There were many hydroponic lettuce researches reported , especially, in terms of ecotoxicological testing, lettuce has been recommended by the Environmental Protection Agency of USEPA and the Food and Drug Administration, USFDA . Modified Hoagland nutrition and 2-fold ozone-treated sludge dilution solutions were used in hydroponic lettuce here, which was simple, rapid and economic. The domestic sludge was treated continuously by ozone until the concentration of fecal Escherichia coli was less than 1000 colony forming units 100 mL-1 , that is the water quality criterion in GB 5084 . The dilution ratio of the ozonetreated domestic sludge in the irrigation was optimized for lettuce growth. This study works on the scientific exploration of vegetable planting fertilized by the low-cost domestic sludge. This experiment was carried out from November 1, 2015 to January 9, 2016 in the greenhouse of Agro-environmental Protection Institute, Ministry of Agriculture, China over a period of 70 d. Lettuce seeds of the cultivar Dasusheng were used in all experiments. The lettuce seeds were grown in the artificial substrate and irrigated with water. Three weeks later, when the seedlings grew to four or five true leaves, they were transplanted . Plant roots were washed with distilled water before planted. The average temperature is 15.6°C in the greenhouse, and the air relative humidity is 41.3%. And the characteristics of ozone-treated domestic sludge on the lettuce growth improvement in the water cultivation process were investigated.The representative five plants were removed from each hydroponics at 15 d intervals, and the leaf number, plant height, root length, root weight, fresh weight and dry weight were determined, respectively. The plant roots were cut off and weighted in a Thousandth Balance after the surface water was absorbed by filter paper. The fresh weight was the weight of the whole plant and measured with Thousandth Balance too. The chlorophyll content was determined directly using the SPAD-502 Plus Portable Chlorophyll Meter .
The soluble sugar was determined by the anthrone colorimetric method, and glucose is used to make the standard curve . One gram of plant samples was ground into homogenate, transferred to 20-mL scale test tube, kept in boiling water for 10 min. After the homogenate was cool, it was filtrated into volumetric flask. Then, 1-mL filtrate was put into 20-mL tube with stopper and mixed with 1 mL water, 0.5 mL anthrone reagent and 5 mL concentrated sulfuric acid ,cannabis square pot then kept in a boiling water bath for 10 min. At last, optical density value was measured using Spectrophotometer under the wavelength of 620 nm . The content of ascorbic acid was determined by 2,6-dichlorophenol titration. A total of 200 g lettuce leaves were crushed and added to the same amount of 2% oxalic acid, then shaked for 5 min. And the solution was fixed to 100 mL with 1% oxalate. Then 10 mL of the above solution was taken and mixed with 10 mL 1% oxalic acid and 1 mL chloroform, then titrated to the end with 2,6-dichlorophenol indophenol dye . The nitrates were determined by Ultraviolet Spectrophotometry . A total of 0.51 g samples of lettuce leaves were ground into homogenate with some distilled water, then the homogenate was washed into 100-mL volumetric flask with 30 mL distilled water. Another volumetric flask with 30 mL distilled water only was used as control. Then, 5 mL ammonia buffer , 30 mL distilled water, and 0.5 g powdered activated carbon were added into both flasks and mixed well. Subsequently, protein precipitating agent was added, mixed, and filtered after the sample and control flasks were kept still for 20 min. The absorbance was determined by the quartz color dish in the UV Spectrophotometer. The number of fecal E. coli was measured using enzyme substrate method . The sample was incubated at 44.5°C for 24 h, then the number of E. coli was got by UV irradiation. And the Colilert Reagent was provided by IDEXX, UK.Using biomass as a performance determinant, the lettuce under CK treatment showed the best performance, followed by the lettuces of T2, T1, T3 and T4 treatments.
This trend was observed for all of the agronomic traits determined with the exception of root weight. Root weight was followed the order of CK>T2>T3>T1>T4. The lettuce under CK had obvious advantages in biomass growth, however, its stalk was spindling, which might be caused by excessive nitrogen supply, small temperature differences and lower phosphorusus supply. The common nitrogen and phosphorus contents were 49–210 and 15–198 mg L–1, respectively . In this study, the nitrogen content in modified Hoagland nutrient solution was excessive in comparison with the phosphorus content. Using an analysis of variance, the lettuce cultured by modified Hoagland nutrition solution showed a significant advantage over the others cultured by ozone-treated sludge dilution treatments at all dilutions in the leaf number, plant height, fresh weight, and dry weight . Among all the ozone-treated sludge dilution treatments, the T2 was significantly better than the other three treatments in the leaf number, plant height, root length, fresh weight, and dry weight. Therefore, from the point of view of lettuce agronomic traits, T2 was the most suitable treatment for lettuce growth and development among all ozone-treated sludge dilution solutions. First, the nitrogen and phosphorus contents in T2 dilution were within suitable range. Second, there were some copper and zinc in the hydroponics solution. The results also indicated that T1 had excessive Cu and Zn that limited the lettuce growth. It was also reported by other researches . The lettuce biomass results implied that the nutrients in the ozone-treated sludge dilution solutions could improve lettuce growth at low dilutions and inhibit lettuce growth at high concentration applications. This result might be attributed to the favorable buffering of the nutrient solution around plant roots in the low dilutions, which was similar to that in modified Hoagland nutrition solution. On one hand, it was deduced that the release of organic matter during sludge degradation caused a lack of oxygen to the root tissue. On the other hand, the heavy metal contents in T1 solution were higher than those in CK.
