Nutritional Relationships and Accumulation Capacity of Broccoli (Brassica Oleracea var. Italica) Grown Under the Stress Caused by Some Heavy Metals Seen in Agricultural Areas

Agricultural soils lose their properties as a result of pollution caused by tra�c, industry, agricultural activities, and urban activities. Among various techniques for the remediation of agricultural soils, phytoremediation is the most cost effective and applicable technique. Phyto extraction technique of phytoremediation has been applied with broccoli (Brassica oleracea var. italica) in this study to remediate some heavy metals (Cd, Cr, and Pb). The study was carried out in controlled conditions in pots according to randomized blocks design using three replicates. Cadmium was applied as only cadmium (30 mg/kg), and together with a chelator as Cd (30 mg/kg) + EDTA, in triplicates. Chromium was applied as only chromium (10 mg/kg), and together with a chelator as Cr (10 mg/kg) +EDTA. Similarly, lead was applied as only lead (100 mg/kg), and together with a chelator as Pb (100 mg/kg) + EDTA. After 30 days of incubation, one broccoli was cultivated in each pot. Plants were harvested after 2 months of experimental period. Agro-morphological traits of the plants were measured. Some macro and micro nutrient elements and Cd, Cr and Pb elemental analyses were carried out from root and shoot pf the plants. Among the agro-morphological traits, plant wet weight was the highest in lead applied pots. This was statistically signi�cant at 5% and they fell into different groups with control pots according to Duncan test. The highest root and shoot dry weights were detected in lead and chromium applied pots. Changes in chlorophyll contents were found to be statistically insigni�cant. The highest lead, cadmium and chromium contents in the body of broccoli (Brassica oleracea var. italica) were found to be 14.0 mg/kg, 6.67 mg/kg and, 5.17 mg/kg, respectively, in the chelated applications. The order of accumulation in the root was Cr>Pb>Cd. The results revealed that this plant played a role in the remediation of lead>cadmium>chromium as a hyper accumulator plant, and broccoli grown in neutral soils could be used as a hyper accumulator plant in the phytoremediation technology.


