Optimizing Acetic Acid Application Strategy Can Effectively Promote the Remediation Performance of Oilseed Sunﬂower on Cd-Contaminated Soils

: Applying exogenous organic acids is an effective method to improve the remediation efﬁciency of Cd-contaminated soils. To investigate the effects of exogenous acetic acid on Cd forms in rhizosphere soils and phytoremediation performance for Cd-contaminated soils, a potted experiment was performed with oilseed sunﬂower as the extractive plant. Acetic acid was applied at 1, 2, 3, 4, 5


Introduction
In recent years, Cadmium (Cd) pollution in soils has become increasingly prominent with the wanton discharge of industrial wastes and the extensive use of chemical fertilizers [1][2][3].Cd does not easily degrade, and is difficult to remove once infiltrated into the soil, resulting in soil fertility decline and irreversibly affecting crop yields [4][5][6].In China, approximately 19.4% of arable land has soil pollution in excess of the standard threshold value, of which, Cd exceeded the standard by 7% [7].Cd in soils is absorbed by crops, and may intrude into the human body through the food chain, ultimately causing irreversible and momentous harm to food safety, human health, and ecological health [8,9].In view of this, an efficient remediation countermeasure for Cd-contaminated soils is imperative.
The remediation methods of Cd pollution in soils mainly include physical remediation, chemical remediation, and biological remediation [10,11].Nevertheless, the current physical and chemical remediation methods have certain limitations [12][13][14], such as high cost, cost of manpower and material resources, damage to soil properties, and an inability to be applied ar a large scale, which can only alleviate the crisis of Cd pollution on a basic level, and may lead to secondary pollution.By contrast, phytoremediation technology has good prospects for the treatment of Cd-contaminated soils because of its low cost, avoidance of damage to soil structure, avoidance of secondary pollution, and wide application range [15][16][17].In recent years, selection for suitable extractive plants has become a hot issue.Some plants such as Noccaea caerulescens and Sedum alfredii have the potential to absorb Cd, but their slow growth rates and limited biomass are unavoidable disadvantages [18].In contrast to these cultivars, some studies suggests that oilseed sunflower is a potential candidate [19][20][21], in consideration of its characteristics of large biomass and considerable root systems.Relevant results showed that Cd accumulation in the shoots of oilseed sunflower was more than 100 mg•kg −1 [22,23].Furthermore, the extraction of Cu, Cd, and As from oilseed sunflower was significantly higher than that from other plants, and its removal effect for Cd was prominent [24][25][26].Meanwhile, among more than 20 kinds of extraction plants, oilseed sunflower has the strongest enrichment effect on Cd [27].Hence, oilseed sunflower is an excellent phytoremediation material for Cd-contaminated soils, from the perspective of ecology and economic application [28].It is of great significance to use oilseed sunflower for soil Cd remediation.
The proportion of Cd mobility in soils determines the magnitude of Cd uptake by plant roots.In most cases, Cd has poor mobility in soils.In view of this, some measures to enhance Cd activity should be highlighted.Indeed, some low molecular weight organic acids released by plant roots have the potential to alter the proportion of Cd mobility in soils [29][30][31].However, organic acids released by plant roots are limited, hence it is necessary to employ exogenous organic acids to improve the remediation effect for Cdcontaminated soils [32].Organic acids combine with activated Cd ions in soils to form chelate, thereby making it easy to be absorbed by roots, thus significantly promoting the enrichment of Cd in plant roots.Concretely, organic acids can reduce rhizosphere pH value and promote the release of Cd adsorbed in soil aggregates, thus enhancing the activity of Cd and improving its bioavailability and phytoremediation efficiency [33][34][35].Moreover, organic acids can also enhance the tolerance of roots to Cd, thereby providing a stable Cd-enrichment environment [36].The effects of different organic acids on the accumulation of Cd were different.Recent research has shown that oxalic acid, acetic acid, lactic acid, malic acid, citric acid, and tartaric acid could improve the ability of plants to absorb Cd in soils [37][38][39][40].Of which, the remediation effect of Cd-contaminated soils with acetic acid was most prominent among various organic acids [41].As a typical exogenous organic acid, acetic acid is accessible and cheap [42].
Previous studies had shown that exogenous organic acids can enhance the availability of Cd by changing the physical and chemical properties of soils, thus enhancing phytoremediation efficiency.Furthermore, the addition of organic acids can also improve microbial activity and enhance soil fertility, thus fixing the effects of Cd [43,44]; therefore, the effects of organic acid application are complex.Improper application of exogenous organic acid may not achieve the ideal reparative effect.In addition, different application times may result in different effects on plant roots.Here, we hypothesized that for acetic acid, there is an optimal application time and concentration, which can effectively assist and promote the remediation effect of oilseed sunflower on Cd-contaminated soils.
In this study, oilseed sunflower was used as the extractive plant, a potted experiment was performed.We added acetic acids to the soils at six concentrations (1, 2, 3, 4, 5, and 6 mmol/kg) at 20, 30, 40, and 50 days after seedling emergence.Soil without acetic acid treatment was used as the control.After harvest, the forms of Cd in the rhizosphere soils, the changes of the related chemical indicators, and the oilseed sunflower biomass were measured.

Test Crops and Soil
The test oilseed sunflower variety "Newrui no.2" was selected.The test soil was taken from a heavily polluted sewage irrigation area in Xinxiang, China, and the content of Cd 2+ was 19.5 mg/kg.The basic physical and chemical properties of soils are shown in Table 1.

