The results of acute toxicity (7-d and 14-d LC50) of PHE toward adult E. fetida in natural medium are exhibited in Table S3. In the control groups, the mortality rate during the test period of exposure was below 5%. As shown in Table S3, PHE caused lethal toxicity at 103.41 mg kg−1 of the LC50 value after 7-d exposure. At the same time, Nam et al. (2017) reported that a higher mortality with 86.20 mg kg−1 of LC50 value in artificial soil after PHE exposure for 7 d. The LC50 of E. fetida in natural soil after 14 d treatment with PHE was 56.68 mg kg−1 (Table S3), which was similar to that of measured values in the natural medium was 40.67 mg kg−1 (Wu et al. 2011). The difference in toxicity between different studies may be due to the types of food supply and the total water-holding capacity of tested soil during the trial period (Nam et al. 2015). Another reason might be the variance among individuals from different populations used. Importantly, we found that the earthworms did not get into the soil above 12.5 mg kg−1 of PHE exposure. More research should be conducted to reveal the relationships between PHE toxicity to earthworms and soil properties.
Effects of phenanthrene on the ROS levels in adult E. fetida
Reactive oxygen species produced in cells and contributed by a disruption of cellular redox balance (Song et al. 2009; Delmastro-Greenwood et al. 2014; Song et al. 2019). Moreover, ROS have been reported to be induced various biological effects, including cell growth, apoptosis, and protein activity, leading to substantial damage to cellular function, and even structure (Choudhury and Panda 2005; Shin et al. 2009). The intracellular amount of ROS is an important biomarker for oxidative stress, and the increased ROS levels can effectively reflect the level of oxidative stress (Song et al. 2009; Zhang et al. 2014). The changes of ROS levels in adult E. fetida when earthworms were treated with PHE at different time intervals are shown in Fig. 1.
As exhibited in Fig. 1, no significant difference was found between the groups exposed to a lower dose of PHE (1.25 mg kg−1) and the control group. However, in higher dose PHE-treated groups (2.5, 5, and 10 mg kg−1), the ROS levels in adult E. fetida was dramatically induced compared to the control treatment on the first day after PHE exposure (Fig. 1). Compared to the control, the ROS levels in all PHE-treated groups (1.25−10 mg kg−1) was observably induced than that of the control group on days 3, 5, 7, and 14, and it increased with the PHE concentration, showing a clear dose-response relationship (Fig. 1). He et al. (2021a) also reported that the ROS levels in coelomocytes of E. fetida was significantly increased, showing a clear dose-effect relationship after 24-h exposure to phenanthrene. These results suggest that PHE exposure can damage the balance between elimination and production of ROS in cells, inducing an increased ROS production and accumulation of ROS-derived damages, eventually leading to oxidative stress in E. fetida.
Antioxidant defense responses in adult E. fetida after phenanthrene exposure
The antioxidant defense systems in living organisms can spontaneously eliminate ROS or repair damage induced by the action of oxidative stress to prevent irreversible cellular oxidative damage in normal physiological conditions (Sies 1997; Ece et al. 2008). When facing exogenous stress, various intracellular antioxidases, and small-molecule antioxidants (e.g., SOD, CAT, GST, and TAC ) exist in E. fetida respond quickly to scavenge excess ROS and free radicals, and lessen cellular oxidative stress and associated cellular damage (Wu et al. 2011; He et al. 2021a). Of the antioxidant system, SOD and CAT provide the first line of defense against oxidative damage caused by excessive ROS in cells (Zhang et al. 2020).
SOD, identified as essential component in antioxidant defense system (El-Mas et al. 2012) that can effectively remove the intracellular oxygen free radicals (O2−) through decomposing them into molecular oxygen (O2) and hydrogen peroxide (H2O2) (Song et al. 2009; Liu et al. 2017). In this study, the SOD activities in earthworms were activated in all PHE-treated groups on days 1 and 3 (Fig. 2A). Increased SOD activity indicates that E. fetida has begun to retard the oxidative damage caused by PHE. The SOD activity in higher treated groups (5 and 10 mg kg−1) was inhibited beginning at day 5 until day 14 (Fig. 2A). The decrease in SOD activity may be attributed to the O2− level exceeding the eliminating ability of SOD under PHE stress, thereby inhibiting SOD activity.