Introduction
Pollution of soil and water sources by toxic heavy metals usually occur as a result of human activities.Technologies for cleaning up soils contaminated by heavy metals require a big capital (Gupta et al. 2013).Phytoremediaiton appears as an economical new technology which can be practically applicated for the elimination of problems caused by pollutants.Phytoremediation has been derived from the words "phyto" which means plant, and "remediation" which means cleaning up; it entered the terminology in 1991 and is de ned as "bioremediation", "botanical remediation", and "green remediaition".Phytoremediation is aneasily applied technology to clean up the environment whose basis is formed by accumulator/hyper accumulator plants (Sadowsky 1999;EPA 2000).Hyper accumulator plants are able to accumulate high number of pollutants in their bodies and can accumulate 10-500 times more pollutants compared to other plants (Ow 1996 The heavy metal cadmium (Cd) is a serious pollutant that affects human health.It can be found in different forms depending on the chemical interaction in soil, bioavalaibility, variability and toxicity (Jinadasa et al. 2016;Tanget al. 2016).Cadmium can enter the soil via natural and antropogenic sources.Volcacanic eruptions, forest res, and rock wheaterings are the natural Cd sources (Khanet al. 2017).Thre is high amount of Cd in ma c and ultrama c rocks.Smelting, wastes of cement production, use of fertilizers with phosphate, ash wastes of fossil fuels are the main antrophological reasons of Cd pollution in soil (Järup 2003;Panet al. 2016).Other Cd sources are plastic stabilizators, batteries, fungicides, wastes of latex and textile productions, motor oil, solar panels and pigments (Khanet al. 2017;Mahmoodet al. 2019).High Cd concentrations casues problems in physiological and biochemical processes in plants.Cd intensity was reported to casue reduction in plant wet weight, leaf length, chlorophyll content, stoma conductivity, seed germination and ATP content (Khanet al. 2017;Rizwanet al. 2017).Cadmium inhibits photosynthesis in plants and causes mitochondrial degradation, therefore inhibits food uptake, delays shoot growth, and casues cell death and chlorosis (Khanet al. 2017).It also interferes with the metabolic processes of the plant, inhibits proton pumps, decreases root growth and harms photosynthetic processes (Mahmoodet al. 2019;Ra qet al. 2014).
The heavy metal lead (Pb) can contaminate by many ways, and it has been found to be in a toxic level in the near-road agricultural areas due to tra c.When it enters the cells, it causes toxicity by changin the permability of the cell membrane, reacting with active metabolic enzyme groups, changing essential ions, and forming complex compounds with ADP and ATP.Lead toxicity causes inhibited ezbtme activities, disrupted mineral nutritions, water imbalance, hormonal disturbance, inhibited ATP production, lipid peroxidation, changes in membrane permeability, and DNA damage due to overproduction of reactişve oxygen species (Kumar et  Chromium toxicity appears in photosynthesis as inhibition of pigment biosynthesis, photosynthetic electron transfer and Calcin cycle, accelerating lipid peroxidation, and distruption of thylakoid membrane.An in vivo examination of inhibition effects of different doses of chromium (Cr +6 ) on light reactions of photosynthesis of Pisum sativum L. was carried out.The results revelaed inhibition of electron carriage between systems, and affecting the level ol b6f complex levels.The inhibitory effects of Cr on PSI were more signi cant than those on PSII.The sensitivity of used kinetic parameters on the functions of photosynthetic reactions was thought to be suitable for the early diagnosis of toxic effects of pollutants on the plants.Many  The family Brassicaceae is an economically important family with its 372 genus and 4060 species.Some members of Brassicaceae are well known hyper accumulators.They have become good candidates for remediation of areas contaminated by various metals/metalloids as they can translocate high amounts of metals from roots to the shoots without a phytotoxic symptom.These plants tolerate by partitioning higher amounts of heavy metals in the vacuoles of the above soil parts.This is carried out by excess expression of some metal carriers in different tissues and affects the storage in the leaves.Studies to understand the hyperaccumulatin and hyper tolerance characteristics related with Brassicaceae, gathering of metals and detoxi cation have been carried out (Daud et al. 2018).The useage of hyperaccumulators results in low biomass rich in metals, which is an easy and economic way of metal recovery and safe elimination.On the other hand, use of non accumulators result in an expensive to safely eradicate, non economical processing to recover metals, large biomass with poor metal accumulation.Many accumulator plants used in the remediation of heavy metals are members of Brassicaceae family (Adiloğlu 2016; Adiloğlu 2018; Adiloğlu and Gürgan

2020; Adiloğlu and Pamay 2021).
There is a need for inexpensive and e cient biological solutions for the problem of heavy metal contamination of agricultural soils.Phytoremediation is the leading of such solutions.The choice of hyperaccumulator plant is very important for this method.That is why in this study the the accumulator capacity of Broccoli (Brassica oleracea var.italica) for the remediation of soil contaminated by chromium, lead and cadmium was evaluated.

Materials And Methods
The soil was taken from 0-30 cm depth.It was air dried, minced and sifted through a 4 mm sieve, and put into the pots.The trial was carried out inthe labs of Soil Science and Plant Nutrition Department, Faculty of AgricultureTekirdağ Namık Kemal University, according to Randomized Blocks Design with triplicates.Three contaminants were used as CdSO 4 , Cr(NO 3 ) 3 and Pb(NO 3 ) 2 in the amounts of 30 mgkg −1 Cd, 10 mgkg −1 Cr (IV) and 100 mgkg −1 Pb, respectively.Constant amount of chelator (10 mmol/kg EDTA) were applied to each contaminant dose in order to increase the solubility.The plants were kept at controlled conditions at 22℃ with an availability of sun light.After 30 days of incubation to naturally pollute the soil and absoroption of pollutants by soil colloids, soil samples were taken from each pot and extractable Cd, Cr and Pb analyses were done.A picture from the experiment is given in Figure 1.

