Water, Air, & Soil Pollution

, 224:1553

Phytotoxicity of Long-Term Total Petroleum Hydrocarbon-Contaminated Soil—A Comparative and Combined Approach

Authors

  • Kanaji Masakorala
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
    • Department of Botany, Faculty of ScienceUniversity of Ruhuna
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
    • State Key Laboratory of Biogeology and Environmental Geology and Sino-Hungarian Joint Laboratory of Environmental Science and HealthChina University of Geosciences
  • Huan Guo
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
  • Radhika Chandankere
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
  • Jingwei Wang
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
  • Minmin Cai
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
  • Haijun Liu
    • School of Civil and Environmental Engineering, and National “International Cooperation Based on Environment and Energy”University of Science and Technology Beijing
    • Department of ChemistryHong Kong Baptist University
Article

DOI: 10.1007/s11270-013-1553-x

Cite this article as:
Masakorala, K., Yao, J., Guo, H. et al. Water Air Soil Pollut (2013) 224: 1553. doi:10.1007/s11270-013-1553-x

Abstract

Petroleum hydrocarbon contamination of soil is an emerging environmental threat on the Earth due to possible toxic impact on different ecological receptors. The present study was mainly carried out to evaluate the phytotoxicity of long-term total petroleum hydrocarbon-contaminated soils by the toxicity end points obtained from three plant species Zea mays, Lactuca sativa L., and Cucumis sativus. The tested soil exerted phytotoxicity for all the evaluated end points of plants with dose-dependent relationship. The determined IC50 indicates inhibition in root elongation as the most sensitive toxicity end point for L. sativa L., while inhibition in cross-section area of meristematic zone as the most susceptible and inhibition in seed germination as the least susceptible end points for both Z. mays and C. sativus. The tested root morphometric parameters confirm their applicability as novel toxicity end points. In addition, microcalorimetric analysis confirmed the applicability of inhibition in metabolic heat emission rate as a toxicity end point. Microcalorimetry can be applied to determine the exerted phytotoxic effect on seedlings. The present combined approach concludes that the phytotoxicity of the tested soil is species-specific and varies as follows: Z. mays < C. sativus < L. sativa L. The findings of this study may have implications in planning comprehensive phytotoxicity assessment for hydrocarbon-contaminated soils or screening plant species for phytoremediation program.

Keywords

Total petroleum hydrocarbonPhytotoxicityMorphometric parametersMetabolic heat

1 Introduction

The demand for the petroleum hydrocarbon is increasing in all over the world in the same pace to the societal and industrial development. Consequently, increased accidental spills and release from anthropogenic activities have been reported resulting in contaminated aquatic as well as terrestrial ecosystems on the Earth. Highly concerning hazardous chemicals such as benzene, toluene, ethylbenzene, xylenes, and naphthalene included in the list of US Environmental Protecting Agency (EPA) are petroleum hydrocarbons (Zou and Crawford 1995; Leibeg and Cutright 1999; Ting et al. 1999; Vasudevan and Rajaram 2001; Bojes and Pope 2007). As these chemicals are highly toxic to plants, microorganisms, and invertebrates, (Andreoni et al. 2004) petroleum contamination may cause highly negative health and ecological impacts on the contaminated ecosystems (Eibes et al. 2006; Al-Mutairi et al. 2008). Since petroleum contamination of soil is one of the major growing environmental threats to the Earth, toxicity characterization of petroleum hydrocarbon-contaminated soil has become immensely important in risk assessment which is vital in the management of contaminated sites.

The toxicity of a hydrocarbon compound may change during the bioremediation process in the soil under natural conditions, and toxic metabolites or transformation by-products could be produced resulting in increased soil toxicity (Loehr and Webster 1996; Loibner et al. 2003). Thus, in the soil quality assessment, reduced contaminant concentration may not always indicate decreased soil toxicity (Al-Mutairi et al. 2008). As chemically analyzed data are not sufficient to predict the potential ecological impacts of contaminated soils, bioassay as a method for ecotoxicity assessment of contaminated soil has become popular. Furthermore, the importance of bioassay specially when predicting the toxic impact of a mixture of chemical compounds such as petroleum has been proven (Banks and Schultz 2005).