Experimental Design
The experiment was carried out in the solar greenhouse of the Field Scientific Observation and Experiment Station of Agricultural Water and Soil Environment, Chinese Academy of Agricultural Sciences.The soil used in the experiment was first air-dried to pass through a 5 mm sieve, mixed and put into plastic pots with a diameter of 25 cm on the upper edge, 15 cm on the bottom, and a height of 15 cm.Each pot was loaded with 3 kg soil.Oilseed sunflower was sown on 2 October 2020, seedlings emerged on 6 October, and harvest occured on 9 January 2021.The test period lasted 100 days.The exogenous organic acid-acetic acid-was applied at 20, 30, 40, and 50 days after seedling emergence (Short for DASE 20, DASE 30, DASE 40, and DASE 50) with six levels of acetic acid, 1, 2, 3, 4, 5, and 6 mmol/kg, respectively, and no acetic acid as control (CK).A total of 25 treatments were performed, and each treatment was repeated 3 times.After harvest, the shoot and root dry weights of oilseed sunflower were measured, soil pH value, soil enzyme activity (catalase activity, amylase activity, sucrase activity), exchangeable Cd, carbonate Cd, Fe-Mn oxide Cd, organic Cd, and Cd removal rate were measured in rhizosphere soils.

Test Indicators and Methods
(1) Shoot and root dry weights of oilseed sunflower: the shoot and root parts of oilseed sunflower were first rinsed with tap water, then rinsed with deionized water, placed in an oven at 105 • C for 30 min, then continued to dry at 75 • C until the weight remained constant, and the shoot and root dry matter weights were measured, respectively.(2) Soil pH value: tested with a pH meter (PHSJ-6L) at a soil to water ratio of 1:5.
(3) Soil enzymatic activity: sucrase activity in soils were determined by the colorimetric method using 3,5-dinitrosalicylic acid, peroxidase activity in soils by potassium permanganate titration, and amylase activity in soils by colorimetric method.(4) Cd in soils: the soil samples were naturally dried, ground, and passed through a 200 mesh sieve; the exchangeable Cd, carbonate Cd, Fe-Mn oxide Cd, and organic Cd in the samples were extracted and determined by the Tessier graded extraction method.The supernatant after extraction was determined by TAS-986 flame atomic absorption spectrophotometer (Sedico, Giza, Egypt).( 5) Cd removal rate: Cd removal rate = (total Cd in soils before planting − total Cd in soils after planting)/total Cd in soils before planting × 100%.
Data were analyzed using Excel 2010 (Microsoft, Redmond, WA, USA) and SPSS 19.0 (SPSS Inc, Chicago, IL, USA) for ANOVA, Origin 2021 (OriginLab, Northampton, MA, USA) for plotting, Canoco 5 (Microcomputer Power, Ithaca, NY, USA) for redundancy analysis (RDA) and the least significant difference (LSD) method was applied for multiple comparisons between treatments at a 95% confidence level.

Cd Forms and Cd Removal Rate in Rhizosphere Soils
Figure 1 shows the contents of exchangeable Cd in rhizosphere soils under different treatments.The contents of exchangeable Cd in rhizosphere soils under treatments were lower than those of CK.When acetic acids were applied at DASE 20 and 50, the contents of exchangeable Cd in rhizosphere soils showed no significant difference compared to CK (p > 0.05).The content of exchangeable Cd in rhizosphere soils of 1, 5, and 6 mmol/kg acetic acids were significantly lower than that of CK at DASE 30 (p < 0.05), which were 22.18%, 25.94%, and 22.78% lower than that of CK.Compared to CK, when 1-6 mmol/kg acetic acids were applied at DASE 40, the contents of exchangeable Cd in rhizosphere soils were significantly decreased by 32.36%-56.33%.
multiple comparisons between treatments at a 95% confidence level.

