Determination method of soil TPHs (C₁₀–C₄₀) content referred to ‘Soil and sediment-Determination of petroleum hydrocarbons (C₁₀–C₄₀)-Gas chromatography’ (HJ 1021-2019) (He et al., 2021). Moisture content of sediment samples was determined according to ‘The specification for marine monitoring—Part 5: Sediment analysis’ (GB 17378.5) (National Standards of the People's Republic of China, 2007). TPHs were extracted by soxhlet extraction method.

The ecotoxicity of petroleum-contaminated soil in remediation process was evaluated with the wheat growth test (based on OECD 208). In brief, 10 wheat seeds were evenly embedded in different soil samples (220 g soil in each sample), with a moisture content of 60% field capacity (FC). Environmental conditions are set according to OECD 208 standard (Organization for Economic Co-operation and Development, 2006). The seed germination rate was recorded on the 5th day, and the plant height, fresh weight of above-ground and root fresh weight of wheat seedlings were measured on the 14th day after 50% of the control wheat emerged. Five plants for each parallel sample were selected. Photosynthetic pigment determination method was determined as previously described (Saric, 1978).

Eisenia foetida were raised 1 week in advance according to the OECD 207. Experiment on earthworm mortality: Earthworms are washed and weighed before being transferred into the soil samples of different remediation periods to be tested (30 organisms per 500 g of soil). The raising conditions are set according to OECD 207 standard (Organization for Economic Co-operation and Development, 1984). After 7 days and 14 days mortalities are registered. Earthworm avoidance experiment: A 25 cm long, 17 cm wide and 13 cm high container was divided into two parts with a partition plate placed in the middle of the container. One part was placed in the clean soil, and the other part was placed in the contaminated soil at different remediation periods. The partition was removed and 10 earthworms were added. The earthworms were cultured in 20°C lightless incubator. After 48 h, the earthworms on both sides were counted. The calculation formula of avoidance rate was described (Achazi, 2002).

Urease (UE) activity: By an indophenol blue method, the soil samples were incubated at 38°C for 24 h. The product NH₃-N reacted with hypochlorite and phenol in strong alkaline medium to form water-soluble dye indophenol blue, which was determined at 578 nm (Feng et al., 2008). Catalase (CAT) activity: By a colorimetric method, CAT catalyzes hydrogen peroxide to produce water and oxygen, residual hydrogen peroxide reacts with a highly sensitive chromogenic probe to produce colored substances, which was determined at 510 nm (Trasar et al., 1999). Sucrase (SC) activity: By a 3, 5-dinitrosalicylic acid (DNS) method, soil samples were incubated at 37°C for 24 h and the reaction product reacted with 3, 5-dinitrosalicylic acid, then the resultant was determined at 540 nm (Zhu et al., 2011). Polyphenol oxidase (PPO) activity: By a colorimetric method, using levodopa as substrate. Soil samples were incubated at 25°C for 60 min and the reaction product red quinones was determined at 475 nm (Bartosz et al., 2009). Soil moisture content was measured by gravimetric method.

Extraction method of water extract from petroleum-contaminated soil referred to ‘Solid waste—Extraction procedure for leaching toxicity—Horizontal vibration method’ (HJ 557-2010) (National Environmental Protection Standards of the People's Republic of China, 2010). The preparation of dichloromethane (DCM)/dimethyl sulfoxide (DMSO) extract from contaminated soil was described (Liu et al., 2007). Determination and calculation of biological toxicity were elaborated (Bundy et al., 2004).

In this study, the germination rate of wheat seeds in clean soils (Group C and Group D) was 83.3% ± 5.8% and 90.0% ± 0.0%, respectively (Supplementary Table S1). The germination rate of wheat seeds in the contaminated soils (PWS and PFS) was not significantly different from those in the clean soils (p > 0.05), which indicated that wheat germination was not affected by the concentration of TPH present in these soils. There are no significant correlation between the germination rate of wheat seeds in the soil at different remediating stages and the TPHs content in the soil, p = 0.254 in Group A and p = 0.312 in Group B. This may be because the nutrients required for seed germination of higher plants are mainly derived from the energy in their endosperm, which makes them less sensitive to soil environmental pollution (Chouychai et al., 2007). At the same time, seed rind plays a protective role on the internal structure of seeds, which can prevent the entry of toxic and harmful substances (Liu et al., 2001).