Phytotoxicity assessment has become mandatory in the ecological risk characterization of petroleum hydrocarbon-contaminated and bioremediated soils since species should be selected for bioassay representing the whole ecosystem (Leitgib et al. 2007). Lettuce (Lactuca sativa L.) is widely used in the toxicity assessment as an indicator plant due to its relatively high sensitivity to toxic chemicals (US EPA 1994). Other plant species such as wheat, millet, radish, red clover (Banks and Schultz 2005), and maize (Ogboghodo et al. 2004) have been used for toxicity characterization of laboratory-spiked soil with petroleum hydrocarbons. Thus, more comprehensive phytotoxicity assessments carried out to characterize the toxicity of long-term total petroleum hydrocarbon (TPH)-contaminated soil are scare today. Although it is vitally important to identify the most suitable plant to be used in petroleum toxicity monitoring, the number of tested plant species is still low compared to the number of tested plant species for herbicide and heavy metal toxicity (Banks and Schultz 2005).

Different types of plant processes are being used as end points in toxicity studies (Wang 1991). An acute toxicity of phytotoxic contaminants is measured by using short-term tests results from germination (Bank and Schultz 2005) and early root growth measurements, and analyzing chlorophyll fluorescence quenching (Masakorala et al. 2010). In most previous studies, the phytotoxicity of petroleum hydrocarbon was mainly characterized on the basis of toxicity end point such as inhibition in seed germination and root growth. Even though the seed germinated in TPH-contaminated soil, there may be changes in root morphology due to negative impact of hydrocarbon-contaminated soil (Reynoso-Cuevas et al. 2008). Root morphology can be described by morphometric parameters such as root cross-section area, pericycle, and endodermis cell area. However, as root growth is determined by the activity of meristematic and elongation zone, it is important to identify specific effect on those different root zones in morphometric studies (Silva et al. 2012). Unfortunately, not much attention has been paid to determining the potential of root morphometric parameters such as inhibition in the root cross-section area to be employed as a toxicity end point to assess the phytotoxicity of TPH-contaminated soil.

Microcalorimetry is a nondestructive technique which can be used to measure very low heat flow. Hence, it has been applied to study the metabolic process of plants (Hansen et al. 1997; Criddle and Hansen 1999). Edelstein et al. (2001) noted the applicability of microcalorimetry to study the influence of the environment or hormone on seed germination through monitoring the heat production. So far, microcalorimetry has not been applied to evaluate the toxicity of petroleum-contaminated soil based on the metabolic heat emission of germinated seeds.

In the present study, a comparative phytotoxicity was conducted by combining typically used and novel toxicity end points taken from previously tested plants L. sativa L., Zea mays, and untested plant Cucumis sativus for hydrocarbon toxicity. The novel toxicity end points such as inhibition in the root cross-section area by applying microscopic method and inhibition of catabolism by applying microcalorimetry were employed with the typical end points such as inhibition in germination, root growth, and root biomass as a combined approach. The primary objective of the study was to comparatively characterize the phytotoxicity of long-term TPH-contaminated soil, and the secondary objective was to investigate the applicability of novel tested end points and novel tested plant in phytotoxicity assessment for TPH-contaminated soil.

2 Materials and Methods

2.1 Plants

Seeds of three plant species including monocotyledon corn (Z. mays), dicocotyledon lettuce (L. sativa L.), and cucumber (C. sativus) were purchased from Kelichang Agricultural Research Centre, Beijing, China. Prior to being used in the tests, all the seeds of the selected species were carefully observed, and apparently healthy undamaged seeds of similar size were selected for the experiments.

2.2 Soil Sampling and Preparation

Petroleum-contaminated soils due to long-term exposure and accidental spills of crude oil were collected in October 2011 from Dagan Oil Field (southeast of Tianjin, northeast China). In the sampling, the surface layer was removed, and the soil was obtained at a depth 5–10 cm. The uncontaminated soil to be used as a control was collected from the undisturbed area located nearest to the oil field. Both collected uncontaminated and contaminated soils had clay loam texture. The collected soils were air-dried and passed through 2-mm sieve to remove root fragments and large particles. Homogenized soils were stored in polyethylene bags at 4 °C for further analyses.