Cd Forms and Cd Removal Rate in Rhizosphere Soils
Figure 1 shows the contents of exchangeable Cd in rhizosphere soils under different treatments.The contents of exchangeable Cd in rhizosphere soils under treatments were lower than those of CK.When acetic acids were applied at DASE 20 and 50, the contents of exchangeable Cd in rhizosphere soils showed no significant difference compared to CK (p > 0.05).The content of exchangeable Cd in rhizosphere soils of 1, 5, and 6 mmol/kg acetic acids were significantly lower than that of CK at DASE 30 (p < 0.05), which were 22.18%, 25.94%, and 22.78% lower than that of CK.Compared to CK, when 1-6 mmol/kg acetic acids were applied at DASE 40, the contents of exchangeable Cd in rhizosphere soils were significantly decreased by 32.36%-56.33%.Figure 2 shows the contents of carbonate Cd in rhizosphere soils under different treatments.Compared to CK, when applying different concentrations of acetic acids at DASE 20, 30, and 40, the contents of carbonate Cd in rhizosphere soils were increased, and when 5 mmol/kg acetic acid was applied at DASE 20, it was significantly increased by 36.74%.When 1, 5, and 6 mmol/kg acetic acids were applied at DASE 30, the contents of carbonate Cd in rhizosphere soils were significantly increased by 45.67%, 151.48%, and 179.83%;When 1-6 mmol/kg acetic acids were applied at DASE 40, the contents of carbonate Cd in rhizosphere soils were significantly increased by 30.68%-169.86%.When acetic acids were applied at DASE 50, the contents of carbonate Cd in rhizosphere soils with different concentrations of acetic acid were generally lower than that of CK (except for the application of 3 mmol/kg acetic acid), but the differences were not significant.
by 36.74%.When 1, 5, and 6 mmol/kg acetic acids were applied at DASE 30, the contents of carbonate Cd in rhizosphere soils were significantly increased by 45.67%, 151.48%, and 179.83%;When 1-6 mmol /kg acetic acids were applied at DASE 40, the contents of carbonate Cd in rhizosphere soils were significantly increased by 30.68%-169.86%.When acetic acids were applied at DASE 50, the contents of carbonate Cd in rhizosphere soils with different concentrations of acetic acid were generally lower than that of CK (except for the application of 3 mmol/kg acetic acid), but the differences were not significant.Figure 3 shows the contents of Fe-Mn oxide Cd in rhizosphere soils under different treatments.Except for the treatment with 1mmol/kg acetic acid at DASE 50, the contents of Fe-Mn oxide Cd in rhizosphere soils under different acetic acid concentrations and application time were generally lower than that of CK.Compared to CK, when 1-5 mmol/kg acetic acids were applied at DASE 20, the contents of Fe-Mn oxide Cd in rhizosphere soils were significantly decreased by 55.98%, 57.37%, 58.53%, 54.74%, and 58.43%.When 1-6 mmol /kg acetic acids were applied at DASE 30 and 40, the contents of Fe-Mn oxide Cd in rhizosphere soils were significantly decreased by 42.04%-60.96%and 37.71%-43.76%,respectively.When acetic acids were applied at 2-6 mmol/kg at DASE 50, the contents of Fe-Mn oxide Cd in rhizosphere soils were decreased by 6.75%-17.75% compared to CK, but there was no significant difference between treatments and CK. Figure 3 shows the contents of Fe-Mn oxide Cd in rhizosphere soils under different treatments.Except for the treatment with 1mmol/kg acetic acid at DASE 50, the contents of Fe-Mn oxide Cd in rhizosphere soils under different acetic acid concentrations and application time were generally lower than that of CK.Compared to CK, when 1-5 mmol/kg acetic acids were applied at DASE 20, the contents of Fe-Mn oxide Cd in rhizosphere soils were significantly decreased by 55.98%, 57.37%, 58.53%, 54.74%, and 58.43%.When 1-6 mmol/kg acetic acids were applied at DASE 30 and 40, the contents of Fe-Mn oxide Cd in rhizosphere soils were significantly decreased by 42.04%-60.96%and 37.71%-43.76%,respectively.When acetic acids were applied at 2-6 mmol/kg at DASE 50, the contents of Fe-Mn oxide Cd in rhizosphere soils were decreased by 6.75%-17.75% compared to CK, but there was no significant difference between treatments and CK.
Figure 4 shows the contents of organic Cd in rhizosphere soils under different treatments.Except for the treatment with 1 mmol/kg acetic acid at DASE 50, the contents of organic Cd in rhizosphere soils under different acetic acid concentration levels and application time were generally lower than CK.When 1-6 mmol/kg acetic acids were applied at DASE 20, the contents of organic Cd in rhizosphere soils were significantly reduced by 26.44%-50.02%compared to CK.When 1-5 mmol/kg acetic acids were applied at DASE 30, the contents of organic Cd in rhizosphere soils were significantly reduced by 23.29%-43.26%compared to CK.When 1-6 mmol/kg acetic acids were applied at DASE 40, the contents of organic Cd in rhizosphere soils were significantly decreased by 36.84%-53.10%compared to CK.At DASE 50, the contents of organic Cd in rhizosphere soils with 2-6 mmol/kg acetic acids decreased by 1.44%-36.70%compared to CK, but there was no significant difference between treatments and CK.Total Cd of rhizosphere soils under different treatments are shown in Table 2. Cd removal rates of rhizosphere soils under different treatments are shown in Figure 5.At DASE 20, the removal rates of Cd in rhizosphere soils with 1-6 mmol/kg acetic acids increased by 22.43% to 51.82% compared to CK, but there was no significant difference between treatments and CK.At DASE 30, the removal rates of Cd in rhizosphere soils with 1-5 mmol/kg acetic acids were significantly increased by 25.66%-37.02%compared to CK.At DASE 40, the removal rate of Cd in rhizosphere soils with 1 mmol/kg acetic acid was significantly increased by 60.45% compared to CK.At DASE 50, the removal rates of Cd in rhizosphere soils with 1-6 mmol/kg acetic acids increased by 4.27%-33.79%compared to CK, but there was no significant difference between treatments and CK. Figure 4 shows the contents of organic Cd in rhizosphere soils under different treatments.Except for the treatment with 1 mmol/kg acetic acid at DASE 50, the contents of organic Cd in rhizosphere soils under different acetic acid concentration levels and application time were generally lower than CK.When 1-6 mmol /kg acetic acids were applied at DASE 20, the contents of organic Cd in rhizosphere soils were significantly reduced by 26.44%-50.02%compared to CK.When 1-5 mmol/kg acetic acids were applied at DASE 30, the contents of organic Cd in rhizosphere soils were significantly reduced by 23.29%-43.26%compared to CK.When 1-6 mmol/kg acetic acids were applied at DASE 40, the contents of organic Cd in rhizosphere soils were significantly decreased by 36.84%-53.10%compared to CK.At DASE 50, the contents of organic Cd in rhizosphere soils with 2-6 mmol /kg acetic acids decreased by 1.44%-36.70%compared to CK, but there was no significant difference between treatments and CK.Total Cd of rhizosphere soils under different treatments are shown in Table 2. Cd removal rates of rhizosphere soils under different treatments are shown in Figure 5.At DASE 20, the removal rates of Cd in rhizosphere soils with 1-6 mmol/kg acetic acids increased by 22.43% to 51.82% compared to CK, but there was no significant difference between treatments and CK.At DASE 30, the removal rates of Cd in rhizosphere soils with 1-5 mmol/kg acetic acids were significantly increased by 25.66%-37.02%compared to CK.At DASE 40, the removal rate of Cd in rhizosphere soils with 1 mmol/kg acetic acid was significantly increased by 60.45% compared to CK.At DASE 50, the removal rates of Cd in rhizosphere soils with 1-6 mmol/kg acetic acids increased by 4.27%-33.79%compared to CK, but there was no significant difference between treatments and CK.

