The Garden Candytuft (Iberis umbellata L.): At the Crossroad of Copper Accumulation and Glucosinolates
, Dario Omanović 2, Azra Đulović 3, Franko Burčul 4, Sandra Svilović 5 and Ivica Blažević 3Round 1
Reviewer 1 Report
Dear Author,
Your study looks interesting but still, it has some drawbacks regarding data interpretations and presentation.
The author is requested to add some physicochemical properties to justify your hypothesis.
Thanks in advance.
Regards,
WOO
Author Response
Changes in the manuscript are marked by yellow text highlight. We would like to take this opportunity to express our gratitude for very useful suggestions.
The answers to the reviewer's questions and remarks can be found below.
Reviewer 1:
The author is requested to add some physicochemical properties to justify your hypothesis.
Revised as suggested. We have added physiochemical properties in the manuscript under section 2.1. Materials and Reagents. The changes are highlighted in yellow through the manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments to Muzek et al.
This ms contains original data from experiments made with the plant Iberis umbellata L., submitted to different Cu concentrations in the soil. Soil for experiments is composed of different substrates that absorb the metal. After a time for germination and growth, plant samples are taken, and metal content was determined by HR ICP-MS. The concentration of glucosinolates is also determined. The conclusion is that the plant accumulates Cu with no sign of phytotoxicity. The conclusions are not entirely consistent with the results because as Cu concentration increases in the soil a clear inhibition of germination and growth rate is observed. These results are recognized by authors (see below) but paradoxically they do not consider them as a harmful effect. These variables can be observed in the picture of figure 1, but neither germination nor growth rates were computed. Number of replicates are not given, nor mean values or variances. Statistical treatment is absent.
Major comments.
-The statement made in the lines 173 and 174 (… no visible harmful effect…) is contradictory with the affirmation made in lines 175 to 178 and with the result obtained in the experiment. In figure 1 is observed a clear inhibitory effect on germination rate and growth with the increment in the free Cu concentration in the soil. These effects are important enough to be considered harmful. This led to inconsistent conclusions in lines 241 and 242.
-Statistical treatment is absent.
-In table 2 authors show the concentration of metals in the plants under different experimental conditions concerning Cu availability. When concentration of Cu in the humus substrate is 10.2 µg g-1, the concentration of Cu in the reference pant sample is 4.3 µg g-1. In the same conditions when concentrations of Mn in the humus is 19.3 µg g-1, the concentration of Mn in the plant reference sample is 171.1 µg g-1. This interesting result (probably) point out this plant as a Mn rather than Cu accumulator.
Minor comments.
Line 25. Please specify which part of the plant shows that concentration of Cu. The same for glucosinolate content.
Abstract and throughout the ms. Concentrations are expressed in µg g-1, for example 27.88 µg g-1. That means the method to measure Cu concentration and the weight of the plant does differentiate concentrations of 0.01 µg g-1? or the second decimal is a result of algebra? Please, consider the use of the first significant non-zero decimal figure only.
Lines 57-58. Accumulators or metal indicator? See Hunt et al. Green Process Synth 2014; 3: 3–22
Lines 74 and 75. “Composana” is a commercial trademark, the composition and characteristics of the humus have to be described. If the company changes the composition or the product disappear no one will be able to reproduce the experiment.
Lines 85, 86 and 87. What is the method for the spectrophotometric determination of Cu?
For Cu determination in water after the absorption process, to eliminate turbidity, water had to be filtered, please explain how and the type (cellulose, fiber glass…) and pore size of the filter used.
Same applies for line 99 and 113.
Lines 102 to 106. What is the pH of the substrate where seed were planted? There were pH differences in the different treatments?
In table 1, the figure 7.95 is confusing is it a control, the initial added concentration or…?
Line 159, please use “control” plant instead “referent”.
Lines 159, 161 and 161. Concentrations are expressed on a total plant biomass? Or leaves?