Plant analyses
Some agro morphological traits of Broccoli (Brassica oleracea var.italica) harvested the trial were determined (Jones et al. 1991).Plants were dried at 65 0 C for 48 hours to determine the plant dry weight (Kacar and İnal, 2010).Extraction was carried out at lab conditions according to EPA 3052 wet burning metahod with microwave (EPA, 1996), and elemental analyses were carried out by ICP-OES (Inductively Couple Plasma Spectrophotometer).

Soil analyses
Soil samples brought to lab were air dried and sieved with 2 mm sieve before analyses.pH of soil samples was measured in 1/2.5 soil/water mixture according to Jackson (1967).
Texture fractions of the study soil were done according to Bouyoucos Hydrometer method (Bouyoucos 1955).Organic matter content was determined according to Walkey-Black method (Kacar 1995).
Available phosphorus was determined according to Olsen method (Olsen 1982).

Some extractable heavy metals
For the analysis of some extractable heavy metals 0.005 M DTPA + 0.01 M CaCl 2 + 0.1 M TEA (pH 7.3) was used (Lindsay ve Norvell, 1978) and Cr, Cd and Pb amounts were detected by ICP-OES.

Statistical analyses
Variance analyses were carried out for plant agro morphological traits, nutrition elements and heavy metals in plants and soils, and signi cant mean values were subjected to Duncan multiple comparison tets in SPSS-17 package program.

Results And Discussion
Some chemical data of the experimental soil showed that the soil pH was neutral.The soil was insu cient in terms of organic matter.This situation is positive for this experiment.Because high organic matter in the soil can inactivate contaminats and can inhibit the uptake of them by the plants (Adiloğlu and Sağlam 2015).The lime content was medium, phosphorus and potassium were su cient in the soil.Available Fe and Mn contents were found to below while Zn and Cu contents were su cient.Extractable Cr, Cd and Pb contents were lower than the toxic level.Some chemical and physical properties of the research soil are given in Table 1.Accumulation of some contaminants (Cr, Cd, Pb) in the roots and shoot of Broccoli ( Brassica oleracea var.italica) and contents of contaminants in the soil after the harvest Remediation of Cd, Pb and Cr pollutants and accumulation in Broccoli (Brassica oleracea var.italica) are given in Table 3.
The accumulation of Pb and Cd are in plant body is higher in pots treated with chelator compared to the control pots.This is the result of increased available form of pollutants in the soil solution.Although there is not a signi cant difference between control and Cr applied with chelator pots, there is a great numerical difference.Broccoli was therefore shown to accumulate chromium pollutants in its body depending on the concentration.The accumulation of lead pollutant in plant shoots rather than roots in the pots where lead was applied drove attention in Table 3.The other pollutants accumulated more in the plant roots as expected.The plants which can accumulate pollutants in their above ground parts are more preferred in phytoremediation, therefore broccoli (Brassica oleracea var.italica) was revealed to be employed in the remediation of lead. ) and Indian mustard (Brassica juncea L.) as the accumulator plants in a study to investigate the phytoremediation of arsenic and lead contamination.The absorption of arsenic and lead were shown to increase by the application of EDTA.As a result, they revealed that as and Pb pollution in soil can be inhibited vy the phytoremediation method.
Effects of some contaminats (Cr, Cd, Pb) accumulated in the shoots of Broccoli ( Brassica oleracea var.italica) on some macro and micro nutrient elements Change in the macro micro nutrient elements which are vital for plant development in broccoli plant (Brassica oleracea var.italica) during the remediation of Cd, Pc and Cr heavy metals from the soil used in the research are given in Table 4.A signi cant decrease in macro and micro nutrient elements in the pots where chromium was applied together with the chelator was observed.Copper and zinc among micro nutrient elements were especially found to increase in the case of lead pollution.The reason of this situation is thought to be increased solubility of these micro elements due to EDTA chelator application.Moreover, a synergistic effect might have occurred between these elements.The other pollutant cadmium positively affected all plant macro elements except Mg signi cantly at 5% signi cance level, and the elements fell into different groups in Duncan test.A synergistic effect among all the micro nutrient elements was observed in plants grew in both cadmium and Cd+EDTA applied pots.That is why the plants continued to develop and were not affected from the contaminats that took up into their bodies.
Calcium is absolutely required for synthesis of cell wall and development of the plants.Approximately 90% of Ca is present in cell walls.It behaves as the cohesion factor for cell binding and keeping the structures together in plant tissues.
In case of Ca insu ciency, new tissue generation in roots and shoots slow down.As a result, the productivity of the plant is adversely affected.Calcium is also one of the keystones of plant defense mechanism and helps the recognition and reaction to stress elements.Since calcium is not mobile in plants, the insu ciency symptoms rst appear in young leaves and and young tissues are the rst to be damaged by insu ciency of calcium (Karaman et al. 2012).