Soil Enzyme Activity
Figure 6 shows the amylase activity of the rhizosphere soils in different treatments.The amylase activity of the rhizosphere soils was generally reduced by the addition of different concentrations of acetic acid at different time periods.Compared to CK, when 1-5 mmol/kg acetic acids were applied at DASE 20, the rhizosphere soil amylase activity was decreased by 10.9%, 13.6%, 12.5%, 11.6%, and 8.90%, respectively.When 1-6 mmol/kg acetic acids were applied at DASE 30, the rhizosphere soil amylase activity was significantly decreased by 7.33%-48.3%.When 1-6 mmol/kg acetic acids were applied at DASE 40, the rhizosphere soil amylase activity was significantly decreased by 47.0%-52.2%.The rhizosphere soil amylase activity was significantly decreased by 7.33% by applying 6 mmol/kg acetic acid at DASE 50, and by 2.29%, 4.81%, and 1.97% by applying 3, 4, and 5 mmol/kg acetic acids, respectively, but there was no significant difference between treatments and CK.

Soil Enzyme Activity
Figure 6 shows the amylase activity of the rhizosphere soils in different treatments.The amylase activity of the rhizosphere soils was generally reduced by the addition of different concentrations of acetic acid at different time periods.Compared to CK, when 1-5 mmol/kg acetic acids were applied at DASE 20, the rhizosphere soil amylase activity was decreased by 10.9%, 13.6%, 12.5%, 11.6%, and 8.90%, respectively.When 1-6 mmol/kg acetic acids were applied at DASE 30, the rhizosphere soil amylase activity was significantly decreased by 7.33%-48.3%.When 1-6 mmol/kg acetic acids were applied at DASE 40, the rhizosphere soil amylase activity was significantly decreased by 47.0%-52.2%.The rhizosphere soil amylase activity was significantly decreased by 7.33% by applying 6 mmol/kg acetic acid at DASE 50, and by 2.29%, 4.81%, and 1.97% by applying 3, 4, and 5 mmol/kg acetic acids, respectively, but there was no significant difference between treatments and CK.
Figure 7 shows the sucrase activity of the rhizosphere soils in different treatments.Compared to CK, there was no significant difference in sucrase activity in rhizosphere soils under different concentrations of acetic acid at DASE 20.When 2-6 mmol/kg acetic acids were applied at DASE 30, the activity of sucrase in rhizosphere soils was increased by 0.43%, 11.40%, 100.71%, 76.55%, and 18.03%, respectively.There were significant differences in sucrase in rhizosphere soils when 4 and 5 mmol/kg acetic acids were applied.The activity of sucrase in rhizosphere soils was increased by 1-6 mmol/kg acetic acids at DASE 40 and 50.Among which, the application of 1, 3, 4, 5, and 6 mmol/kg acetic acids at DASE 40 significantly increased soil sucrase by 94.37%, 81.82%, 110.55%, 103.56%, and 92.80%.At DASE 50, 1-6 mmol/kg acetic acids increased the sucrase in rhizosphere soils by more than 32%, but there was no significant difference between various treatments.Figure 7 shows the sucrase activity of the rhizosphere soils in different treatments.Compared to CK, there was no significant difference in sucrase activity in rhizosphere soils under different concentrations of acetic acid at DASE 20.When 2-6 mmol/kg acetic acids were applied at DASE 30, the activity of sucrase in rhizosphere soils was increased by 0.43%, 11.40%, 100.71%, 76.55%, and 18.03%, respectively.There were significant differences in sucrase in rhizosphere soils when 4 and 5 mmol/kg acetic acids were applied.The activity of sucrase in rhizosphere soils was increased by 1-6 mmol/kg acetic acids at DASE 40 and 50.Among which, the application of 1, 3, 4, 5, and 6 mmol/kg acetic acids at DASE 40 significantly increased soil sucrase by 94.37%, 81.82%, 110.55%, 103.56%, and 92.80%.At DASE 50, 1-6 mmol/kg acetic acids increased the sucrase in rhizosphere soils by more than 32%, but there was no significant difference between various treatments.Figure 8 shows the catalase activity of the rhizosphere soils in different treatments.The application of different concentrations of acetic acid at DASE 20, 30, 40, and 50 increased the catalase activity of rhizosphere soils.Compared to CK, when 2 mmol/kg acetic acid was applied at DASE 20, the catalase activity in rhizosphere soils was significantly increased by 61.47%.When 1 mmol/kg acetic acid was applied at DASE 40, the catalase activity in rhizosphere soils was significantly increased by 90.48%.When 6 mmol/kg acetic acid was applied at DASE 50, the catalase activity in rhizosphere soils was significantly increased by 85.86%.There was no significant difference between other treatments and CK.applied at DASE 20, the catalase activity in rhizosphere soils was significantly increased by 61.47%.When 1 mmol/kg acetic acid was applied at DASE 40, the catalase activity in rhizosphere soils was significantly increased by 90.48%.When 6 mmol/kg acetic acid was applied at DASE 50, the catalase activity in rhizosphere soils was significantly increased by 85.86%.There was no significant difference between other treatments and CK.

