3.1.1 Oxidative stress: ROS content and enzyme activities
Reactive oxygen species (ROS) are highly reactive chemical species formed due to the electron acceptability of oxygen. Excessive ROS has a destructive effect and promotes oxidative stress (Li et al., 2019). Over a 56-day period, the variation of ROS level was assessed following exposure to TCP (Fig. 1).
In all four soils, the ROS significantly increased in the TCP exposed group in different soils during the experimental period. In the OECD artificial soil and black soil, a dose-response relationship was observed whereby increasing ROS content was observed with increasing TCP concentration. In fluvo-aquic soil, a similar dose-response relationship was observed except at day 56 where ROS content of 0.5 mg/kg dose group decreased compared to 0.1 mg/kg dose group. In red clay, no significant difference in ROS response was observed between 0.01 and 0.1 mg/kg dose groups at day 7, 14, 42 and 56 as well as 0.1 and 0.5 mg/kg dose groups on day 28.
In the present study, the result indicates that TCP exposure induces excessive ROS production in Eisenia fetida and, in most cases, a dose-response relationship was observed. A similar result was observed in our previous study (Zhu et al., 2020) where TCP’s parent chlorpyrifos exposure resulted in excessive ROS production in earthworms in the same four soils. Excessive ROS production in Eisenia fetida after exposure to TCP and its parent chorpyrifos indicates that they caused oxidative stress to Eisenia fetida. From the point of inducing excessive ROS, the toxicity of TCP and chlorpyrifos was similar.
In order to mitigate oxidative stress caused by excessive ROS, Eisenia fetida could produce a battery of enzymes like SOD, CAT and GST. SOD and CAT, the first defense line of cellular protection (Wang et al., 2018), are produced by organisms to inactivate ROS preventing oxidative stress and consequent damage. SOD could transform O2− into H2O2, which could be detoxified by CAT (Liu et al., 2020b). These two enzymes (SOD and CAT) constitute the antioxidant enzyme system to jointly combat oxidative stress. GST contributes greatly to oxidation protection and xenobiotic metabolism, as it also detoxifies ROS in cells. (Zhu et al., 2011).
The detailed changes in SOD, CAT, GST enzyme activities are illustrated in Fig. S1-S3. Unlike with ROS content, the dose-response was not observed for the biomarkers SOD, CAT, and GST with some values higher than the control group following TCP exposure and some lower than the control group. The SOD activity was significantly activated in the early stage of exposure and gradually decreased to the control level in the later stage in all four soils. The CAT and GST activity was activated in most periods.
To clearly evaluate Eisenia fetida oxidative stress in different soils caused by TCP exposure, the IBR index calculated using SOD, CAT and GST (IBR index of SCG) activity was used to describe an integrate biomarkers responses. The IBR index could indicate the toxicity of pollutants and able to assess environmental pollution risk (Wang et al., 2011; Shao et al., 2019). The normalized calculated IBR index of SCG is illustrated in Fig. 2.
The variation of Eisenia fetida enzyme activities including SOD, CAT, and GST shows the early oxidative damage caused by TCP. The oxidative damages suffered by earthworms in 4 soils are different. During the experimental period, the IBR index of SCG in red clay were higer than that in the other three soils. On day 7 and 14, the IBR index of SCG in black soil was little higher than that in fluvo aquic soil and much higher than that in artificial soil. On day 28, 42 and 56, the IBR index of SCG in fluvo-aquic soil was much higher than that in artificial and black soil.
Organic matter (Gebremariam et al., 2012), pH, cation exchange capacity and clay content could interact with chemical substances (Stepnowski et al., 2007). The IBR index of SCG indicates that oxidative stress caused by TCP in red clay and fluvo-aquic soil was higher than that in artificial and black soil. This may due to that the organic carbon of red clay and fluvo-aquic soil is lower than that of artificial soil and black soil. Zhu et al. (2020) demonstrated that chlorpyrifos was more toxic to Eisenia Fetida in red clay with high clay content (71.3%). Xu et al. (2021) stated that azoxystrobin had more lasting adverse effects on earthworms in soils with low organic matter content and low pH. These results are consistent with that in the present study, TCP also has a greater influence on SCG in red clay than in the other three soils. In general, the higher toxicity of TCP in red clay than the other three soils may be due to the fact that red clay has lower organic matter and pH, and higher clay content (71.3%) than the other three soils.