Lines 164 and 165. Cu is absorbed by the substrates by ion exchange process, no doubt, eliminate “probably” and use the appropriate references.
Line 195. The use of any common chemical speciation program, as the freeware MINTEQ would have allowed to know the Cu free ion concentration in the soil and hence the available Cu.
Lines 214 to 218. The problem here is that Cu induces toxicity in the plant shows clear signs of toxicity (inhibition of germination and growth).
Lines 241 and 242. This assessment is not true.
Author Response
Changes in the manuscript are marked by yellow text highlight. We would like to take this opportunity to express our gratitude for very useful suggestions.
The answers to the reviewer's questions and remarks can be found below.
Reviewer 2:
Major comments:
-The statement made in the lines 173 and 174 (… no visible harmful effect…) is contradictory with the affirmation made in lines 175 to 178 and with the result obtained in the experiment. In figure 1 is observed a clear inhibitory effect on germination rate and growth with the increment in the free Cu concentration in the soil. These effects are important enough to be considered harmful. This led to inconsistent conclusions in lines 241 and 242.
We have slightly changed this paragraph 173 to 178 to be more understandable for readers. First part of this paragraph is related to different substrates, while lines from 175 to 178 are related only to the seedlings watered with copper solution, page 6 line 202-208: “Although the concentrations of copper in these seedlings are slightly higher than the normal concentration in plant (1 - 30 µg g-1) there are no visible harmful effects on seedlings growth and development in accordance to control (Fig. 1. a1), a2), a3), a5), b1), b2), b3), b5, c1), c2), c3), c5). The highest copper concentration in the garden candytuft was detected when seedlings were watered with a CuSO4∙5H2O solution (514.63 μg g-1). Higher concentration of copper decreased seed germination as well as the seedlings growth and development (Fig. 1 a4), b4), c4).”
Also, the conclusion is rewritten, page 8 line 242-244: “The garden candytuft has shown no differences in seedlings appearance after forty-days of cultivation regardless of the soil or the watering solutions used. The differences were noticed in the seed germination and the seedling growth rate when pots were watered with CuSO4∙5H2O solution.”
-Statistical treatment is absent.
We have added statistical data in the Table 2, page 7 line 235.
-In table 2 authors show the concentration of metals in the plants under different experimental conditions concerning Cu availability. When concentration of Cu in the humus substrate is 10.2 µg g-1, the concentration of Cu in the reference pant sample is 4.3 µg g-1. In the same conditions when concentrations of Mn in the humus is 19.3 µg g-1, the concentration of Mn in the plant reference sample is 171.1 µg g-1. This interesting result (probably) point out this plant as a Mn rather than Cu accumulator.
The authors are very grateful for this comment. However, the garden candytuft needs to accumulate much higher concentrations of Mn (10 000 µg g-1) to be considered as indicator or hyperaccumulator plant, and further experiments should be carried out.
Minor comments:
Line 25. Please specify which part of the plant shows that concentration of Cu. The same for glucosinolate content.
Revised as suggested. All the copper concentrations and glucosinolate content were detected in the total seedlings biomass, page 1 line 24: “…was detected in the total seedlings biomass…”
Abstract and throughout the ms. Concentrations are expressed in µg g-1, for example 27.88 µg g-1. That means the method to measure Cu concentration and the weight of the plant does differentiate concentrations of 0.01 µg g-1? or the second decimal is a result of algebra? Please, consider the use of the first significant non-zero decimal figure only.
We used two significant decimal figures because of the uniformity of all data presented.
Lines 57-58. Accumulators or metal indicator? See Hunt et al. Green Process Synth 2014; 3: 3–22
According to the Boyd (Plant Soil 2007; 293:153–176) they are called accumulators and according to the Hunt et al. (Green Process Synth 2014; 3: 3–22) they are called metal indicators. We used both names and relevant citations, page 2 line 57-58: “Plants with elevated but not extraordinary levels of heavy metals in plant tissues are known as metal indicators or accumulator plants.“
Lines 74 and 75. “Composana” is a commercial trademark, the composition and characteristics of the humus have to be described. If the company changes the composition or the product disappear no one will be able to reproduce the experiment.