However, H2O2 is similarly a kind of ROS, which has a strong ability to induce cell oxidative damage, and it can also stimulate ROS generation (Liu et al. 2017; He et al. 2021b). CAT is a specific H2O2 scavenger, which can remove the excess H2O2 by converting it into O2 and H2O (Wu et al. 2012a; Liu et al. 2018). In PHE amended soil, the CAT activity in earthworms showed an upward trend on the first day and third day (Fig. 2B). However, the activity of CAT begun to decrease as the time of exposure was extended (7 and 14 d) (Fig. 2B). According to the theory of ROS (Mittler 2002; Zhang et al. 2020), the increased CAT activity may be induced by external factors, which lead to an increase in the amount of ROS produced in earthworms, so the biosynthesis of CAT was significantly increased. The decrease of CAT activity may be due to PHE exposure increases the levels of oxidative stress, resulting in the removal rate of ROS lower than the production rate of ROS. Another reason may be that the enzyme superoxide dismutase in cells converts O2− into H2O2, and excessive H2O2 can inhibit the activity of CAT (Zhang et al. 2021).
As another important antioxidant enzyme, POD involves in the defense mechanism against exogenous stresses by removing the excess H2O2 in cells (Song et al. 2009; Zhang et al. 2014). Fig. 2C exhibits that the POD activity in adult E. fetida after PHE exposure at different time intervals. In all PHE-treated groups, the POD activity in earthworms was activated compared to the control. It increased with the PHE dose and the treatment time, showing a clear dose-response relationship (Fig. 2C). The enhanced POD activity may be that the oxidative stress to E. fetida increases with increasing PHE concentration, and POD assists CAT to eliminate excess H2O2 in earthworms under PHE stress (Qiao et al. 2019).
GST is the most important phase II detoxifying metabolic enzyme in cells (Saint-Denis et al. 1998; Liu et al. 2017) that can remove ROS caused by oxidative stress through accelerating the combination of ROS with electrophilic reagents and glutathione to metabolize contaminants (Pickett and Lu 1989). Besides that, GST can also eliminate the lipid peroxidation metabolites such as malondialdehyde and reduce DNA damage in organisms (Song et al. 2019; Yao et al. 2020; 2021). The enzymatic activities of GST in adult E. fetida exposed to PHE at various time intervals are shown in Fig. 2D. As illustrated in Fig. 2D, no significant difference was observed between PHE-treated groups and the control group at 1st day. Comprehensive results showed that the activity of GST in earthworms increased at early periods of exposure (3 and 5 d) and then decreased after treatment with different doses of phenanthrene on days 7 and 14 (Fig. 2D). This finding indicates that the increased GST activity was needed to effectively detoxify the PHE-induced ROS and oxidative damage to reduce the toxicity of harmful compounds against earthworms. The deceased GST activity may be due to the excessive ROS and free radicals induced by PHE beyond the normal detoxification or repair capacities in earthworms.
CarE, an enzyme responsible for the detoxification of exogenous toxicants, which is involved in xenobiotic metabolisms in organisms (Wang et al. 2019). Additionally, previous studies have reported that the CarE activity in earthworms shows the slowest recovery rate among other organisms (Collange et al. 2010; Ečimović et al. 2019). Hence, CarE is an excellent biomarker for assessing the environmental impacts of pollutants. The activity of CarE in adult E. fetida after exposure to PHE in natural soil medium is presented in Fig. 2E. The CarE activity in E. fetida in treatment groups exposed to PHE was not significantly different from that in the control group on 1st day (Fig. 2E). In the higher dose treatment groups (5 and 10 mg kg−1), the CarE activity in earthworms was significantly activated compared to the control on days 3 and 5. In addition, the CarE activity in E. fetida of all PHE-treated groups was observably lower than that in the untreated group, and it decreased with the PHE dose, exhibiting a clear dose-response relationship (Fig. 2E). This result shows that exposure to PHE can inhibit the activity of CarE, resulting in the detoxification function in E. fetida was impaired, especially in the high-dose and long-time exposure.
