The Effect of Dibutyl Phthalate (DBP) on Soybean Seed Germination

: Industrial plasticizer phthalate esters (PAEs) is commonly utilized in PVC products. One of the most widespread plastic additives, dibutyl phthalate (DBP), is a known endocrine disruptor in the environment. The high volatility and low durability of DBP mean that it is present in soil, water, and air and can be taken up by the roots of plants, where it may stunt their development. Soybean seeds were used in a series of experiments in which the effects of DBP on germination were measured at doses of 0, 0.1, 0.5, 1.0, 5.0, and 10.0 mg/L. Soluble sugar content, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), isocitrate lyase (ICL), and malate dehydrogenase (MDH) levels were evaluated on days 1, 3, 5, and 7. The results demonstrated that the concentration and duration of DBP treatment both contribute to the observable effect on soybean seeds. Soybean germination, antioxidant enzyme activity, and glyoxylate cycling enzyme activity were all stimulated by DBP at concentrations below 1 mg/L, and the stimulatory impact was negatively linked with increasing DBP concentration. Soybean seed germination, antioxidant enzyme activity, and glyoxylate cycling enzyme were all suppressed at concentrations of DBP >1mg/L. Inhibitory action was improved with an elevation of DBP concentration.


Introduction
One of the most used industrial plasticizers is a phthalic acid ester (PAE).Vegetable, fruit, and fungal plant development have been the primary focus of studies on the impacts of phthalates.This study provides a novel perspective for future investigations into the impacts of PAEs on plants by using a common oilseed crop, soybean, to examine the impact of dibutyl phthalate on germination.One of the most popular plasticizers is dibutyl phthalate (DBP), a phthalate ester (PAE) that is also one of the most easily released from polymers and into the environment, where it can harm living things due to its instability [1] .Plantbased endocrine disruptor DBP can accumulate in humans through the food chain, where it can interfere with normal hormone synthesis and other processes in the endocrine system.This is why many national governments have included DBP and other PAEs on their lists of pollutants that need to be tracked [2] .Hence, researching the impacts of DBP on antioxidant enzymes and glyoxylate cycle enzymes in soybean seed germination, from both the stress response of the cellular reactive oxygen system under adversity and the intracellular sugar metabolism route, can give a more comprehensive and systematic analysis of the harmful effects of DBP on plant seed growth, thereby advancing the study of the relationship between DBP pollution and crop yield in agricultural production applications.The theoretical insights gained from this research will be invaluable for improving the quality of life for all living things.

Material
Specifically, the soybean seeds used in this study came from the Weihai Agricultural Market in the Chinese province of Shandong.The whole soybean seeds were chosen, steeped for 6 hours to increase their water content, and then rinsed in distilled water.As experimental materials, we chose soybean seeds that were both consistent in size and had developed entire pods.DBP levels were adjusted to 0 mg/L, 0.1 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L, and 10 mg/L.Initially, an analytically pure mother liquor containing 1 g/L DBP was made.For better solubilization, we added a trace amount of Tween-80 (0.01%) [3] and diluted the mixture many times in rapid succession.Any leftover mother liquor or preparation liquor was kept in the fridge at 4 degrees Celsius.

Design
The materials for the experiment were distributed uniformly among Petri dishes (a small amount of cotton and filter paper had been laid out beforehand).Each petri dish contained 20 seeds.Each DBP concentration was assigned to three separate groups (including a control group), for a total of fifteen groups, which were then incubated at 25 ° C in the presence of natural light.
After a 1-day, 3-day, 5-day, and 7-day intervention with varying doses of DBP solution, samples were collected at each time point.Each time, five seeds that had germinated were chosen at random.Each sample's embryos were extracted by removing the seed coat and then documented in a labeled, standardized clear sealed bag of the proper size.
(2) Using the nitrogen blue tetrazolium technique to evaluate SOD activity, 50% inhibition of photoreduction was expressed as one enzyme activity unit [5] ; this value was then used to compute SOD activity.
(3) Determining the POD's activity with the guaiacol technique.One unit of peroxidase activity [6] corresponds to a change of 0.01 in OD470 per minute.
(4) Using the H2O2 UV spectrophotometric technique, the activity of CAT was measured as a unit of enzyme activity with a 0.1 per minute change in OD240 [7] .
(5) Calculation of ICL by measuring the amount of glyoxylate being generated using UV spectrophotometry [8] ; MDH was measured according to Meijer et al (2007).'s protocol [9] .

Data analysis
The data were analyzed using SPSS 22.0 and the S-N-K test (significance level P = 0.05).The mapping tool utilized was Origin 8.