We have added information about Compo Sana from the declaration of product, page 2 line 77-81: “According to the specification found on the package humus is declared as a soil for sowing and medicinal plants. It is a high-value special soil for growing all kinds of plants in pots and gardens. The composition is also suitable for growing medicinal plants with pH value (CaCl2): 5.0-6.5, salt content (g dm-3): 1.0-1.5, nutrient content: 50-250 N, 80-300 P2O5, 100-350 K2O. It consists of raw materials such as high-value peat (H5-H7) (96%), green compost (4%), Agrosil, real COMPO Guano, lime and nutrients.”
Lines 85, 86 and 87. What is the method for the spectrophotometric determination of Cu?
Revised as suggested, page 3 line 93-94: “… at wavelength of 810 nm using an absorbance-concentration calibration curve abs(c)=1.025∙10-4+0.0118∙c having correlation coefficient 0.9999. Calibration curve was obtained by measuring absorbance of previously prepared solutions with known copper concentrations in the range from 0.60 - 21 mmol dm-3.”
For Cu determination in water after the absorption process, to eliminate turbidity, water had to be filtered, please explain how and the type (cellulose, fiber glass…) and pore size of the filter used.
Filter paper used for filtration process was Whatman 42. The authors added that information in the manuscript, and made a change in this paragraph, page 3 line 97-100: “After 10 days of mixing the suspensions were filtered through filter paper Whatman 42. Obtained filtered copper solutions were used for measuring the Cu ions concentration remained in the solutions by UV/Vis spectrophotometer.”
Same applies for line 99 and 113.
The authors made some changes in these lines, page 3 line 106-125. The changes are highlighted in yellow.
Lines 102 to 106. What is the pH of the substrate where seed were planted? There were pH differences in the different treatments?
The authors added pH values of the prepared substrate, page 3 line 107-109: “The adsorbent addition changed the original pH value of humus (pH=5.62) depending on the adsorbent added, as follows: humus with zeolite NaX addition (pH=6.59); humus with extra humus addition (pH=5.34); humus with egg shells addition (pH=6.54).”
In table 1, the figure 7.95 is confusing is it a control, the initial added concentration or…?
The number 7.95 refers to the initial concentration of the copper solution. To make it more understandable the authors have added an explanation below the Table 1, page 4 line 167-168: “c0 is the initial concentration of Cu in the solution (mmol dm-3); c10 is concentration of Cu in the solution after 10 days (mmol dm-3); q10 is the amount of the copper retained on the adsorbent (mmol g-1).”
Line 159, please use “control” plant instead “referent”.
Revised as suggested. The authors replaced referent plant with control seedling throughout the whole manuscript.
Lines 159, 161 and 161. Concentrations are expressed on a total plant biomass? Or leaves?
All concentrations presented in Table 2 are expressed on a total seedling biomass. The authors added that information in the table name, page 7 line 234: "Table 2. Measured heavy metals concentration in the garden candytuft seeds, and the cultivated garden candytuft (total seedlings biomass), respectively.”
Lines 164 and 165. Cu is absorbed by the substrates by ion exchange process, no doubt, eliminate “probably” and use the appropriate references.
Revised as suggested. The authors added appropriate references, page 5 line 193: “…[6, 28-30].”
References added:
- Svilović, S., Rušić, D., Žanetić, R. Thermodynamics and Adsorption Isotherms of Copper Ions Removal from Solutions Using Synthetic Zeolite X. Chem. Biochem. Eng. Q. 2008, 22, 299–305.
- Svilović, S., Mužek, M.N., Nuić, I., Vučenović, P. Taguchi design of optimum process parameters for sorption of copper ions using different sorbents. Water Sci. Technol. 2019, 80, 98–108.