Total antioxidant capacity (TAC) including non-enzymatic antioxidant defense systems and antioxidant enzymes that can supply a comprehensive antioxidant assessment (Sies 1997; Meng et al. 2019). Its levels are considered a key evaluation index that reflects the scavenging capacity of free radicals and ROS in living organisms (Chen et al. 2015; Hu et al. 2019). The T-AOC levels in adult E. fetida after exposure to PHE in natural soil soil are exhibited in Fig. 2F.
As indicated in Fig. 2F, the T-AOC levels in earthworms were higher in all PHE-treated groups at all time points, except for a lower dose treatment group (1.25 mg kg−1) on 1st day in the natural medium. The increase in the total antioxidant capacity indicated that E. fetida have begun to remove damage caused by PHE pressure. However, a decreased tendency of the TAC levels in E. fetida was observed at a high concentration (10 mg kg−1) on the last day (14 d) of PHE exposure. This result indicated that the antioxidant system in E. fetida could not completely counteract the oxidative stress induced by PHE, and increased ROS can lead to oxidative damage to E. fetida, especially exposure at higher doses and longer times.
Effects of phenanthrene on the degree of oxidative damage in adult E. fetida
Upon exposure to exogenous stress, the production of ROS in cells is an inevitable process (Song et al. 2009). Meanwhile, the endogenous antioxidant system in cells is not sufficient to remove excess ROS under most oxidative stress conditions (Sies 1997; Jiang et al. 2020). The accumulation of excess ROS can result in oxidative damage to macromolecules, such as lipids, proteins, and DNA, thus accelerating the development of LPO, protein carbonylation, and DNA damage, ultimately trigger cell death (Wu et al. 2012b; Liu et al. 2017).
Malondialdehyde (MDA) is known as the cell membrane lipid peroxidation end-products, which is widely considered as an oxidative stress indicator in cells (Song et al. 2009; Zhu et al. 2020). Also, its contents can indirectly reflect the damage level of LPO and cell membranes (Qiao et al. 2019). The changes in MDA content in E. fetida exposed to PHE in natural soil matrix are exhibited in Fig. 3A. Similar to the ROS results, the MDA levels in E. fetida was beyond the normal levels in all PHE exposure treatments throughout the exposure trial period. Moreover, the MDA content was greatly enhanced as the concentration of PHE increased, showing an explicit dose-response relationship (Fig. 3A). The increased MDA content indicated that PHE exposure could induce more ROS in E. fetida and cause LPO and oxidative damage to earthworms. Previously, He et al. (2021a) also reported that PHE exposure can induce lipid peroxidation in coelomocytes of E. fetida. These findings indicate that PHE could trigger oxidative damage to cell membranes in adult E. fetida, especially at high concentration levels.
Protein carbonylation is an irreversible and non-enzymatic post-translational modification induced by excess ROS, which can cause a change of protein structure, make it lose its original biological function (Weng et al. 2017). The PCO level is widely used as a marker of oxidative damage in proteins (England et al. 2006; Liu et al. 2018). No significant change in PCO content on 1st day for lower concentrations (1.25 and 2.5 mg kg−1) of PHE compared to the control (Fig. 3B). However, a significant increase in PCO content in E. fetida was found in 5 and 10 mg kg−1 treatment groups on days 3, 5, 7, and 14 compared to the control (Fig. 3B). These findings show that environmental contaminants, including PHE, can cause oxidative damage to the protein in E. fetida.