Shoot and Dry Weight of Oilseed Sunflower
Figure 9 shows the shoot dry weight of oilseed sunflower under different treatments.Compared to CK, applying 1-6 mmol/kg acetic acids at DASE 20 increased shoot dry weight.Notably, applying 5 mmol/kg acetic acid increased shoot dry weight by 96.4%, which was a significant difference.Application of 1, 2, 3, 4, and 6 mmol/kg acetic acids increased shoot dry weight by 22.3%, 75.9%, 70.8%, 59.6%, and 72.6%, respectively.When 2 mmol/kg and 4 mmol/kg acetic acids were applied at DASE 30, the shoot dry weight of oilseed sunflower was significantly increased by 92.4% and 85.5%.When 1 mmol/kg acetic acid was applied at DASE 40, the shoot dry weight of oilseed sunflower was significantly increased by 99.7%, and the shoot dry weight of oilseed sunflower under other treatments was increased by 43.8%-91.5% compared to CK.At DASE 50, the shoot dry weight of 6 mmol/kg acetic acid was significantly increased by 83.2% compared to CK, but there was no significant difference between other treatments and CK.

Shoot and Root Dry Weight of Oilseed Sunflower
Figure 9 shows the shoot dry weight of oilseed sunflower under different treatments.Compared to CK, applying 1-6 mmol/kg acetic acids at DASE 20 increased shoot dry weight.Notably, applying 5 mmol/kg acetic acid increased shoot dry weight by 96.4%, which was a significant difference.Application of 1, 2, 3, 4, and 6 mmol/kg acetic acids increased shoot dry weight by 22.3%, 75.9%, 70.8%, 59.6%, and 72.6%, respectively.When 2 mmol/kg and 4 mmol/kg acetic acids were applied at DASE 30, the shoot dry weight of oilseed sunflower was significantly increased by 92.4% and 85.5%.When 1 mmol/kg acetic acid was applied at DASE 40, the shoot dry weight of oilseed sunflower was significantly increased by 99.7%, and the shoot dry weight of oilseed sunflower under other treatments was increased by 43.8%-91.5% compared to CK.At DASE 50, the shoot dry weight of 6 mmol/kg acetic acid was significantly increased by 83.2% compared to CK, but there was no significant difference between other treatments and CK. Figure 10 shows the root dry weight of oilseed sunflower with different treatments.Compared to CK, applying 1-6 mmol/kg acetic acids at DASE 20 increased root dry weight.Notably, applying 5 mmol/kg acetic acid increased root dry weight by 179.7%, which was a significant difference.when 2 mmol/kg acetic acid was applied at DASE 30, the dry weight of root was significantly increased by 82.3%.The dry weight of oilseed sunflower was increased by 16.5%-75.9%at DASE 40 with 1-6 mmol/kg acetic acids, but the differences were not significant.When 1-6 mmol/kg acetic acids were applied at DASE 50, there was no significant difference between the treatments and CK. Figure 10 shows the root dry weight of oilseed sunflower with different treatments.Compared to CK, applying 1-6 mmol/kg acetic acids at DASE 20 increased root dry weight.Notably, applying 5 mmol/kg acetic acid increased root dry weight by 179.7%, which was a significant difference.when 2 mmol/kg acetic acid was applied at DASE 30, the dry weight of root was significantly increased by 82.3%.The dry weight of oilseed sunflower was increased by 16.5%-75.9%at DASE 40 with 1-6 mmol/kg acetic acids, but the differences were not significant.When 1-6 mmol/kg acetic acids were applied at DASE 50, there was no significant difference between the treatments and CK.

pH Value in Rhizosphere Soils
The pH values of oilseed sunflower rhizosphere soils under different acetic acid application times and different concentrations are shown in Figure 11.The pH values of 4 mmol/kg acetic acid at DASE 20 and 2 mmol/kg acetic acid at DASE 50 were significantly different from those of CK, which were 1.17% and 1.63% lower than those of CK, respectively, while the pH values of other treatments were not significantly different

pH Value in Rhizosphere Soils
The pH values of oilseed sunflower rhizosphere soils under different acetic acid application times and different concentrations are shown in Figure 11.The pH values of 4 mmol/kg acetic acid at DASE 20 and 2 mmol/kg acetic acid at DASE 50 were significantly different from those of CK, which were 1.17% and 1.63% lower than those of CK, respectively, while the pH values of other treatments were not significantly different from those of CK.However, adding different concentrations of acetic acid at DASE 50 can reduce pH value, and the reduction effect was more obvious than that at other times.