Many researches have reported that low levels may not have been high enough to have phytotoxic effects at this stage of wheat growth. Tang et al. (2011) also showed that contaminated soils from the Shengli oilfield with a TPH level of around 0.1% had little effect on wheat germination, whereas at 0.5%–3.0% TPH they observed significant inhibition of wheat germination (10%–60%). The group A (PWS) soil in our study had TPH levels of 0.16% and in the group B soils (PFS) had TPH levels of 0.4%. Other researchers have shown that the inhibition rate was 48.4% at TPH content of 3%, suggesting strong tolerance of wheat on TPH (Oboh et al., 2007). Studies have shown that only TPHs concentrations higher than 0.5% can significantly reduce seed germination (Gaskin et al., 2008). So some studies have speculated that high concentrations of TPHs will affect the germination rate of seeds (Varjani et al., 2020). Meantime, the phytotoxicity of TPHs may be affected by differences in absorption, nutrient availability and cell wall structure of petroleum compounds in different plant species or tissues (Banks and Schultz, 2005). High concentrations of salt and organic pollutants inhibit seed germination (Kumari et al., 2016; Kaboosi, 2017). There was no change in the germination rate of wheat in the soil of group E and F (83.3% and 86.7%) at different remediation stages.

The results of toxicity experiment of higher plants showed a significant ecotoxicity of the soil during the whole period of bioremediation (Table 2). The plant height of Group B wheat in the 7th and 14th days of soil remediation showed significant inhibition compared with the non-pollution control group (p < 0.01). There were also significant differences (p < 0.01) in the above-ground fresh biomass between the 7th and 14th days of remediation and the non-polluting soil control. The above-ground fresh weight was significantly lower than that of the pollution control (p < 0.01), and it was significantly inhibited (p < 0.05) in the 21st day. Root fresh weight in 0 days, 28 days, 35 days and 42 days soil, compared with non-pollution control showed a significant increase (p < 0.01), and in 7 days and 14 days soil showed a significant inhibition (p < 0.01). At the same time, the growth index of wheat in Group B was slightly higher than that of Group A, probably because the farmland soil had greater nutrients P, organic carbon and less salt, so more suitable for plant growth, the extra nutrients probably lead to enhanced plant growth that compensated for the slightly higher levels of TPH. TPHs will be degraded by plants and generate energy for their own growth (Košnář et al., 2020). The correlation analysis between the growth and development indexes of wheat seeds in the soil at different restoration stages and the TPHs content in the soil was carried out, and the P values were all greater than 0.05. In general, a higher sensitivity of root elongation was found when compared with shoot length and seed germination test (Khan et al., 2014). There was greater root growth of the wheat in the soil samples after 28 days of bioremediation than at the beginning of the bioremediation process, indicating some reduction in phytotoxicity.

TPHs can interfere with chloroplast arrangement, thereby interfering with electron transport and photosynthesis (Singh Tomar and Jajoo, 2013; Jajoo et al., 2014). Some high molecular hydrocarbon substances in petroleum can enter cells through the cell wall, affecting chlorophyll synthesis, thereby affecting the photosynthesis process, and producing serious toxic effects on plants (Plaza et al., 2010). It can be seen that the synthesis of light and pigments is affected. Chlorophyll a content of wheat showed inhibitory effect in soil at different remediation time-points. Group A and Group B had the greatest inhibitory effect on soil on day 21, had the least inhibitory effect on soil on day 7 (Table 2). Carotenoid content of wheat was also inhibited throughout the remediation process. Carotenoid content of Group A was significantly inhibited, most severely inhibited in soil on day 14, and Group B was most severely inhibited in soil on day 21. After statistical analysis, the chlorophyll a content and carotenoid content of wheat had a very significant positive correlation, r = 0.917 (p < 0.01) in Group A and r = 0.541 (p < 0.01) in Group B, indicating that the content of chlorophyll a and carotenoid content had good consistency under petroleum pollution stress. In the correlation analysis of wheat photosynthetic pigment content and TPHs content in soil at different restoration stages, only the carotenoid content of wheat in Group B was extremely significantly correlated with TPHs content, r = 0.897 (p < 0.01).

Studies have shown that, petroleum pollutants into the soil will reduce the bioavailability of soil nutrients, weaken the uptake of soil nutrients by plants, thereby affecting the synthesis of photosynthetic pigments in plants (Karthikeyan et al., 2014); on the other hand, petroleum in the soil and intermediate metabolites during microbial remediation destroy the chloroplast structure (Xie et al., 2014) and inhibit some enzyme functions in the cells, resulting in a decline in photosynthetic pigment content in plants. Some components of TPHs, other than metabolite, have direct toxic effects, such as the discovery in wheat that fluoranthene inhibits both light and dark photosynthesis (Tomar and Jajoo, 2014). Research has demonstrated that PAHs inhibit OEC and electron transport chain of PS-II donor side and acceptor side (Kummerová et al., 2008). The toxicity increased and then decreased during the remediation process, probably due to the production and accumulation of toxic metabolites during petroleum degradation, and then the intermediate products were partially degraded. Or while the bioremediation removed many of the organic compounds, less degradation occurred for fluoranthene and other polycyclic aromatic hydrocarbons that affect leaf photosynthesis.