2.3 Chemical Characterization of Soil

Soil pH was measured with a pH meter (OHAUS Starter 2C) in distilled water extract (1:5 w/v) of soil. Moisture content was determined gravimetrically (Koponen et al. 2006). Soil organic matter (OM) was analyzed by applying hydrogen peroxide digestion method (Nelson and Sommers 1996). Soil extractable phosphorus (P) was determined according to the Mehlich (1984) method. Soil NO3-N was analyzed following the method as described by Kaneko et al. (2010). Heavy metals lead (Pb) and cadmium (Cd) were extracted by applying ultrasound-assisted acid digestion and then analyzed by atomic absorption spectrometry (Kazi et al. 2009). TPH content in soil was measured following the method described by Tang et al. (2012). Table 1 summarizes the soil physicochemical characteristic of control and contaminated sample.
Table 1

Soil properties of uncontaminated control soil and TPH-contaminated soil

Soil property

Control soil

Contaminated soil

TPH (mg kg−1)

0

29,244

pH

7.34

8.3

OM (%)

3.68

6.7

GWP (%)

34.94

15.32

N-NO3 (mg kg−1)

32.74

9.5

P (mg kg−1)

22.47

14.21

Cd (mg kg−1)

ND

ND

Pb (mg kg−1)

ND

45

TPH total petroleum hydrocarbon, OM organic matter, GWP gravimetric water potential, N-NO3 nitrogen nitrate, P extractable phosphorus, Cd cadmium, Pb lead, ND not detected

2.4 Preparation of Soil for Phytotoxicity Studies

The collected contaminated soil with 29,244 mg kg--1 (2.9 %) TPH content was used as a stock contaminated soil. Other concentrations (1.0, 1.5, 2.0, and 2.5 % w/w TPH) of the contaminated soil were then prepared by diluting the stock soil with the uncontaminated ones. All these five contaminated soils together with the uncontaminated soil (control) were applied for this study. All the experiments were designed according to complete randomized block.

2.5 Seed Germination

Seed germination experiment at prepared soils with different TPH concentrations and control were carried out according to the ISO (1993b) and OECD (2000) methods. One hundred fifty grams of each contaminated soil and the control, respectively, was put in 150-mm Petri dishes, and the water holding capacity of the soil was adjusted to 70 % and maintained over the test period by adding distilled water. Twenty seeds from a selected species were placed evenly at the surface of the substrate and pressed into the soil, and finally, Petri dishes were covered with the lids and incubated at 25 °C under dark conditions for 48 h for Z. mays and C. sativus and 72 h for L. sativa L. seeds. At the end of the respective incubation periods, the number of germinated seeds was recorded. The seeds with the measurable root or shoot were considered as germinated seeds. The percentage of inhibition of seed germination (GI) was calculated as follows (Oleszczuk and Hollert 2011):
$$ \mathrm{GI}=\frac{A-B }{A}\bullet 100 $$
(1)
where A is the mean seed germination in the control soil and B is the mean seed germination in the tested contaminated soil. When the GI is equal to 50 %, the equivalent contamination level was considered as IC50.

2.6 Root Growth and Root Biomass

In this experiment, the seeds were germinated in the abovementioned manner, and the root length was measured following a 72-h incubation period for Z. mays and C. sativus and 120 h for L. sativa L. Three seedlings were randomly selected from each treatment, and the length of the main root was measured. The length from the tip to the radicle was considered as the root length. Dry weight of the root system of Z. mays and C. sativus were measured. In there, the root systems of selected seedlings were removed and cleaned carefully, and then the root dry weight was measured after keeping the samples at 60 °C in an oven for 24 h. Finally the percentage of inhibition of root elongation (REI) and the percentage of inhibition in root biomass (RBI) were calculated as follows:
$$ \mathrm{REI}/\mathrm{RBI}=\frac{A-B }{A}\bullet 100 $$
where A is the mean root length/biomass in the control soil and B is the mean root length/biomass in the tested contaminated soils. When REI/RBI is equal to 50 %, the corresponding contamination level was considered as IC50.

2.7 Root Morphometric Parameters

The seed germination experiment was repeated as the abovementioned method under the root growth and root biomass. At the end of the incubation period, cross sections from the meristematic zone (MZ, 3.0 mm behind the root cap) and the elongation zone (EZ, 10–15 mm behind the root cap) of the main root were taken by using a razor blade, and the cross-section area was measured by using an inverted microscope (DSZ5000X) equipped with an Arcam Measure 2 software. Finally, the percentage inhibition of the cross-section area of meristematic zone (MZAI) and elongation zone (EZAI) were calculated as follows:
$$ \mathrm{MZAI}/\mathrm{EZAI}=\frac{A-B }{A}\bullet 100 $$
where A is the mean root cross-section area of the control soil and B is the mean root cross-section area of the MZ or EZ in the tested contaminated soils. When MZAI/EZAI is equal to 50 %, the corresponding contamination is considered as IC50.