In other words, Cu and Zn contents in the nutrition solution of CK were lower than 100 and 200 μg L–1, respectively. The root elongation bioassay was one of the most straightforward test methods used for environmental monitoring in terms of simplicity, rapidity and economy . Nitrogen and phosphorus contents in T1 nutrition solution was higher than those in CK, but the root length of lettuce in T1 was lower than that in CK. The result was caused by the higher heavy metal contents in T1 solution. There was no significant difference between CK and T2 on the root length, however, the dry biomass of CK was higher than that of T2. It was as a result of lower nitrogen content in T2 than in CK. Additionally, high heavy metal concentrations in the sludge were also harmful, resulting in the decrease of root enzyme activity and the inhibition of root growth. The soluble sugar contents in the lettuce cultured with the ozone-treated sludge dilution solutions and the CK nutrition solution was determined . The soluble sugar contents of lettuce leaves in T2 treatment were the greatest, then followed by T3, T4, T1 and CK. According to the ANOVA analysis, the soluble sugar content of lettuce in T2 treatment showed significantly higher than that of other treatments ; while the soluble sugar contents in the T3 treatment showed significantly higher than that of CK, T1 and T4. There was no significant difference for CK, T1 and T4 treatments.As was known to all, chlorophyll absorbed energy from the light, and then used the energy to turn carbon dioxide and water into carbohydrates. The high soluble sugar content of lettuce in T2 may be the result of an optimal sludge concentration causing overall improvements in plant metabolic activity, transport of trace elements and enzyme activity. These improvements could accelerate adenosine tri-phosphate synthesis and other enzymatic reactions, leading to increased photosynthesis where soluble sugar was the primary by product from this process.
Cu and Zn were the most likely toxicant elements in lettuce when exposed in the sludge dilution solution . The biological toxicity of ozone-treated sludge dilution solution in T1 was presented, which was caused from the higher toxic compounds . The Cu and Zn contents were over the range of control standards, which resulted in the decrease of soluble sugar contents in T1 when compared with that in T2.The Vc content in lettuce cultured by various ozone-treated sludge dilution solutions and the CK nutrition solution were calculated by ascorbic acid measurements . The Vc content of lettuce in T2 was the highest among all the treatments, then followed by T4, T1, T3 and CK. And the Vc content of lettcue in T2 was significantly higher than that of other treatments. No significant difference was observed among the T1, T3, and T4 ozone-treated sludge dilution treatments. The trend of Vc was similar to the trend of chlorophyll and soluble sugar contents for all treatments. Vc was especially found in vegetables and fruits and an essential nutritional element for human and cannot be synthesized within the human body. This vitamin must be obtained from external sources such as food. If the Vc storage content in human body was less than 300 mg, some symptoms would appear, such as increased capillary fragility and subcutaneous haemorrhaging to some degree . Therefore, Vc was an important index for lettuce quality evaluation. The Vc content in the lettuce from the T2 treatment was significantly higher than that of other treatments, demonstrating the advantages of the nutritional value in ozone-treated sludge applications when compared to the modified Hoagland nutrient solution.The nitrate contents in the lettuce leaves cultured by the ozone-treated sludge dilution and the CK nutrition solutions were determined. The results demonstrated that the nitrate content in the lettuce leaves under CK was the highest among all the treatments, followed by T1, T2, T3, and T4 treatments .
Based on the ANOVA results, there were significant differences between CK and the ozone-treated sludge dilution treatments on the nitrate content in lettuce. Among the ozone-treated sludge dilution treatments, nitrate content in T1 showed significantly higher than that in the T2, T3, and T4 treatments . The nitrate contents of the cultured lettuce in the T1, T2, T3 and T4 were 34.3, 53.93, 55.34, and 68.60% lower than that in CK treatment, respectively. Nitrate content is one of the most important indices in vegetable safety evaluation. Normally, trim tray a certain amount of nitrate in vegetables is harmless to human health. However, nitrate can be converted into nitrite by plants, fungi and human gut bacteria, resulting in the formation of compound nitrosamine that is a strong carcinogen . In addition, excessive absorption of nitrate can lead to mental and developmental retardation, low levels of methemoglobin, or even death . According to the requirements of criterion in GB18406.1 , the nitrate content must be below 600 mg kg–1 in fruits and vegetables, 1200 mg kg–1 in root vegetables, and 3000 mg kg–1 in leafy vegetables. Because 80% of the nitrogen in modified Hoagland nutrition solution is nitrate, the cultivated plant will absorb and accumulate excessive nitrate after the ammonium nitrogen is exhausted . While, the plant can utilize ammonium nitrogen directly when cultivated by the ozone-treated sludge dilution solution due to the main nitrogen source of this solution . Thus, the nitrate contents of lettuce treated by the ozonetreated sludge dilution solution satisfied the nitrate limit in leafy vegetables set by the GB18406.1- 2001 . As shown in Fig. 3-D, nitrate contents in all the treatments of this study were within security scope, and the nitrate contents of lettuce in T1-T4 were significantly lower than that in CK, which indicated that the lettuces cultivated with demestic sludge were security when evaluated with nitrate index only. The result was consisted with the nitrate contents in the nutrition solution, and the nitrate in CK was the highest. However, the leaves of lettuce in T4 are pale green or yellow, which is typical of nitrogen deficiency. Therefore, T2 and T3 were suitable solutions in terms of nitrogen requirements and security of plant.In this hydroponic experiment, we found that 2-fold ozonetreated sludge dilution solution could improve the quality of hydroponic lettuce more compared to the modified Hoagland nutrient solution.