Among the ecological toxicity evaluation indexes of higher plants in this study, the growth and development indexes of wheat seedlings (plant height, above-ground fresh weight, root fresh weight) indicated that the soil had the strongest toxicity on the 7th and 14th days of remediation. The biochemical indexes of wheat leaves (chlorophyll a and carotenoids) showed that the ecological toxicity of soil in the early to middle stages of remediation increased. The soil toxicity was the strongest on the 21st day, and then decreased, but there was a certain ecological toxicity in the soil during the whole remediation period.

As a biological indicator, earthworm mortality bioassay is an important tool for ecotoxicological research (Khan et al., 2012). Survival of earthworms in the soils at different time-points in the remediation process was examined. Earthworms were placed into the clean soils from both sites (CWS and CFS) survived until the end of the experiment. There were death and poisoning symptoms of earthworms in contaminated soil at different remediation periods. The poisoning symptoms were as follows: body dehydration, in vivo peristalsis and stretching ability was significantly weakened.

It can be seen that the survival rate of E. foetida (Savigny, 1826) decreased significantly with the prolongation of exposure time, indicating a chronic toxicity of petroleum-contaminated soil on the earthworms. There was a gradual decrease in the survival rate between 7-day exposure and 14-day exposure in both group A and B soils probably due to the bioaccumulation of toxic compounds over time (Figure 2A). There was an increase in earthworm survival rates in Group A soils after more than 7 days of bioremediation. Whereas in the Group B soils survival rates initially increased but then decreased at around day 21 but by the end of the bioremediation experiments the survival levels were much greater than at the beginning. This dip in survival rate could be due to increase levels of toxic metabolites that were later degraded. The survival rate of earthworms increased at the end of remediation of both contaminated soils, suggesting that bioremediation ultimately succeeded in reducing soil toxicity. While the mortality of earthworms in the earthworm toxicity experiments at different petroleum concentrations showed a dose-effect relationship (Lucia et al., 2012), it is different from the results of this paper, indicating that some toxic intermediates will be produced in the process of soil remediation, and these intermediates cannot be simply expressed by chemical units, but their toxicity will be reflected in organisms (Dallinger and Horn, 2014; Zhao and Zhu, 2017).

In the correlation analysis between earthworm survival rate and TPHs content in soil at different restoration stages, only earthworm survival rate in Group A was significantly correlated with TPHs content, r = −0.967 (p < 0.01) at 7 days and r = −0.958 (p < 0.01) at 14 days.

TPH may cause death, swelling, body lesions, stiffening, coiling and low reproduction of earthworm (Oboh et al., 2007). In the avoidance test for 48 h, no earthworm death was observed in the control group C and D and the treatment group, but the avoidance rates of E. foetida in all treatment groups were positive after 48 h (Figure 2B). In the avoidance test throughout the remediation period, most earthworms chose the clean soil, and those which chose to enter the TPH contaminated soil would escape and re-enter, indicating that E. foetida are very sensitive to the toxicity of petroleum pollutants. With the decrease of petroleum pollution concentration, the avoidance rate also decreased. In Group A, even if the TPHs content in the soil at the late restoration stage had dropped to a low level (1.00 g/kg), E. foetida still have a 6.67% avoidance rate, indicating that E. foetida are very sensitive to soil toxicity. When the number of earthworms in clean soil accounted for 80% of the total number of earthworms added in the experiment, it showed that contaminated soil was not suitable for earthworm survival, that is, there was avoidance behavior (ISO) (International Organization for Standardization, 2004). Therefore, in Group B, only the soil on the 35th day (1.97 g/kg) and the 42nd day (1.94 g/kg) was suitable for earthworm survival, and the E. foetida avoidance rate was 73.3%. The avoidance rate is higher in the Group B (PFS) soil with the higher (4 g/kg) TPH level. In the correlation analysis of earthworm avoidance rate and TPHs content in soil at different restoration stages, the earthworm avoidance rate of group A and group B were extremely significantly correlated with TPHs content, r = 0.947 (p < 0.01) in group A, r = 0.910 (p < 0.01) in group B. The data shows that avoidance rates in both contaminated soils dropped as the bioremediation time delayed, again indicating that bioremediation reduced the ecotoxicity in these soils.