9/16
According to Wallace et al. (1976) Cr contents in chromium accumulating plants are really high.For instance, Cr content in the leaves of Cr toxicated plants were 1-4 mg/kg, and even more in the roots.
Effects of some contaminats (Cr, Cd, Pb) accumulated in the roots of Broccoli ( Brassica oleracea var.italica) on some macro and micro nutrient elements The interactions and accumulation of heavy metals in the Broccoli (Brassica oleracea var.italica) in roots with the absolutely required macro and micro nutrient elements are given in Table 5. * Cd: 30 mgkg −1 ** Pb: 100 mgkg −1 *** Cr: 10 mgkg −1 +: % ++: mgkg −1   There was a signi cant increase of the macro nutrient elements except potassium and phosphorus, and micro elements except manganese in the pots where chromium was applied together wih the chelator.Increases in the amounts of all macro and micro nutrietnts except potassium were observed in the case of lead pollution.The reason of this situation is thought to be increased solubility of these micro elements due to EDTA chelator application.Moreover, a synergistic effect might have occurred between these elements.The other pollutant cadmium positively affected all plant macro elements except Ca signi cantly at 5% signi cance level, and the elements fell into different groups in Duncan test.A synergistic effect among all the micro nutrient elements except Zn was observed in plants grew in cadmium and Cd+EDTA applied pots.That is why the plants continued to develop and were not affected from the contaminats thet took up into their bodies.
When chromium (Cr +6 ) enters the root cell, it is easily reduced to Cr (III) form by the Fe (III) reductase enzyme.Since chromium immobilizes in the voids of root cells, a high Cr accumulation is observed in roots (Adhikari et  In root ltration method, pollutants adsorb to the roots or taken by the roots depending on the biotic and abiotic processes. During these processes, pollutants can be uptaken by the soils and can be transported.The immobilization of pollutants on or in the roots is substantial at this point.The pollutants can later be extracted from the plants by various methods.This method can be applied to underground waters, ground wasters and wastewaters (Adiloğlu 2021).
Effects of some contaminants (Cr, Cd, and Pb) on some macro and micro nutrient elements in soil where Broccoli ( Brassica oleracea var.italica) was grown The effects of contaminats and EDTA chelator on some macro and micro nutrient elements in the soils are given in Table 6.All the macro and micro nutrient elements in the soil of cadmium applied pots were signi cantly lower (at 5% signi cancy level).This shows that EDTA chelator increased the uptake of nutrient elements by the plants.
All the macro and micro nutrient elements, except Ca, in soil increased after the application of EDTA in the pots polluted with lead.This was also obvious in plant results and EDTA application to the soil polluted with Pb increased the uptale of nutrient elements from the soil.Plant available nutrient elements were found to be higher in EDTA applied soils after the experiment.These results were statistically signi cant at the level of 5%.
The effects of EDTA applications on some macro and micro nutrient elements of soils after the chromium polluted pots differ.Mg and P of the macro nutrient elements and Fe, Zn, and Mn micro nutrient elements were found to be higher after the application of EDTA.The solubilities of the mentioned elements were signi canly (at 5% level) and positively affected.The relationship between Cr absorption from soil solution to the plant bodies and orgabnic acids in the soil solution was determined, and together with the increased organic acid concentration in soil, the plants were shown to absorb more Cr from soil solution (Srivastava et al. 1999).