Correlation between Soil Indicators and Dry Weight of Oilseed Sunflower
Taking the dry weight of shoot (S−W) and root (R−W) of oilseed sunflower as response variables; pH, amylase (AL) activity, sucrase (SC) activity, catalase (CAT) activity, exchangeable Cd (E-Cd) content, carbonate Cd (C−Cd) content, Fe-Mn oxide Cd (FM−Cd) content, organic Cd (O−Cd) content, and total Cd (T−Cd) content in rhizosphere soils are explanatory variables.A redundancy analysis (RDA) was performed for the two variables (Figure 12).The Axis1 and Axis2 explain 52.47% of the influence degree of abovementioned soil indicators on the dry weight of shoot and root of oilseed sunflower, among which the Axis1 accounts for 45.95%.The red arrows in Figure 12

Correlation between Soil Indicators and Availability of Cd
E-Cd is the main available Cd in soils, followed by C-Cd.AL was positively correlated with E-Cd; SC, pH, and CAT were negatively correlated with E-Cd.On the contrary, SC, pH, and CAT were positively correlated with C-Cd, and AL was negatively correlated with C−Cd (Figure 12).

Discussion
It has been found that the application of organic matter acids can reduce soil pH and activate Cd in soil [45,46].This study showed that there was no significant difference between different concentrations and application times, but the addition of different concentrations of acetic acid at DASE 50 reduced the pH, and the reduction effect is more obvious than other times.This is consistent with the previous studies [39,[47][48][49].This result may be due, on the one hand, to the fact that the plant growth cycle was 100 days and the application points were 20th, 30th, 40th, and 50th day, setting a relatively early application time, and the fact that the soil used in the trial was alkaline and inherently buffered [50].On the other hand, it may be due to the fact that exogenous organic acids can be used as a carbon and energy material for soil microorganisms, resulting in the degradation of organic acids by soil microorganisms [51,52].
Soil enzymes are directly related to a variety of important metabolic activities in the soil.Studies have shown that the content of Cd in soils has a negative correlation with

Correlation between Soil Indicators and Availability of Cd
E-Cd is the main available Cd in soils, followed by C-Cd.AL was positively correlated with E-Cd; SC, pH, and CAT were negatively correlated with E-Cd.On the contrary, SC, pH, and CAT were positively correlated with C-Cd, and AL was negatively correlated with C−Cd (Figure 12).