Catalase is a kind of oxidoreductase, which is formed during the biochemical oxidation reaction of biological respiration and organic matter. Its activity can characterize the soil humus intensity and the conversion rate of organic matter (Zhou and Zhang, 1980). It can be seen from Figure 3A that the catalase activity of Group B was higher than that of Group A, indicating that the increase in organic matter content would enhance the enzyme activity, there is a very significant positive correlation between soil catalase activity and soil organic matter content (Wang et al., 2012). The increase in TPH content would not inhibit the catalase activity within a certain range. The growth rate of catalase activity in contaminated soil with microbial agents at the early stage of soil remediation was significantly higher than that at the middle and late degradation stages, and the catalase activity steadily increased over 28 days and then plateaued, which was consistent with the results of Margesin and Schinner (2000). The enhancement of catalase activity is due to the large-scale reproduction of microorganisms after adapting to the environment in the early stage of remediation, which increases the decomposition of H₂O₂ generated during the degradation of TPHs, promotes the secretion of catalase, and increases the enzyme activity. The production of toxic intermediates and the accumulation of excess H₂O₂ can inhibit the growth and activity of microorganisms, and reduce the secretion of enzymes (Sarkar et al., 2005).

Urease is an amide enzyme that promotes the hydrolysis of carbon-nitrogen bonds (CO-NH) in organic matter. It can reflect the status of soil nitrogen transformation and nutrient level in bioremediation of petroleum-contaminated soil. It can be seen from Figure 3B that the urease activity in soil increased with the extension of remediation time, possibly due to the inoculated microorganisms using the TPH as a carbon source for reproduction and growth after adapting to the soil environment, which promoted the transformation of soil nitrogen and thus promoted the secretion of urease (Makoi and Ndakidemi, 2008). At the same time, some studies showed that soil urease activity was not only closely related to nitrogen level, but also positively correlated with soil organic matter content, total nitrogen and available phosphorus (Niu et al., 2021).

The degradation and metabolism of TPHs will also produce a certain amount of polyphenols, which are the substrates for PPO enzymes. The PPO activity in soil can be used to characterize the biodegradation of some TPHs (Riah et al., 2014). It can be seen from Figure 3C that during the remediation of petroleum-contaminated soil, the activity of PPO increased first and then decreased continuously, with the highest activity on the 7th day of remediation. The increase of PPO activity within 0–7 days may be due to the addition of microbial agents and supplement of nitrogen, which is conducive to the degradation of TPHs (Xu et al., 2020). Chen et al. (2017) also observe a similar PPO profile. However, as the TPHs content decreased and became less available for microbial degradation then PPO activity decreased. Previous studies showed that there is a certain correlation between PPO activity and petroleum degradation rate (Wang et al., 2011).

Sucrase is involved in the decomposition of organic matter in soil, which is closely related to soil carbon cycle (Chen et al., 2010). In this study, the sucrase activity (Figure 3D) of Group A continued to decrease and the sucrase activity during day 0–14 of Group B was higher than that before remediation, and then continued to decrease. The increase of sucrase activity in the early stage of Group B remediation may be because low concentrations of organic pollutants in petroleum-contaminated soil would increase sucrase activity (Dindar et al., 2015). And sucrase and microorganisms use hydrocarbons as substrates and carbon sources (Baran et al., 2004; Labud et al., 2007). A sharp reduction in the activity of this enzyme in the contaminated soil may indicate reduced content of sucrose and other sugar-based organic molecules. The increase respiration rate, in the remediated soil have led to rapid utilization of the organic matter in the soil, and so to a drop in sucrase activity.

Through the correlation analysis between soil enzyme activity and petroleum residue in the remediation process of petroleum-contaminated soil, the results are shown in Table 3. In Group A and Group B, catalase activity was significantly negatively correlated with TPHs content (p < 0.01), urease activity was significantly negatively correlated with TPHs content (p < 0.05), and PPO activity was significantly positively correlated with TPHs content (p < 0.05). Sucrase activity was positively correlated with TPHs content in Group A (p < 0.05), but not in Group B (p > 0.05).

TPHs can cause changes in soil enzyme activity. Firstly, TPHs affects the growth and reproduction of microorganisms in soil, thereby affecting the synthesis and secretion of enzymes in microorganisms. Secondly, TPHs affect the growth and metabolism of plants and the secretion of root exudates and related enzymes are also affected. Thirdly, petroleum pollutants directly act on soil enzymes, changes the structure of enzymes, and inhibits or promotes the activity of enzymes (Tabatabai and Dick, 2002; Andreoni et al., 2004). From the perspective of the four enzyme activities in the six soils during the whole microbial remediation process, the indigenous microorganisms in the soil have little effect on relieving the ecological toxicity of petroleum-contaminated soil, which is manifested by the fact that the enzyme activity in the contaminated soil without the addition of the microbial agent changes little throughout the remediation process. This also verifies the consistency of the previous test: the test exogenously added composite microbial agents play a leading role in the removal of petroleum pollutants. The difference of enzyme activity between Group A and Group B indicated that soil enzyme activity was significantly affected by topography, land use and soil type (Alvarenga et al., 2008).