Conclusion And Suggestions
Health problems related with environmental pollution increase day by day.Uncontrolled polluted agricultural soils and the unability of remediation form the basis of these problems.The studies revealed that heavy metals reached signi cant levels in soils of our country.This study investigated the potential of phytoremediation method for cleaning up of soils and to carry out a healthy agricultural production.High quality soils will improve public health and life quality through the plants and animals.The usability of Broccoli (Brassicaoleracea var.italica) a hyperaccumulator plant to remediate cadmium, chromium and lead from the soils contaminated due to industrial activities, intense population and intense tra c was revealed by this study.This plant is especially important for the removal of heavy metal lead.Because this contaminant was accumulated especially in the upper parts of the plant and this plant was showed to be a hyperaccumulator to phytoextract the contaminant.Chromium and cadmium accumulated more in the roots of the plant.For these heavy metals, rhizo ltration can be suggested as a phytoremediation method.
Remediation of heavy metal contaminated agricultural soils is vital for the sustainable productivity of soils.Because, contaminated soils negatively affect the health of the organisms, especially humans.Use of hyperaccumulator plants for the remediation of such soils has become popular recently and heavy metals can therefore be cleaned up successfully from the agricultural soils.This study revealed the potential of broccoli (Brassica oleracea var.italica) to remediate the heavy metals Cr, Cd and Pb from the soils.The use of hyperaccumulator plants such as broccoli has been suggested to phytoremediate the ongoing heavy metal pollution in agricultural elds for healty and quality crop production.
Cadmium (Cd) toxicity affects the photosystem II (PS II) (Baker 1991) and stress damage can be easily detected by chlorophyll change in uorescent structures(Maxwell and Johnson 2000).Ribulose-1,5 diphosphate carboxylase (BuBisCo) and phosphoenol pyruvate carboxylase, the two enzymes that take place in CO 2 xation, are the main targets of Cd.It reduces the activity of RuBisCoby changing its structure, by changing the Mg ion which is the vital cofactor of carboxylation reactions, and by directing the Mg ions to oxidation reactions (Shanmugarajet al. 2019).Water oxidation complex of photosystem II is affected by the modi cations of Qb binding sites and replacement of Ca 2+ in Ca/Mn clusters.Stimulation of calmodulin likes proteins by structural changes to regulate mechanisms such as gene regulations, stress tolerance, ion exchange by interacting with Ca 2+ ions signi cantly increase during Cd stress.Concentration signi cantly increases during of Cd stress (Geikenet al. 1998; Yang andPoovaiah 2003; Sigfridssonet al. 2004; DalCorsoet al. 2008; Shanmugarajet al. 2019).Various physiological of plants such as the transpiration ratio, stoma movements, plant water absorbtion, enzyme activity, seed germination, protein synthesis in plant, cell membrane stability, hormonal balance of the plant are negatively affected by chromium contamination (Asri and Sönmez 2006).Salidoet al. (2003) employed Chinese fern (Pteris vittata L.

Table 1
Some physical and chemical datas of the experimental soil Some agro morphological traits of Broccoli (Brassica oleracea var.Italica) used in the experiments and grown on soils contaminated by 30 mgkg −1 Cd, 10 mgkg −1 Cr IV and 100 mgkg −1 Pb are given in Table2.