- Yurekli, Y. Determination of adsorption characteristics of synthetic NaX nanoparticles. J. Hazard. Mater. 2019, 378, 120743.
- Schenkel, R. Sorption of small polar molecules on micro- and mesoporous zeolitic materials, Dissertation, Fakultät für Chemie, München, 2004.
Line 195. The use of any common chemical speciation program, as the freeware MINTEQ would have allowed to know the Cu free ion concentration in the soil and hence the available Cu.
The authors would like to thank the reviewer for this suggestion. In our case we investigated cultivation of the plant on different types of the soil with various factors influencing copper availability. In order to investigate the influence of free copper ions, it would be better to use an experiment with hydroponic cultivation, where less factors could influence the copper availability than in our case (M. Klimek-Szczykutowicz, et al., Food Chem. 2019, 300 125184).
Lines 214 to 218. The problem here is that Cu induces toxicity in the plant shows clear signs of toxicity (inhibition of germination and growth).
Lines 241 and 242. This assessment is not true.
After observing the garden candytuft growing for forty-days period of cultivation the garden candytuft has shown no visible signs of phytotoxicity regardless of the soil or the watering solution used. The only difference noticed was in the total amount of seedlings grown in the cultivation period. This is especially visible for seedlings watered with CuSO4∙5H2O solution which is due to the decreased seed germination and development. The evidence is added in the form of Figure 1. c1)-c5) that can be found on page 5. These lines were changed, page 8 line 242-244: “The garden candytuft has shown no differences in seedlings appearance after forty-days of cultivation regardless of the soil or the watering solutions used. The differences were noticed in the seed germination and the seedling growth rate when pots were watered with CuSO4∙5H2O solution.”
Author Response File: Author Response.pdf
Reviewer 3 Report
Your Manuscript ID: processes-902790 describes the copper accumulation capability correlate to glucosinolates content on forty-days grown seedlings of candytuft (Iberis umbrellata L.). The detection of heavy metals and glucosinolates sound correctly performed. In general, the manuscript, in present form, requires essential technical correction in almost all section of it. In some points od the manuscript an accurate English revisioni s recommended. Some suggestions are reported below.
Some more recent bibliographic references could be added in the introduction regarding the absorption of heavy metals in plants for example De Caroli et al., 2020, Plants 9, 482, doi: 10.3390 / plants9040482.
It might be useful to add a photo of the forty-days seedlingd in order to evaluate their morphological state.
Pag. 1 line 24: determinated in the plant should be detected in seedling
line 25: (27.88 µg g-1)… (27.88 µg g-1DW)
line 26: the highest concentration… the highest copper concetration
line 26: plant: seedlings
line 27: (514.63 µg g-1)… (514.63 µg g-1DW)
line 27: with unfavorable impact… with a sharp decrease on the…
line 39: metabolism of plants… development of plant.
line 41: metabolism… development.
line 43: with the normal concentration in the range of 1-30 µg g-1… in the range of physiological concentration (1-30 µg g-1)…
Pag. 2 line 49: papers… studies
line 54: up large amounts… up by the roots large amounts
line 54: translocate to the… translocate them to the…
line 64: This plant family… This family…
line 81: egg shell… egg shells
line 90: were agitated… how?
line 100-101: and the growth… and germination, growth and…
line105: reference sample… control sample
line 109: is it possible to dry seedlings at 25°Cfor two days?