3.1.2 Lipid peroxidation: MDA contents
Excessive ROS can cause lipid peroxidation (LPO), which damages cell membranes and causes cell damage. MDA content could reflect the degree of LPO (Box and Maccubbin, 1997). Figure 3 illustrates the changes in Eisenia fetida MDA content influenced by TCP in soils.
In artificial soil, the MDA contents of each concentration exposure group were significantly higher than that of control group except for 7th, 42nd day 0.01 mg/kg group. In fluvo-aquic soil, the same trend was observed but no significant discrepancy was observed between 7th, 14th day 0.01 mg/kg group and control group. In black soil, the MDA contents at diverse concentration were significantly higher than that of the control group except for 7th day 0.01 mg/kg group. In red clay, on day 7 and 28, the MDA contents of medium and high concentration (0.1 and 0.5 mg/kg) group were significantly higher than that of control group but no significant discrepancy was observed between low concentration (0.01 mg/kg) and control group. On day 14, only the MDA contents of high concentration (0.5 mg/kg) group were significantly higher than that of control group. On day 42 and 56, the MDA contents of each concentration group were significantly higher than that of the control group.
As we previously studied (Zhu et, al., 2020), TCP’s parent chlorpyrifos exposure significantly increased the MDA content in Eisenia fetida over a 4 weeks exposure. Li et al. (2019) demonstrated that another organophosphorus insecticide tolclofos-methyl could also significantly incresed the MDA content in Eisenia fetida. Uniformly, the MDA content in Eisenia fetida was significantly increased after exposed to TCP and the increase was more obvious in the later stage of the experiment. This indicated that TCP exposure caused lipid peroxidation to Eisenia fetida.
3.1.3 DNA oxidative damage: 8-OHdG contents
The product generated when ROS attacking DNA (Guo et al., 2014), 8-hydroxy-2-deoxyguanosine (8-OHdG) could indicate the degree of oxidative and DNA damage (Zhang et al., 2014). Figure 4 illustrates changes in 8-OHdG content in different soils after exposure to TCP.
In artificial soil, the 8-OHdG contents of each concentration group were significantly higher than that of control group. However, the significant difference between the 0.1 and 0.5 mg/kg exposure group was not observed on day 42. Furthermore, on day 28, the 8-OHdG content in the 0.1 mg/kg exposure was significantly lower than that of 0.01 and 0.5mg/kg exposure. In natural soils, the 8-OHdG in TCP concentration groups were significantly higher than that of 0 mg/kg. A dose-response relationship was observed.
The increase in 8-OHdG content indicates that TCP induced DNA damage to Eisenia fetida. As we previously studied (Zhu et, al., 2020), chlorpyrifos treatments also significantly increased the earthworm’s 8-OHdG content. Zhang et al. (2014) also demonstrated that Dechlorane Plus could induce an increase of earthworm’s 8-OHdG content. Besides, 1-methyl-3-(tetrahydro-3-furylmethyl) urea and 1-methyl-3-(tetrahydro-3-furylmethyl) guanidium dihydrogen, which are two main metabolites of the insecticide dinotefuran, were stated that induced DNA damage in Eisenia fetida cells (Liu et al., 2018). Based on the response of 8-OHdG content to TCP exposure, TCP has a certain effect on DNA oxidative damage to Eisenia fetida in all four soils.
In summary, the result shows that TCP is a toxic pollutant to earthworms because TCP can induce excessive ROS, alter enzyme activity and induce lipid peroxidation as well as DNA damage. In addition, the effects on Eisenia fetida of TCP in red clay was higher than that in the other three soils, followed by fluvo-aquic soil and black soil, the lowest was artificial soil. This may due to the low organic matter content in red clay and fluvo-auic soil and the high clay content in red clay. We believe that artificial soil toxic experiment may not correctly evaluate the toxicity of TCP in natural soil including fluvo-aquic soil and red clay.
TCP has a similar effect on earthworms compared to the parent chemical chlorpyrifos. However, the toxicity of TCP and chlorpyrifos to earthworms may further be elucidated by calculating the IBR index.