Table 2
Some agro-morphological measurements* of Broccoli (Brassica oleracea var.italica) *: values are the mean of three replicates +Cd: 30 mgkg −1 ++Pb: 100 mgkg −1 +++Cr: 10 mgkg −1 WW: wet weight DW: dry weight The applied contaminants did not negatively affect the chlorophyll contents of the plant (Table2).It is seen that the plant physiologically defended itself by accumulating heavy metals in its body and also changed the cental Mg atom in the chlorophyll so that heavy metals could not affect the chlorophyll(Karaman et al. 2012).Negative effects of Pb, Cd and Cr were shown on mnay plants in the literature.Cadmium was shown to inhibit photosynthesis in Brassica napus, Helianthus annus, Thlaspica erulescens, Zea mays, Pisum sativum, Hordeum vulgare, Vignar adiata and Triticum which were exposed to Cd toxicity (Baryla et al. 2001; DiCagno et al. 2001; Küpper et al. 2007; Moussa and El-Gamal 2010; Popova et al. 2008; Wahid et al. 2008).The accumulation of lead in the plants has many direct or indirect effects on morphological, physiological and biochemical funcitons of the plants.The typical symptoms of lead toxicity are inhibition of photosynthetic pigment content and photosynthetic activity (Singh et al. 2010).Increasing doses of chromium application were shown to decrease dry weight in corn (Zea mays L.) (Adiloğlu and Göker 2021).

Table 4
Effects of some contaminats (Cr, Cd, Pb) accumulated in the shoots of Broccoli (Brassica oleracea var.italica) on some macro and micro nutrient elements * Cd * Cd + EDTA ** Pb Adiloğlu 2017; Shanmugaraj et al. 2019; Adiloğlu 2020; Adiloğlu 2021).The reduction in the photosynthetic activity in plants exposed to lead is a result of destruction of chlorophyll subunits, inhibition of plastoquinone and carotenoid synthesis, de ciency in basic elements such as Mn and Fe, substitution of bivalent cations with Pb, and destruction of electron transport system (Pourrut et al. 2011; Sharma and Dubey 2005).

Table 5
Accumulation of Cd, Pb and Cr pollutants in the roots of broccoli and effects on some nutrient elements al. 2020; Hayat et al. 2012).Chromium (Cr +6 ) toxicity can change enzyme activities, reduce resistance to pathogenic organisms, causes changes in behaviouralmodi caitons, population structure and species diversity and inhibition of photosynthesis (Pradhan et al. 2019; Sethuraman and Balasubramanian 2010).If chromium (Cr +6 ) accumulation in plants exceeds metabolic capacity for detoxi cation, seed germination, root and shoot development and biomass production slow down (Hayat et al. 2012; Sharma and Dubey 2005).Moreover, chromium (Cr +6 ) affects nutrient uptake and interferes with antioxidant systems and intercellular membrane structures.This metal harms the photosytnethic process as it decreases lipid peroxidation in proteins and photosynthetic pigments, (Diwan et al. 2012; Panda and Choudhury 2005; Singh et al. 2013; Vajpayee et 2000).Electron transport, CO 2 xaiton and photophosphorylation are affected by chromium (Cr +6 ) (Ali et al. 2006; Diduret al. 2013; Mathur et al. 2016; Pandey et al. 2013; Susplugas et al. 2000; van Assche and Clijsters 1983).

Table 6
Accumulation and effects of Cd, Pb and Cr on some plant nutrient elements in soil where broccoli was grown *Cd: 30 mgkg −1 ** Pb: 100 mgkg −1 *** Cr: 10 mgkg −1 +: % ++: mgkg−1 (Adhikari et al. 2020;Avudainayagam et al. 2003)etals and abounds in the eath crust.It enters the food chain via the plant absorption.It is found as chromite (FeCr 2 O 4 ) or as a complex with other metals in ultramaphic and serpentine rocks and in nature(Oliveira 2012).Moreover, Cr is released into the ecosystesms as a result of antropogenic activities such as inconscious use of chemical fertilizers, melting of metals, municipality waste waters, and wastes of different industries such as textile, ceramic, leather, steel and galvanic industries(Adhikari et al. 2020;Avudainayagam et al. 2003).Low a nity sulfate carriers mediate the absorption of chromium (Cr +6 ) (Ske ngton et al. 1976).
Soil lead content depends heavily on soil pH, colloid size and cation exchange capacity.Moreover, root surface area, root excretions and transpiration degree affect the absorption and usability of Pb.Plant roots absorb Pb via Ca 2+ channels or in an apoplastic way (Kumar et al. 2019; Pourrutet al. 2011).(