Coelomocyte DNA damage in E. fetida exposed to phenanthrene
DNA damage is one of the most harmful toxic effects of exogenous toxicants. One target of reactive oxygen species is DNA, which is more vulnerable to oxidative stress in organisms (Turillazzi et al. 2017; Song et al. 2019). 8-hyoxy-2-deoxyguanosine (8-OHdG) is the product of the 8th carbon atom of guanine base in DNA molecules frequently attacked by ROS (e.g., hydroxyl radicals and singlet oxygen) (Agnihotri and Mishra 2009; Xu et al. 2021). Accordingly, the 8-OHdG level is regarded as a promising indicator to evaluate the degree of oxidative stress and DNA damage of endogenous and exogenous factors to earthworms (Zhang et al. 2020; Zhu et al. 2020). In this study, the level of DNA oxidative damage was determined by measuring the content of 8-OHdG in adult E. fetida. The results showed that the 8-OHdG level in E. fetida all PHE-treated groups was significantly induced compared to the control (Fig. 3C). This result demonstrated that PHE could cause DNA oxidative damage to coelomocytes of E. fetida. This result indicates that PHE exposure can cause oxidative stress in E. fetida, and finally result in DNA damage.
The comet assay is a simple and rapid method for detecting the DNA damage and repair at the individual-cell level (Song et al. 2009; Gajski et al. 2019). In the control group, the nuclear DNA aggregated into a dense round shape, with little or no DNA migrating to the periphery (Fig. 3F1). The comet head of coelomocytes appeared fluffy, mushy, and scattered, and longer comet tails were observed when the E. fetida earthworms were exposed to high-dose PHE (Fig. 3F2 and F3). Several studies have demonstrated that a good linear relationship between the OTM value and DNA damage (Olive et al. 2012; Song et al. 2019). Thus, the value of OTM can be used to quantitatively evaluate the DNA damage in organisms after exposure to exogenous contaminants. In this study, the degree of DNA damage of coelomocytes in E. fetida after PHE exposure at different doses and various time intervals was assessed in natural soil. As exhibited in Fig. 3D, the OTM value in lower PHE treatments (1.25 and 2.5 mg kg−1) did not differ as compared to the control group on days 1, 3, and 5. However, the OTM values in high-PHE concentration treatments (5 and 10 mg kg−1) were significantly higher than the control during the whole exposure period (Fig. 3D). This indicates significant DNA damage of coelomocytes in earthworms after exposure to PHE, especially at high dose levels.
The tail DNA% is the percentage of DNA in the comet tail, which is used to quantify the degree of DNA damage in individual cells (Zhu et al. 2020). As illustrated in Fig. 3F, the results showed that the values of tail DNA% in coelomocytes had the same variation trends with the OTM values after PHE exposure. All PHE-treated groups are minimal/low damage to the DNA of coelomocytes during the entire exposure period, except 10 mg kg−1 treatment for the 14th day, where PHE are middle damage to the coelomocyte DNA (Fig. 3F). Hence, PHE exposure can cause DNA damage of coelomocytes in earthworms, especially at high doses. Results from the comet assay are consistent with the changing trend of 8-OHdG level in the above research. Meanwhile, the value of OTM and Tail DNA% of coelomocytes exhibited the same trend as that of the ROS level in adult E. fetida. The overall results indicate that PHE can induce DNA strand breaks through the formation of ROS and exhibit strong genotoxic potential to the coelomocytes in E. fetida.
Effect of phenanthrene on avoidance behavior of adult E. fetida
Avoidance tests can offer a rapid and low-mortality method for earthworm risk assessments of environmental contaminants (Garcia et al. 2008; Tang et al. 2016). The results of the avoidance behavior of adult E. fetida are exhibited in Fig. 4. No avoidance effects and earthworm escape were observed in the lowest PHE-treated groups (1.25 mg kg−1) and control groups (Fig. 4). However, the observed avoidance effects (NR between 20 and 80%) were found in the 2.5 mg kg−1 treatment groups. Earthworms displayed habitat function loss and avoidance behavior (NR > 80%) in the highest PHE-treated groups (10 mg kg−1) in higher treatment groups (5 and 10 mg kg−1) of PHE (Fig. 4). This result was accordant with the acute toxicity test that earthworms prefer to stay on or near the surface of soil substrate (Section 3.1). These results show that avoidance behavior is an essential endpoint in toxicological assessment and can provide extrapolations at the ecosystem level, even at low doses (Saggioro et al. 2019; Junior et al. 2020).