2.8 Microcalorimetric Analysis

Microcalorimetric analysis was performed to investigate the influence of petroleum-contaminated soil on seedlings. The catabolic activity of germinated seeds was measured as heat production rate (in microwatt) by using a TAM III multichannel thermal activity microcalorimeter (Thermometric, Jarfalla, Sweden). All steel ampoules (4.5 mL) were cleaned thoroughly and sterilized in an oven at 100 °C for 1 h. The selected Z. mays and C. sativus seeds were incubated for 48 h and L. sativa L. for 72 h in the TPH-contaminated soils and control at 25 °C according to the ISO (1993b) and OECD (2000) methods. Twenty seeds were used for one replicate. After the respective incubation periods, representing each treatment, one germinated seed from Z. mays and from C. sativus and two germinated seeds from L. sativa L. were used in microcalorimetric analysis. The analysis was carried out separately for each species, and the selected seedling/seedlings from each treatment were transferred into each ampoule. Prior to transferring the germinated seed into the ampoule, 25 μL of distilled water was added into the ampoule to prevent the seedlings from desiccation. All the experiments were carried out at 28 °C, and the released metabolic heat due to metabolism in seedling was recorded by the microcalorimeter. The metabolic heat emission rate (MHER) of the germinated seeds was calculated (μW 0.1 g−1 DW) (Kupidlowska et al. 2006). The percentage of metabolic heat emission rate inhibition (MHERI) both at the peak and at the end of the experimental period was calculated as follows.
$$ \mathrm{MHERI}=\frac{{\mathrm{MHE}{{\mathrm{R}}_0}-\mathrm{MHE}{{\mathrm{R}}_{\mathrm{c}}}}}{{\mathrm{MHE}{{\mathrm{R}}_0}}}\bullet 100. $$
where MHER0 and MHERc are the metabolic heat emission rate of seedling in the control and TPH-contaminated soil at concentration c, respectively. The phytotoxic influence of the TPH-contaminated soil on the metabolism of the seedling was deduced from the calculated percentage of MHERI of treatments.

2.9 Statistical Analyses

Statistical analyses were performed by using standard statistical software package MINITAB version 16. All data except microcalorimetric data were analyzed using one-way analysis of variance, and significant differences between individual means were ascertained by Tukey’s post hoc test. The statistical significance in all analyses was defined at p < 0.05. TPH contamination levels responsible for the 50 % inhibitory effect (IC50) were calculated on the basis of the best fitting models of the linear regression. The values for which 50 % inhibition did not result in the phytotoxicity tests were extrapolated.

3 Results and Discussion

3.1 Seed Germination

Seed germination is a highly important stage of plant growth where there is a particular sensitivity to contaminants (Maila and Cloete 2002; Banks and Schultz 2005). Further, phytotoxicity evaluations are mostly relevant when possible phytotoxic contaminants are in the soil (Frische 2003). Adam and Ducan (2002) have noted that the variations in the scale of seed germination inhibition are due to differences in species, concentration, and type of oil. Thus, seed germination experiments were carried out to investigate the phytotoxicity of long-term TPH-contaminated soil. Seed germination of all tested plant species Z. mays, C. sativus, and L. sativa L. in uncontaminated soils (data not shown) which was used as the control was of 100 %. Fig. 1 shows the increasing trend of seed GI with the increase in TPH level in all the three tested species. However, the tested species displayed apparent differences in seed germination inhibition at the different TPH doses. In soil with the lowest TPH contamination level (1.0 %), L. sativa showed 41 % of GI. Even though Z. mays and C. sativus responded in the same way (inhibition rate 8.33 %) at the lowest contamination level, differences in GI were observed at high TPH doses. In the highest tested TPH level (2.9 %), L. sativa showed 86 % of GI, and at the same level, there were only 47 and 55 % of inhibitions, respectively, for Z. mays and C. sativus. The resulted inhibition rate for each species at different TPH levels was significant (p < 0.05) from the control, and the results of high majority of treatments were significant from each other. Therefore, these results infer that the susceptibility of the tested species to seed germination inhibition depends on the TPH contamination level. The calculated IC50 values were 1.09, 2.47, and 3.12 % for L. sativa L., C. sativus, and Z. mays, respectively. Furthermore, the results indicate that the sensitivity of seed germination for toxic influence is species-specific and varies as L. sativa L. > C. sativus > Z. mays.
https://static-content.springer.com/image/art%3A10.1007%2Fs11270-013-1553-x/MediaObjects/11270_2013_1553_Fig1_HTML.gif
Fig. 1