Effect of DBP on the Soluble Sugar Content of Soybean Seeds
Notes: a, b, c, d, e refer a conspicuous difference, P<0.05.sugar content in soybean seeds showed a trend of increasing first and then decreasing with the increase in DBP concentration, and with the increase in the mass concentration of DBP, and there were very significant differences in the contents of each group (P0.01), with 0.1 mg/L being the maximum concentration.The concentrations of soluble sugar in the 0.1 and 0.5 mg/L groups were 14.0 and 6.6 percent greater, respectively than in the control group, but the concentration of 1 mg/L had no discernible effect.When compared to the control group, the soluble sugar concentration in the treatment groups with 5 and 10 mg/L decreased by 11.15% and 20.33%, respectively (P0.05).

Effect of DBP on the Activity of SOD, POD and CAT in Soybean Seeds
Figure 2A demonstrates that SOD activity increased with increasing DBP concentration over the first three days, and continued to rise in the 0.1 mg/L, 0.5 mg/L, and 1 mg/L group on day 5; in contrast, SOD activity declined dramatically in the 5 mg/L and 10 mg/L groups.All groups showed significant differences from the control group by 7 days (P0.01), with the 0.1 mg/L and 0.5 mg/L groups showing the greatest increases (44.84% and 46.57%, respectively) compared to the control group.The SOD activity began to decline, however, in the 1 mg/L group.In comparison to the control group, SOD activity continued to drop in both the 5 mg/L and 10 mg/L treatment groups (3.03% and 17.48%, respectively).Figure 2B displays the overall trend of POD activity decreasing, increasing, and then decreasing over time.There were statistically significant deviations (P0.01) between every group and the control group.POD activity in soybean seeds from each group was lower than that of the control group after 24 hours, and it continued to decline with increasing DBP concentration.On day 3, however, POD activity spiked in all groups and was higher than in the control group, indicating a tendency of rising activity with increasing concentration.In the low-concentration group, POD activity kept rising on day 5.The POD activity in the high-concentration group, on the other hand, leveled out after initially being much higher than in the control group.By day 7, POD activity had dropped across the board, with no significant difference between the 5 mg/L groups and the control group (P>0.05).
Soybean CAT activity increased and then decreased as indicated in Fig. 2C when the DBP concentration was between 0.1 and 5 mg/L.Soybean seed CAT activity gradually decreased when the DBP concentration was 10 mg/L.The 0.1-5 mg/L group had significantly lower CAT activity than the control group on day 1 (P0.01).CAT activity in the 10 mg/L groups was 2.5% greater than in the control group.On day 3, the CAT activity of the 0.1-1mg/L group increased by 2.73 percent, 4.72 percent, and 7.93 percent, respectively, compared with the control group, while the CAT activity of the 10 mg/L groups fell by 13.02 percent.When compared to the control group, there was a statistically significant (P 0.05) drop in CAT activity on day 5.It was shown that the amplitude fell by 3.62%, 8.40%, 16.93%, 31.40%, and 41.30%, respectively, when the DBP concentration increased from day 1 to day 7.

Effect of DBP on the Activity of ICL and MDH in Soybean Seeds
Figure 3A demonstrates that on day 1, ICL activity was significantly decreased in all groups compared to the control group (P0.05).Day 3 saw a general upward trend in ICL activity across all three groups (0.1mg/L, 0.5mg/L, and 1mg/L).Increased ICL activity was observed in all three groups on day 5 (0.1mg/L, 0.5mg/L, and 1mg/L).When compared to the control group, the ICL activity in the 0.1mg/L and 0.5mg/L groups was considerably higher by 14.66% and 8.69%, respectively, while the rise in the 1mg/L group was not statistically different from the control group (P > 0.05).Even though ICL activity dropped across the board by day 7 (even in the control group), it remained statistically significant in the 0.1mg/L and 0.5mg/L groups (P 0.05).
Figure 3B demonstrates an uptick in MDH activity followed by a decline in its level of expression.At first, as DBP content rose, so did MDH activity in soybean seeds.On day three, all groups had significantly higher MDH activity than the control group (P 0.05), with the 0.1mg/L group showing the greatest rate of increase (44.63 percent higher than the control group).On day 5, MDH activity in the 0.1-1 mg/L group reached a maximum, increasing by 53.57percent, 47.22 percent, and 37.70 percent, respectively, relative to the control group.On day 7, MDH activity was reduced in all groups and tended to diminish with the increase in DBP concentration; nevertheless, it mained substantially higher than that in the control group (P0.05).