Growth inhibition and reproductive toxicity of adult E. fetida after phenanthrene exposure
Earthworm weight dynamics is an extremely sensitive marker of short- and long-term exposure to chemical toxicants (Sadeghi et al. 2018; Yao et al. 2021). In this study, the weight of adult E. fetida showed no significant changes between all PHE-treated groups on days 7 and 14 and the control group. However, exposure to higher PHE concentrations (> 1.25 mg kg−1) significantly reduced the earthworm weight on days 21 and 28 compared to the control (Fig. 5A and C). Severe cases lead to the earthworm weight of E. fetida exposed to 10 mg kg−1 at 21 d, and 10 and 1 mg kg−1 at the last exposure (28 d) was even lower than before the experiment (Fig. 5A and C).
Studies have reported that exposure to toxic chemicals can lead to the growth of earthworms was inhibited, with reduced weight being a common response to stress (Xiao et al. 2006; Liu et al. 2018; Qiao et al. 2019). The decreased weight is likely to be associated with the detoxification mechanism of earthworms involves the removal of external toxicants by energy metabolism (Yao et al. 2021). Moreover, a reduction in their energy reserves, such as protein, lipid, and glycogen in earthworms caused by toxic chemicals, might be another reason for this (Ye et al. 2016; Yao et al. 2020). Thus, we can conclude that PHE exposure disturbs the normal metabolism and physiological function of biomacromolecules in E. fetida, with detoxification leading to excessive metabolism, resulting in energy substance was excessively consumed, especially at high concentration levels have potential toxicity to E. fetida.
Reproductive toxicity can influence the population development of any organism, and it is an essential indicator of ecological risk assessment after long-time exposure to toxicants or stresses (Zheng et al. 2008; Qiao et al. 2021). The effects of PHE on E. fetida reproduction in two soil is exhibited in Fig. 5B and D. The cocoon production and the number of juvenile of E. fetida significantly reduced with increasing the PHE concentrations on days 28 and 56 (Fig. 5 Band D). Previous studies found that exposure to environmental contaminants can damage the reproductive systems of earthworms, leading to abnormal sperm, loss of fertilization ability, and finally infertility (Liu et al. 2018; Qiao et al. 2019; Yao et al. 2020; He et al. 2021b). Consistent with these findings, we also believe that PHE can induce reproduction toxicity in E. fetida, especially in the case of exposure to high-dose treatments.
Histopathological changes in adult E. fetida after exposure to phenanthrene
Histopathology is a popular tool to assess the environmental impact of possible exogenous contaminants on various living organisms, including earthworms (Li et al. 2020a). The body wall of earthworms is mainly composed of the epithelium (epidermis), outer circular muscle, and inner longitudinal muscle layer (Zhang et al. 2015; Sun et al. 2022). In this study, the microstructure of the epidermis, circular muscle, and longitudinal muscle layer in the control treatment maintained their normal architecture after 28 days of PHE exposure (Fig. 6A1 and B1). However, in PHE-treated soils, the histological structure of E. fetida changed in varying degrees. A visible exfoliation of the cuticular layer was found in 1.25 and 10 mg kg−1 PHE treatment. Also, the circular muscle and longitudinal muscle layer were significantly damaged upon PHE exposure at day 28 (Fig. 6A2, A3, B2 and B3).
The inner layer of the E. fetida intestine is intestinal epithelial tissue, and the outer layer of the earthworm intestine is chlorogenic tissue, which separates the intestine from the body wall (Li et al. 2020b). No obvious damage to the intestinal epithelial and chlorogenic tissues was found in the 1.25 mg kg−1 exposure group compared to the control (Fig. 6A1 and A2). However, the chlorogenic and the intestinal epithelial tissues of E. fetida were severely damaged in the 10 mg kg−1 PHE-treated group (Fig. 6A3). Also, PHE exposure caused obvious degradation of intestinal tract, and this was particularly true with high concentrations (10 mg kg−1) (Fig. A3). Usually, the intestines and epidermis of earthworms are the main tissues exposed to pollutants in the soil environment through direct contact, digestion, and absorption. Thus, significant lesions may occur after these tissues are heavily damaged, eventually leading to death (Li et al. 2020a). Our results suggest that high-dose PHE exposure can injure the intestinal tract and epidermal tissues of E. fetida.