Pag.4 line 155: (98.36%; 95.22%)… (98.36% and 95.22% respectively)…
line 155: efficiency… efficiency (49.81%)
Table 1: Humus COMPO SANA… Humus
line 159: The lowest measured… The lowest concentration…
line 159: in plants… in seedlings…
line 160: (4.31 µg g-1)… (4.31 µg g-1 DW)
line 161: (27.88 µg g-1)… (27.88 µg g-1 DW) (Table 2).
line 159-160: plants..plant ARE PLANTS OR SEEDLINGS?
line 167: (39.82 µg g-1)… (39.82 µg g-1 DW) (Table 2).
line 172-173: (44.45 µg g-1)… (44.45 µg g-1 DW) (Table 2).
line 174: plants… seedling
Pag. 5 line 175: reference… control (seedlings)
line 175: the largest… the highest copper concentration
line 176: is present when the plant was watered… was detected when seedlings were watered
line 177: in plant has decreased seed germination and also had a repressive impression in plant growth and development (Fig.1 a4), b4)… in seedlings decreased seed germination as well as growth and development (Fig.1°4), b4)
lines 189-208: OUT it is a repetition of the text
Pag. 7 line 217: (514.63 µg g-1)… (514.63 µg g-1 DW) (Table 2)
line 235-236: 2865.02 µg g-1 DW (1.08 µg g-1)…3103.33 µg g-1 DW (XX µg g-1 Dw) (Table 2). Please check the counts!
line 246: Contrary… On the contrary
line 247: unfavorable impact… a sharp decrease.
Author Response
Changes in the manuscript are marked by yellow text highlight. We would like to take this opportunity to express our gratitude for very useful suggestions.
The answers to the reviewer's questions and remarks can be found below.
Reviewer 3:
Pag. 1 line 24: determinated in the plant should be detected in seedling
Revised as suggested. The changes are highlighted in yellow.
line 25: (27.88 µg g-1)… (27.88 µg g-1DW)
Revised as suggested. The changes are highlighted in yellow.
line 26: the highest concentration… the highest copper concentration
Revised as suggested. The changes are highlighted in yellow.
line 26: plant: seedlings
Revised as suggested. The changes are highlighted in yellow.
line 27: (514.63 µg g-1)… (514.63 µg g-1DW)
Revised as suggested. The changes are highlighted in yellow.
line 27: with unfavorable impact… with a sharp decrease on the…
Revised as suggested. The changes are highlighted in yellow.
line 39: metabolism of plants… development of plant.
Revised as suggested. The changes are highlighted in yellow.
line 41: metabolism… development.
Revised as suggested. The changes are highlighted in yellow.
line 43: with the normal concentration in the range of 1-30 µg g-1… in the range of physiological concentration (1-30 µg g-1)…
Revised as suggested. The changes are highlighted in yellow.
Pag. 2 line 49: papers… studies
Revised as suggested. The changes are highlighted in yellow.
line 54: up large amounts… up by the roots large amounts
Revised as suggested. The changes are highlighted in yellow.
line 54: translocate to the… translocate them to the…
Revised as suggested. The changes are highlighted in yellow.
line 64: This plant family… This family…
Revised as suggested. The changes are highlighted in yellow.
line 81: egg shell… egg shells
Revised as suggested. The changes are highlighted in yellow.
line 90: were agitated… how?
The authors additionally explained the adsorption studies, page 3 line 96-97: " The suspensions were kept in a thermostatic orbital shaker (200 rpm) for 10 days order to achieve the maximum adsorption.”
line 100-101: and the growth… and germination, growth and…
Revised as suggested. The changes are highlighted in yellow.
line 105: reference sample… control sample
Revised as suggested. The changes are highlighted in yellow.
line 109: is it possible to dry seedlings at 25°Cfor two days?
The authors additionally explained the drying process, page 3 line 118-120: “…and left to dry at room temperature (ca. 25°C) over a period of two days and afterwards in the laboratory dryer oven at 75°C to constant mass in order to obtain the dry weight.”
Pag. 4 line 155: (98.36%; 95.22%)… (98.36% and 95.22% respectively)…
Revised as suggested. The changes are highlighted in yellow.
line 155: efficiency… efficiency (49.81%)
Revised as suggested. The changes are highlighted in yellow.