Discussion
It has been found that the application of organic matter acids can reduce soil pH and activate Cd in soil [45,46].This study showed that there was no significant difference between different concentrations and application times, but the addition of different concentrations of acetic acid at DASE 50 reduced the pH, and the reduction effect is more obvious than other times.This is consistent with the previous studies [39,[47][48][49].This result may be due, on the one hand, to the fact that the plant growth cycle was 100 days and the application points were 20th, 30th, 40th, and 50th day, setting a relatively early application time, and the fact that the soil used in the trial was alkaline and inherently buffered [50].On the other hand, it may be due to the fact that exogenous organic acids can be used as a carbon and energy material for soil microorganisms, resulting in the degradation of organic acids by soil microorganisms [51,52].
Soil enzymes are directly related to a variety of important metabolic activities in the soil.Studies have shown that the content of Cd in soils has a negative correlation with soil enzyme activity [53,54], and the scientific application of exogenous organic acids significantly increases soil enzyme activity [55,56].Amylase in soils is mainly of microbial origin and is an important enzyme involved in the natural carbon cycle; sucrase converts sucrose into glucose and fructose, which, in turn, improves the biological activity of the soils and increases the nutrients in the soil, and can be used to assess the fertility of the land.Usually, the higher the activity of sucrase, the greater its fertility; biological respiration and the oxidation of organic matter can produce hydrogen peroxide, which has a strong effect on fertility.Hydrogen peroxide is an oxidoreductase enzyme that breaks down hydrogen peroxide into water and oxygen, reducing its toxicity to both organisms and the soils [57,58].This study showed that the application of different concentrations of acetic acid at different time points reduced the amylase activity of rhizosphere soils to a certain extent, and showed a trend of first decreasing and then increasing with the time of addition, and the amylase activity of rhizosphere soils reached the lowest at DASE 40.The sucrase activity and catalase activity in rhizosphere soils were improved by acetic acid at different time points, and the effect of 1 mmol/kg acetic acid at DASE 40 was the most obvious, which was consistent with the results of a previous study [59].This is partly due to the fact that acetic acid provides carbon and nutrients to the microorganisms in the soil, thus accelerating their growth and reproduction, while the secreted metabolites of the microorganisms increase the soil enzyme activity [60]; and partly, is probably due to the fact that the functional groups of the applied exogenous acetic acid prevent the binding of Cd to the sulfhydryl groups of the enzyme by forming acetic acid-Cd complexes [61], thus increasing the enzyme activity in soils compared to CK.The results of this study showed that the addition of acetic acid activates soil nutrients and contributes to soil fertility, self-purification, and detoxification.
The addition of organic acids to Cd-contaminated soils can reduce the toxicity of Cd to a certain extent and benefit plant growth [62].The present study showed that the application of different concentrations of acetic acid at different time points promoted the increase of shoot and root dry weight, indicating that the addition of acetic acid was beneficial to the increase of the biomass of oilseed sunflower.Except for the DASE 50 application, the shoot and root dry weights of sunflower showed a trend of increasing and then decreasing with different concentrations of acetic acid at all time points, which is consistent with the previous studies [63][64][65].It can be seen that moderate levels of acetic acid are the most beneficial for increasing the shoot and root dry weights of oilseed sunflower.Studies have shown that the application of high concentrations of organic acids can result in a significant decrease of plant biomass [66,67], which may be due to the fact that high concentrations of organic acids exceed the plant's tolerance range, impairing its growth and leading to the inhibition of lateral root development near the plant's root tip, as well as to an inability of the plant's root system to absorb nutrients properly due to the combination with Cd ions in the soil, thus resulting in a decrease of biomass [68].Therefore, the effects of acetic acid on plant growth and biomass are different under different application times and concentrations.Scientific application of organic acids under Cd stress can effectively regulate plant root, stem, and leaf development, and can significantly improve plant biomass.
Cd contamination in soils is long-term and irreversible.Cd can only be transformed from one state to another after entering the soil, and the activity varies greatly between different forms of Cd.The content of Cd in the active state in the soil largely determines the effect of phytoremediation on Cd-contaminated soils.Studies have shown that the application of organic acids increases the content of Cd in the active state and has a significant activating effect on Cd in soils [31,69,70].This study showed that the application of different concentrations of acetic acid at different time points decreased the exchangeable Cd content, Fe-Mn oxide Cd content, and organic Cd content, while the carbonate Cd content increased in the rhizosphere soils compared to CK, which is similar to the results of previous studies [33,71].The exchangeable form of Cd was the most mobile and readily taken up by plants, while the Fe-Mn oxide bound and organic matter bound forms were less mobile in the soil.Acetic acid contains functional groups that can complex with Cd to form soluble acetic acid-Cd complexes, while the application of acetic acid will acidify the rhizosphere environment of the soils in the short term, promoting the release of Cd from the soil and increasing the solubility of Cd, transforming it from a relatively stable state to an effective state that can be easily absorbed by plants, and promoting the transformation of the Fe-Mn oxide bound and organic matter bound states to the exchangeable state [71].It has been shown that organic acids can also reduce the toxicity of Cd by promoting the expression of enzymatic and non-enzymatic antioxidants and certain tolerance genes in plants to maintain normal cell forms [72], thus enhancing the uptake of Cd ions and complexes by plants, improving the ability of plants to remediate Cd-contaminated soil, and reducing the exchangeable Cd content, Fe-Mn oxide Cd content, and organic Cd content in the rhizosphere soil.The results showed that the applications of acetic acid at DASE 30 and 40 were more beneficial to reduce the contents of exchangeable Cd, the contents of Fe-Mn oxide Cd, and the contents of organic Cd in rhizosphere soils.Meanwhile, application of 5 and 6 mmol/kg acetic acid at DASE 30 and 40 promoted an increase of the contents of carbonate Cd in rhizosphere soils.This is similar to the research result of a previous study [71].It may be due to the undeveloped root system of the plants in the early stage, which was easily affected by the application, as well as to the moderate time of addition.The root system of the plant was developed and the enrichment capacity of Cd was enhanced, which led to the reduction of the total amount of Cd in the rhizosphere soils and to the increase of the removal rate.
The relationship between soil indicators and the dry weight of oilseed sunflower was investigated in this paper.The study showed that CAT, SC, C-Cd, and pH were positively correlated with the shoot and root dry weight of oilseed sunflower.O-Cd, FM-Cd, T-Cd, E-Cd, and AL were negatively correlated with the shoot and root dry matter weight of oilseed sunflower, among which O-Cd and FM-Cd had significant effects on the dry weight of oilseed sunflower, and CAT also had obvious effects.The application of acetic acid significantly increased the soil CAT activity, and the increase of CAT effectively reduced soil toxicity, thus promoting the growth of oilseed sunflower, which is consistent with the results of a previous study [47].In addition, there were numerous studies which showed that the higher the content of Cd, the more detrimental the soil was to the growth of plants.In this study, the content of O-Cd, FM-Cd, and T-Cd in soils had a negative correlation with the change in dry weight of oilseed sunflower.The Cd in soils inhibited plant growth, and the application of acetic acid significantly alleviated this inhibition, which is similar to the results of previous studies [37,41].
In addition to the current study, we previously discussed other organic acids..For example, the optimal application strategy of citric acid was to apply 1 mmol/kg at DASE 40 and 50 [40], and the optimal application strategy of oxalic acid was to apply 4 mmol/kg at DASE 30 [71], which may be because the high concentrations of citric acid and acetic acid were more conducive to an increase of the microbial activity, leading to a remedy of a portion of the Cd content [43,44], reducing the remediation effect of oilseed sunflower.In general, adding acetic acids increased the biomass of oilseed sunflower and decreased the concentration of Cd in soils, and was a good low molecular organic acid for repairing Cd pollution.However, this experiment only studied some basic soil indexes related to Cd and the biomass of oilseed sunflower, and ignored the measurement of soil nutrient indexes, such as organic matter, N, P, K, etc.In addition, this experiment only studied a series of rhizosphere soil changes through a pot experiment; the experimental conditions were well controlled, but the promotion to the field may be different due to uncertain factors such as rainfall, which could make it difficult to obtain accurate measures of the concentration of acetic acid or other experimental conditions.At the same time, field soil may easily form macropores after rainfall or irrigation, resulting in the nutrient loss [73], etc., which needs further research in the future.