Figure 6C shows the histopathological observations of the seminal vesicle in E. fetida after 28 days of exposure. As illustrated in Figure 6C1, the seminal vesicles of E. fetida had a clear outline and complete structure in the control group. In the 1.25 mg kg−1 PHE-treated group, the seminal vesicles began to shrink, showing a slight degree of damage to its structure (Figure 6C2). When earthworms were exposed to 10 mg kg−1 PHE, the seminal vesicles became dissipate and shrink to an indistinguishable level (Figure 6C3). Our results are similar with Sun et al. (2021), who found that pyrene exposure could cause heavy damage to seminal vesicles of E. fetida. Similar histopathological changes were also found when earthworms were exposed to petroleum hydrocarbons (Li et al. 2020b). Liang et al. (2017) reported that the health of seminal vesicle tissue was related to the reproduction rate of earthworms. It may be the reason for E. fetida with poor reproductive performance (decreased cocoons and juveniles) after exposure to PHE.
Integrated assessment of biochemical response in adult E. fetida after exposure to phenanthrene
In order to compare the toxicity of PHE on adult E. fetida at different doses and exposure times, a comprehensive biomarker response index (IBRv2) was employed (Sanchez et al. 2013). This method has the advantage of visualization and can clarify the relationship between various biological indicators after exposure to pollutants and distinguish the pollution degree of multiple biomarkers in earthworms (Li et al. 2020a; Zhang et al. 2021). IBRv2 is the sum of the standardized values of all indicators, with higher values represent greater toxicity. The greater standardized value means the greater impact of PHE on earthworms (Normalized value greater than 0 indicates activated, less than 0 indicates suppressed) (Sanchez et al. 2013; Zhu et al. 2020).
The standardized values of all biochemical response biomarkers in E. fetida earthworms exposed to PHE at different doses and exposure times are exhibited in Fig. 7. These values can provide a visual illustration of the toxic effects of PHE on E. fetida earthworms. Compared to other biomarkers, the change of OTM values was higher in all treatment groups, except in the groups treated with 1.25 mg kg−1 PHE at 1 and 5 d (Fig. 7), thus indicating that DNA in E. fetida would be the most negatively affected in the presence of PHE. Also, the activities of the detoxification enzymes (GST and CarE) were in a repressive state for the majority of the time, especially a longer exposure (7 and 14 d), indicating GST and CarE are involved in detoxification and metabolism processes of PHE. Additionally, SOD, MDA, TAC, and 8OHdG were also susceptible to damage in earthworms. Hence, these biomarkers can be used as indicators to reflect the toxic effects of toxic substances including PHE.
In general, the values of IBRv2 are proportional to the comprehensive toxicity of exogenous contaminants to earthworms (Zhu et al. 2020). The changes in the sum of IBRv2 index value in all PHE-treated groups at different exposure times are shown in Fig. 7. As depicted in Fig. 7, PHE at higher concentration (5 and 10 mg kg−1) exhibited a greater impact on all biomarkers than that in lower treatment groups (1.25 and 2.5 mg kg−1) at different exposure times (1, 3, 5, 7, and 14 d). Also, long-term exposures (7 and 14 d) exhibited a larger impact on multiple biomarkers in E. fetida compared to short-term exposure (1 and 3 d), showing a clear time- and dose-effect after exposure to PHE. These results indicated that PHE exposure cause more serious negative effects on E. fetida, especially in the high-dose and long-term exposure groups. A more important finding was that the comprehensive toxicity of PHE to E. fetida earthworms showed a tendency to decrease after 14 d of exposure. The result suggested that the influence of PHE on E. fetida gradually reduced with increasing exposure time. It may be likely due to earthworms adapting to the soil ontaminated with this pollutant. Further research is needed to clarify the soil parameters and types that influence the toxicity of PHE to earthworms.