Table 1: Humus COMPO SANA… Humus
Revised as suggested. The changes are highlighted in yellow.
line 159: The lowest measured… The lowest concentration…
Revised as suggested. The changes are highlighted in yellow.
line 159: in plants… in seedlings…
Revised as suggested. The changes are highlighted in yellow.
line 160: (4.31 µg g-1)… (4.31 µg g-1 DW)
Revised as suggested. The changes are highlighted in yellow.
line 161: (27.88 µg g-1)… (27.88 µg g-1 DW) (Table 2).
Revised as suggested. The changes are highlighted in yellow.
line 159-160: plants..plant ARE PLANTS OR SEEDLINGS?
Revised as suggested. According to the plant growth stages after forty days of cultivation our plant reached the plant growth stage somewhere between vegetative and bud stage. But we decided to change plant with the seedlings through the manuscript as we cultivated the garden candytuft in the pots from the seeds. Changes are highlighted in yellow.
line 167: (39.82 µg g-1)… (39.82 µg g-1 DW) (Table 2).
Revised as suggested. The changes are highlighted in yellow.
line 172-173: (44.45 µg g-1)… (44.45 µg g-1 DW) (Table 2).
Revised as suggested. The changes are highlighted in yellow.
line 174: plants… seedling
Revised as suggested. The changes are highlighted in yellow.
Pag. 5 line 175: reference… control (seedlings)
Revised as suggested. The changes are highlighted in yellow.
line 175: the largest… the highest copper concentration
Revised as suggested. The changes are highlighted in yellow.
line 176: is present when the plant was watered… was detected when seedlings were watered
Revised as suggested. The changes are highlighted in yellow.
line 177: in plant has decreased seed germination and also had a repressive impression in plant growth and development (Fig.1 a4), b4)… in seedlings decreased seed germination as well as growth and development (Fig.1°4), b4)
Revised as suggested. The changes are highlighted in yellow.
lines 189-208: OUT it is a repetition of the text
Revised as suggested.
Pag. 7 line 217: (514.63 µg g-1)… (514.63 µg g-1 DW) (Table 2)
Revised as suggested. The changes are highlighted in yellow.
line 235-236: 2865.02 µg g-1 DW (1.08 µg g-1)…3103.33 µg g-1 DW (XX µg g-1 Dw) (Table 2). Please check the counts!
Revised as suggested. The changes are highlighted in yellow. The authors checked all the counts again.
line 246: Contrary… On the contrary
Revised as suggested. The changes are highlighted in yellow.
line 247: unfavorable impact… a sharp decrease.
Revised as suggested. The changes are highlighted in yellow.
Round 2
Reviewer 1 Report
Dear Author,
The revised version looks okay.
Author Response
Changes in the manuscript are marked by yellow text highlight. We would like to take this opportunity to express our gratitude for very useful suggestions.
The answers to the reviewer's questions and remarks can be found below.
Reviewer 1:
The revised version looks okay.
We thank the reviewer for this comment.
Reviewer 2 Report
There are still two comments concerning this ms,
-The problem with Cu accumulation in candytuft (again) is when Cu concentration in the soil is 10.2 µg g-1 and 4.3 µg g-1 in the plant. This is a clear exclusion of Cu in the control conditions (that is the point), hence, these values give a negative concentration factor. To compare, please look at the numbers for Mn, 19.3 in the soil and 171.1 in the plant, CF=8.9. In the context of the ms, authors have to explain that. This is an important concern because the plant is proposed as Cu accumulator.
-Statistical treatment is something beyond write down mean values and variances in a table (table 2 in this case). A statistical test has to be applied to estate if two figures are equal or significantly different, for instance, is 39.8±0.59 significantly different from 44.45±3.05? That depends (as authors know) on the number of replicates, the statistical significance and, in part, the type of statistical test applied. The number of replicates has not been indicated and no statistical test has been even mentioned. Details about statistics have to be included in the M&M section.
Author Response
Changes in the manuscript are marked by yellow text highlight. We would like to take this opportunity to express our gratitude for very useful suggestions.
The answers to the reviewer's questions and remarks can be found below.