Effect of DBP on the Soluble Sugar Content of Soybean Seeds
Oil seeds rely on soluble sugar, a byproduct of fat metabolism, for the energy and nutrients they need to grow and develop during the germination process. [10].Li Jiahua et al. (2005) looked at how the soluble sugar content of bittercress changed with the DBP concentration and found no strong association between the two. [11].This, however, ran counter to the findings of this particular investigation.A decrease in soluble sugar content was seen across all concentration groups as DBP concentration was increased (see Fig. 1).Possible explanations include the fact that bittercress is an aquatic plant whose photosynthesis continues unabated in the presence of DBP contamination and the plant's ability to decompose and transfer soluble carbohydrates.Soybean seeds, on the other hand, are oil crops that do not engage in photosynthesis but are instead subject to DBP pollution during the germination process.The soluble sugar level in soybean seeds is inversely associated with DBP concentration, leading researchers to believe that enzymes in GAC are more sensitive to DBP pressure.

Effect of DBP on the Activity of SOD, POD and CAT in Soybean Seeds
Accumulation of reactive oxygen species (ROS) and its metabolites during plant stress is harmful because it exceeds the plant's typical tolerance range for ROS and its metabolites and causes severe oxidative damage to cells.Antioxidant enzymes, another type of enzyme with a protective impact on the plant, are able to efficiently scavenge ROS and its byproducts, limit the damage to the cell membrane system caused by reactive oxygen species and keep the plant growing normally [12] .Wheat seedling SOD, POD, and CAT activities were found to increase at low DBP and MBP concentrations and decrease at high concentrations.As expected, the results showed that increasing concentrations of DBP inhibited SOD, POD, and CAT activity in wheat seedlings [13] .Soybean seed SOD, POD, and CAT enzyme activity also varied with DBP stress concentration, and the pattern was comparable (as shown in FIG. 2).Soybean seed germination and antioxidant enzyme activity benefit greatly from DBP at concentrations below 1 mg/L.Soybean seed development was inhibited more rapidly and dramatically at higher DBP concentrations (>1 mg/L).As a result, it appeared that soybean seeds were more resistant to lower concentrations of DBP than they were to larger concentrations of DBP throughout the same time frame.Consequently, the effect of DBP on soybean seeds is a function of both the concentration of DBP and the length of time the seeds are exposed to it.Antioxidant enzymes may have a threshold of tolerance to DBP concentrations of 1 mcg/L.More than this concentration, the soybean seed's ROS clearance rate drops dramatically, and the resulting damage is much more severe.

Effect of DBP on the Activity of ICL and MDH in Soybean Seeds
A great deal of fat is stored in soybean seeds; this fat is broken down during germination into glycerol and fatty acids, and then into sugars for the embryo's growth and development.Both ICL and MDH are essential for the synthesis of soluble sugars through their functions in the uronic acid pathway and the citric acid cycle.Furthermore, the effect of DBP on soluble sugar concentration during the germination of soybean seeds can be further explained by evaluating changes in the activity of ICL and MDH.Previously, research by Carvalho et al. (2014) demonstrated that arsenic's effects on ICL activity in soybean seed embryos ranged from an increase at low concentrations to a considerable decrease at high doses [14] .The findings from this experiment are consistent with this theory.Figure 3A shows that the pattern of change in MDH activity in soybean seeds is quite similar to that of ICL.When concentration is low, the boost is larger.Increases in ICL and MDH activities of soybean seeds were inhibited at concentrations over 1mg/L, with a clear declining trend, showing that DBP's inhibitory impact improved with increasing concentration.

Conclusion
During the germination of soybean seeds, the researchers examined the variations in soluble sugar content and enzyme activity of SOD, POD, CAT, ICL, and MDH.The discussion above suggests that the effect of DBP on soybean seed germination is not only proportional to DBP concentration, but also to treatment duration.The low concentration of DBP (1mg/L) improved the germination of soybean seeds and raised the activities of antioxidant enzymes, ICL and MDH, with a negative connection between concentration and promotion impact.The activity of antioxidant enzymes and sugar metabolizing enzymes in soybean seeds was decreased at high concentrations of DBP (>1mg/L), despite a short-term trend of mild enhancement.In the meantime, it is hypothesized that 1 mg/L may represent a tolerance barrier for DBP in soybean seeds, at which the inhibitory effect of DBP on soybean seeds begins to increase significantly.

Fig. 1 .
Fig. 1.Effect of DBP on the soluble sugar content of soybean seeds Figure 1 displays the results of the changes in soluble sugar content in soybean seeds after one week of incubation with different concentrations of DBP.The soluble

Fig. 2 .
Fig. 2. Effect of DBP stress on SOD, POD and CAT activity in soybean seeds

Fig. 3
Fig.3 Effect of DBP stress on ICL and MDH activity in soybean seeds Group B Malate dehydrogenase (MDH) Group A Isocitrate lyase (ICL)Isocitrate lyase Time(d) Notes: a, b, c, d, e, f refer a conspicuous difference, P<0.05.