates .Earthworms ameliorate agricultural soil structure through the formation of new aggregates and macropores; improving soil tilth, aeration, infiltration, and drainage . Furthermore, earthworms consume plant litter, recycle organic matter and aid in nutrient cycling . Earthworms dominate soil fauna with an average biomass of 10 – 200 g m-2 . Due to their ecological importance and abundance, earthworms are a good candidate for ecotoxicity testing . Herein we sought to understand some of the potential consequences of CECs exposure in earthworms. For this study, we selected the earthworm species Eisenia fetida as the test organism due to their widespread use in the scientific literature and extensive habitat range. For CECs, we considered three pharmaceuticals, i.e., naproxen, diazepam, and sulfamethoxazole,raspberry container growing and one cosmetic preservative, i.e., methyl paraben. These four compounds were selected due to their range of physicochemical properties and frequent detections in the environment . Naproxen is a commonly consumed nonsteroidal anti-inflammatory drug that has been often found in TWW and bio-solids . Diazepam is a psychoactive compound from one of the most commonly prescribed classes of pharmaceuticals of wastewater treatment plants.

Diazepam has also been frequently detected in TWW due to poor removal efficiency . Sulfamethoxazole is an antibiotic and has garnered significant scientific interest due to the growing concern over antibiotic resistance . Methyl paraben is a preservative that is widely used in various cosmetic products, is amongst the most frequently detected parabens in TWW and bio-solids, and is a known endocrine disruptor . Several studies have examined the fate and toxicity of these compounds in aquatic organisms . The observed adverse effects on aquatic organisms have raised concerns about the unintended consequences from widespread consumption and, ultimate release of the CECs into the aquatic environment . With the increasing use of TWW and bio-solids for agriculture, it is crucial to also understand the effects of these compounds on terrestrial organisms. In this study, we carried out laboratory experiments to assess the potential uptake, bio-transformation, and biochemical effects of CECs in earthworms. Eisenia fetida was exposed to the four CECs in an artificial soil, and kinetics of the parent compound, uptake and metabolite formation were evaluated. Changes in enzymes associated with oxidative stress and lipid peroxidation were assessed as biochemical markers of potential toxicity.Preliminary experiments were carried out to assess any potential mortality from the test compounds on E. fetida . The uptake and bio-accumulation of the test compounds followed OECD guideline on “Bio-accumulation in Terrestrial Oligochaetes.” Tests were performed in glass jars painted black and then white to reduce light and heat absorption. Jars contained 150 g ± 0.5 of artificial soil with 3 worms in each container. The worms were allowed to acclimate to the test conditions for 24 h before exposure to the test compounds. The incubation was carried out at room temperature. Prior to spiking, earthworms were removed from jars and soils were spiked with different volumes of the standard stock solutions to arrive at initial concentrations of 70, 50, 275, and 200 ng g-1 for sulfamethoxazole, diazepam, naproxen and methyl paraben, respectively.

Soils were moistened with deionized water, homogenized by stirring, and the earthworms were then reintroduced. Concentrations were selected based on environmental concentrations previously reported in the literature. Moisture content of 50% was selected based on preliminary experiments and maintained during the incubation. Jars containing the spiked soil without E. fetida and non-spiked soil with E. fetida were prepared and maintained simultaneously. At the start of the incubation, individual mature worms were maintained in separate jars to assess treatment-induced weight changes, if any. Samples were taken at 0 h, 1 d, 3 d, 7 d, 14 d, and 21 d of incubation. At each sampling time point, four treatment and four control jars were harvested for a total of twelve worms per time point per treatment. Worms were collected, rinsed with deionized water and placed in Petri dishes with a moistened paper towels for 24 h to purge their gut content. They were then weighed, frozen in liquid nitrogen and stored at -80 °C until extraction. The earthworms were homogenized with 8 mL acetonitrile:H2O for 5 min using a Kinematica™ Polytron PT 10/35 GT Benchtop Homogenizer . The CECs were extracted from the homogenized using 10 min of sonication, followed by 15 min of centrifugation at 15000 g. The supernatants were collected, dried under nitrogen, and reconstituted using 1.5 mL methanol:H2O . The reconstituted extracts were placed in LC-vials for analysis. Porewater was collected using 20 g of soil by centrifugation at 15000 g for 20 min, after which 2 mL of water was withdrawn and further centrifuged at 12000 g for 15 min. The resulting supernatant was used for instrument analysis. The soil was extracted by vortexing 10 g of soil with 10 mL acetonitrile:H2O for 5 min, followed by sonication for 20 min. Samples were centrifuged at 15000 g for 20 min, and the supernatant was collected, dried under nitrogen and reconstituted in 1.5 mL methanol:H2O .