Reviewer 2:
-The problem with Cu accumulation in candytuft (again) is when Cu concentration in the soil is 10.2 µg g-1 and 4.3 µg g-1 in the plant. This is a clear exclusion of Cu in the control conditions (that is the point), hence, these values give a negative concentration factor. To compare, please look at the numbers for Mn, 19.3 in the soil and 171.1 in the plant, CF=8.9. In the context of the ms, authors have to explain that. This is an important concern because the plant is proposed as Cu accumulator.
We agree with the reviewer that the garden candytuft can be Mn accumulator/hyperaccumulator which would be an interesting point for the further research.
However, we cannot agree that the plant is acting in exclusion mechanism in the control soil sample concerning Cu in comparison to Mn for several reasons:
- Both metals were taken up by the plant in normal ranges (normal range for Mn in plants is 20-400 µg g-1 and normal range for Cu in plants is 1-30 µg g-1) (Reeves, R.D., Baker, A.J.M. Metal-accumulating plants. In: Phytoremediation of toxic metals: using plants to clean up the environment, Raskin, I., Ensley, B.D., Eds.; John Wiley and Sons: New York, 2000, pp 193–229. Boyd, R.S., The defense hypothesis of elemental hyperaccumulation: status, challenges and new directions. Plant Soil 2007, 293, 153–176. Marschner, P. Marschner’s Mineral Nutrition of Higher Plants, 3rd ed.; Elsevier: Amsterdam, Netherlands, 2012).
- Exclusion mechanism is a plant defence strategy to survive in metal polluted soils (like in our experiments where we added Cu-saturated NaX, eggshells and humus to normal control soil) unlike control experiment with non-polluted soil (Pongrac, P., Tolrà, R., Vogel-Mikuš, K., Poschenrieder, C., Barceló, J., Regvar, M. At the Crossroads of Metal Hyperaccumulation and Glucosinolates: Is There Anything Out There?, Sherameti, I., Varma, A., Eds.; Springer-Verlag: Berlin, Germany, 2010; Volume 19, pp. 139–161.).
After this mechanism is surpassed, as in our last experiment (when watering the plant /soil with copper solution), plant begins to accumulate higher concentrations of heavy metal (Pongrac, P., Tolrà, R., Vogel-Mikuš, K., Poschenrieder, C., Barceló, J., Regvar, M. At the Crossroads of Metal Hyperaccumulation and Glucosinolates: Is There Anything Out There?, Sherameti, I., Varma, A., Eds.; Springer-Verlag: Berlin, Germany, 2010; Volume 19, pp. 139–161.). According to the results obtained in the last experiment where candytuft managed to accumulate 514.63 µg g-1 of Cu (i.e. >100 µg g-1), we pointed out that the garden candytuft may be considered as Cu-accumulator.
-Statistical treatment is something beyond write down mean values and variances in a table (table 2 in this case). A statistical test has to be applied to estate if two figures are equal or significantly different, for instance, is 39.8±0.59 significantly different from 44.45±3.05? That depends (as authors know) on the number of replicates, the statistical significance and, in part, the type of statistical test applied. The number of replicates has not been indicated and no statistical test has been even mentioned. Details about statistics have to be included in the M&M section.
Revised as suggested. We have added a new subsection about statistics in the section 2. Materials and Methods, page 4 line 164-168: “2.5. Statistical Analysis
Analysis of variance (one-way ANOVA) was used to assess the statistical difference between data reported in Table 2, followed by a least significance difference test to evaluate differences between sets of mean values at significance level set at p < 0.05. Analyses were carried out using Statgraphics Centurion-Ver.16.1.11 (StatPoint Technologies, Inc., Warrenton, VA, USA) [28].”
We also added statistical data in Table 2, as well as the number of replicates, page 7 line 232-235: “Results are presented as mean ± SD (n = 3). a−f Different letters in the same column denote statistically significant difference (p < 0.05) between samples.”
The changes are highlighted in yellow.