Conclusions
(1) When 4 mmol/kg acetic acid was applied at DASE 20 and 2 mmol/kg acetic acid was applied at DASE 50, the pH value in rhizosphere soils decreased significantly compared to CK. Acetic acid promoted the activity of rhizosphere soil sucrase and catalase, and amylase activity tended to decrease and then increase with the time of application.
(2) The application of acetic acid significantly promoted the increase of shoot and root dry weight of oilseed sunflower.Except for the application of acetic acids at DASE 50, the trend of increasing shoot and root dry weight of oilseed sunflower at different concentrations of acetic acid at all other time points firstly increased and then decreased, which showed that a moderate concentration of acetic acid was more suitable for the increase of the dry weight of all parts of oilseed sunflower.(3) Application of different concentrations of acetic acid at different times reduced the exchangeable Cd, Fe-Mn oxide Cd, and organic Cd contents and increased the carbonate Cd content of the rhizosphere soil.In comparison, the application of acetic acids at DASE 30 and 40 were more beneficial to the reduction of rhizosphere soils' exchangeable Cd, Fe-Mn oxide Cd and organic Cd, and the application of 5 and 6 mmol/kg acetic acids at DASE 30 and 40 were beneficial to the increase of rhizosphere soils' carbonate Cd.Data Availability Statement: Not applicable.

Figure 1 .
Figure 1.Exchangeable Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.Figure 1. Exchangeable Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 1 .
Figure 1.Exchangeable Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.Figure 1. Exchangeable Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 2 .
Figure 2. carbonate Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 2 .
Figure 2. Carbonate Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Minerals 2022, 12 , x 6 of 22 Figure 3 .
Figure 3. Fe-Mn oxide Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 3 .
Figure 3. Fe-Mn oxide Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 4 .
Figure 4. organic Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 4 .
Figure 4. Organic Cd content in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 5 .
Figure 5. Cd removal rate in rhizosphere soils under various application concentrations and application periods.

Figure 5 .
Figure 5. Cd removal rate in rhizosphere soils under various application concentrations and application periods.

Figure 6 .
Figure 6.The amylase activity in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 6 . 22 Figure 7 .
Figure 6.The amylase activity in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.Minerals 2022, 12, x 10 of 22

Figure 7 .
Figure 7.The sucrase activity in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 8
Figure 8 shows the catalase activity of the rhizosphere soils in different treatments.The application of different concentrations of acetic acid at DASE 20, 30, 40, and 50 increased the catalase activity of rhizosphere soils.Compared to CK, when 2 mmol/kg acetic acid was

Minerals 2022, 12 , x 11 of 22 Figure 8 .
Figure 8.The catalase activity in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 8 .
Figure 8.The catalase activity in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 9 .
Figure 9. Dry weight of oilseed sunflower shoot under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 9 .
Figure 9. Dry weight of oilseed sunflower shoot under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Minerals 2022, 12 , x 13 of 22 Figure 10 .
Figure 10.Dry weight of oilseed sunflower root under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Figure 10 .
Figure 10.Dry weight of oilseed sunflower root under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Minerals 2022, 12 , x 14 of 22 Figure 11 .
Figure 11.pH in rhizosphere soils under various application concentrations and application periods.
indicate the 9 explanatory variables of rhizosphere soils and the blue arrows in Figure 12 indicate the shoot and root dry weight of oilseed sunflower.The angles between CAT, SC, C−Cd, and pH and the arrows of the shoot and root dry weight of oilseed sunflower are all acute angles, indicating that there were positively correlated with the shoot and root dry weight of oilseed sunflower.On the contrary, the angles between O−Cd, FM−Cd, T−Cd, E−Cd, and AL and the arrows of the shoot and root dry weight of oilseed sunflower are obtuse.This means that they were negatively correlated with the shoot and root dry weight of oilseed sunflower.Among them, O−Cd and FM−Cd have significant effects on the dry weight of oilseed sunflower.

Figure 11 .
Figure 11.pH in rhizosphere soils under various application concentrations and application periods.

Figure 12 .
Figure 12.Redundancy analysis among soil chemical characteristics, oil sunflower biomass, and availability of Cd.
(4) CAT, SC, C-Cd, and pH were positively correlated with the shoot and root dry weight of oilseed sunflower; O-Cd, FM-Cd, T-Cd, E-Cd, and AL were negatively correlated with the shoot and root dry matter weight of oilseed sunflower.Among them, O-Cd and FM-Cd had significant effects on the dry weight of oilseed sunflower, while CAT also had obvious effects.(5)Combining the dry weight of oilseed sunflower and soil indicators, the application of 1 mmol/kg acetic acid at DASE 40 had the best effect on the remediation of Cdcontaminated soil under the conditions of this experiment.Author Contributions: Conceptualization, D.Q.; Methodology, Y.W.; Software, Y.W.; Formal analysis, Y.W.; Investigation, Y.H. and D.Z.; Resources, Y.H.; Data curation, D.Q., Y.H. and D.Z.; Writingoriginal draft preparation, Y.W.; Writing-review and editing, D.Q. and Y.H.; Supervision, D.Q.; Project administration, D.Q.; Funding acquisition, D.Q.All authors have read and agreed to the published version of the manuscript.Funding: This work was jointly funded by the Natural Science Foundation of China (grant numbers 51879268).

Table 1 .
The physical and chemical properties of soil.

Table 2 .
Total Cd in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.

Table 2 .
Total Cd in rhizosphere soils under various application concentrations and application periods.Different lowercase letters show significant differences for p < 0.05.