All extracts were filtered using 0.2 µm PTFE syringe filters before instrument analysis. Extraction efficiencies were assessed using deuterated standards and are given in the Supplementary Information . The concentrations of CECs were monitored in three phases, soil, soil pore water, and earthworm tissue, in both the presence and absence of earthworms throughout the 21 d incubation. To determine the potential effect of earthworms’ presence on the partitioning of the four CECs amongst the soil and soil pore water the distribution coefficient was calculated at each time point and the differences between earthworm treatment and the non-earthworm controls were compared . No significant differences in the Kd values were observed between the earthworm treatment and non-earthworm controls for any of the CECs, indicating that earthworms did not significantly affect the association of these CECs to the solid phase of the artificial soil. For diazepam, the Kd values were calculated to range between 0.84 to 6.56 mL g -1 throughout the incubation. These Kd values were lower than those previously reported fordiazepam in batch and field sorption measurements using agriculture soils  but were in agreement with the low Kd values observed for pharmaceuticals in sandy soils . The low Kd values indicated that diazepam was not strongly adsorbed to the solid matrix of the artificial soil. The Kd for naproxen ranged between 0.87 to 11.0 mL g -1 throughout the incubation. The low Kd values were consistent with those previously reported for naproxen in sandy soils [0.49 mL g -1 ] The Kd values for sulfamethoxazole were similarly very low throughout the incubation, ranging from 0.71 to 1.75 mL g.-1 These values were consistent with those previously reported in the literature for grassland soils and arable land soils ,container grown raspberries indicative of its high mobility in the soil environment . The derived Kd values, for methyl paraben in the earthworm treatment and non-earthworm controls could be calculated only for the initial sampling point as it rapidly disappeared from the soil and soil pore water. This may be due to rapid bio-degradation in the soil and/or rapid metabolism in E. fetida . To verify active uptake of CECs by earthworms, a range of controls were used, including soil blanks and non-earthworm controls. None of the parent CECs were detected in the earthworm or soil blanks. However, degradation of both methyl paraben and sulfamethoxazole was observed in the non-earthworm soil, indicating that microbial and/or abiotic degradation of these compounds occurred in the media . The parent compounds of diazepam, sulfamethoxazole, and naproxen were detected in earthworms throughout the 21 d incubation, with the concentration of both diazepam and naproxen increasing to 53.8 ± 24 ng g-1 and 110 ± 25 ng g-1 , peaking at 14 d. These results suggested that both diazepam and naproxen were being taken up and accumulated in E. fetida . Sulfamethoxazole, on the other hand, appeared to have a relatively stable concentration in the earthworm tissues throughout the incubation .

However, this could be due to active metabolism of sulfamethoxazole in earthworm instead of limited uptake or accumulation. Methyl paraben was not quantifiable in earthworm tissues and was rapidly lost in the artificial soil. For each of the three quantifiable CECs in the earthworm tissues , the bio-concentration factor and bio-accumulation factor were calculated and compared for each time point. No significant differences in the BAF were observed for sulfamethoxazole or naproxen over the course of the incubation . However, the BAF for diazepam did significantly increase over time , indicating that the increased exposure time to soil pore water resulted an increased concentration of diazepam in the earthworm tissues, likely due to slower metabolism or time needed for reach equilibrium. The BCF for diazepam and naproxen did not significantly change throughout the incubation period . For sulfamethoxazole the a significant difference in BCF was observed between the 3 d and the 14 d sampling points, but no clear pattern in BCF values over time was discernable. A significant difference between BAF the BCF values were observed for sulfamethoxazole at 7 d . There was a trend towards a significantly higher BCF than BAF for diazepam throughout the incubation. The trend may be due to increased uptake of the compounds from the soil porewater by E. fetida, which was consistent with several previous studies that showed dermal absorption via water to be the primary route for uptake of contaminants by worms . However, due to a lack of quantifiable replicates in soil or soil porewater statistical significance could not be assessed. Further research is necessary to understand the exposure pathways for polar CECs for invertebrates such as in earthworms in soils. Intriguingly, it was also observed that all three quantifiable CECs displayed an similar pattern where there was an initial increase in BAF or BCF up to 3 or 7 d, followed by a decrease at 7 or 14 d, and followed by increases again till the end incubation . This pattern may be indicative of early uptake and metabolism, followed by an insufficient response from detoxification enzymes, resulting in storage and accumulation of the compounds in the earthworm tissues, as was previously observed in aquatic organisms . To the best of knowledge, this was the first time the BCF and BAF have been calculated in earthworms for naproxen, diazepam and sulfamethoxazole. While many studies have considered the bioconcentration/accumulation of different CECs in plants and earthworms , very few studies have considered the changes to these values over time. The results from this study showed that it may be valuable to evaluate the BCF or BAF of CECs in earthworms over time, as there may bean increased potential for biomagnification at a given time point. However, more research is needed to determine if these differences persist for a longer time scale and if they occur in different soils or for other CECs. Out of four major metabolites examined in this study only N4- acetylsulfamethoxazole was quantifiable in the earthworm tissues. N4- acetylsulfamethoxazole was seen to increase to a peak concentration of 4.39 ± 0.4 ng g-1 at 3 d and then decreased to 2.62 ± 0.01 ng g-1 by the end of the 21 d incubation . Further, N4-acetylsulfamethoxazole was also detected in the earthworm soil with the highest concentration observed at 7 d , indicating that earthworms was capable of metabolizing these CECs and excreting the transformation product into its surrounding environment. N4-acetylsulfamethoxazole is the primary metabolite of sulfamethoxazole in humans and has been previously detected in wastewater effluent, environmental samples and plant tissue . However this was, to the best of our knowledge, the first time that N4-acetylsulfamethoxazole has been observed to form in earthworms.