Inhibition of a seed germination and b root elongation of Z. mays, C. sativus, and L. sativa L. over the tested range of TPH-contaminated soils. Error bars represents the standard deviation of three independent measurements

Bank and Schultz (2005) reported a great reduction in germination of L. sativa L. in artificially TPH-contaminated soil, and the soil with 2.4 % dose displayed only 5 % of germination. They recommended using L. sativa L. germination test to assess the TPH-contaminated soil toxicity. Since our findings in the present study are in agreement with their results, the calculated IC50 for germination inhibition approved the sensitivity of L. sativa L. seed germination test to be employed in the toxicity evaluation of long-term petroleum-contaminated soil. Ogboghodo et al. (2003) quoted an increase in germination inhibition of Z. mays as a response to rising oil contamination level in artificially contaminated soil with two different types of crude oils. Even though the trend in germination inhibition here agreed with the resulted germination inhibition trend of the present study, the calculated IC50 infer that the sensitivity of Z. mays seed germination test might be suitable only for the characterization of toxicity of long-term petroleum-contaminated soil with much higher doses than the tested ones. Although toxicity of hydrocarbon-contaminated soil on seed germination of C. sativus has not been studied previously, the present study indicates that the suitability of seed germination test of C. sativus to characterize the phytotoxicity of long-term TPH-contaminated soil as 50 % of inhibition was observed for the tested range.

During the germination under the normal condition, hydrolysis of plant storage carbohydrates in seeds is started with imbibitions which reduce the osmotic potential of the radical cells leading for rapid increase in water uptake and vacuolar volumetric growth (Bewley and Black 1994) marking the germination. In the presence of TPH or their microbial metabolites in the soil, these compounds may enter into seeds with imbibition. Then, as a consequence of toxic influence, inhibition in the hydrolysis and the mobilization of hydrolyzed product might take place, resulting in germination inhibition or alterations. We can speculate this as the reason for the resulted seed germination inhibition since the exact mechanism of seed germination inhibition due to the toxic effect of hydrocarbon is not available in the literature. Differential responses of the tested species for toxic influence on germination are acceptable due to species-specific differences in susceptibility to TPH contaminants and their microbial metabolites.

3.2 Root Elongation and Biomass

The root growth is a very complex process resulting from a number of mutually connected molecular–biological, biochemical, and physiological processes (Kummerova et al. 2013). In the literature, various authors (Xu and Johnson 1995; Merkl et al. 2005) have reported the inhibitory influence of petroleum hydrocarbons on root growth. Seed germination is the test with low sensitivity for toxicity evaluation of oil-contaminated soil as compared with plant growth as an end point (Dorn et al. 1998). Reynose-Cuevas et al. (2008) found the differences in the negative influence of hydrocarbon-contaminated soil on root growth of tested grasses species. Therefore, in the present study, root elongation of all tested three species and RBI, except L. sativa L., were used as toxicity end points. The RBI of L. sativa L. was not measured due to poor root growth in the TPH-contaminated soils. The upward trend of REI in all tested species was noticed with increasing doses of TPH in soil from 1.0 to 2.9 % (Fig. 2a). At the lowest TPH dose (1.0 %), the root elongation inhibitions were 48.38, 17.55, and 11.77 % for L. sativa L., C. sativus, and Z. mays and at the highest TPH level (2.9 %) and the corresponding values were 90.48, 63.75, and 58.16 %, respectively. All the data were significantly different from the control, and high majority of measurements were significantly different from each other. The TPH-contaminated soil had significant phytotoxic effect on the root growth of the plants even at the lowest contamination level, marking a clear dose–response relationship. The calculated IC50 were 0.68, 2.34, and 2.76 % for L. sativa L., C. sativus, and Z. mays, respectively. Therefore, the susceptibility of root growth to phytotoxic influence of long-term TPH-contaminated soil is as follows: L. sativa L. > C. sativus > Z. mays, demonstrating the species-dependent influence. Our results are in complete agreement with those of a previous work (Liste and Prutz 2006) that the concentration of hydrocarbon contaminants affecting the root growth was dependent on plant species. When comparing the calculated IC50 for seed germination inhibitions (1.09, 2.47, and 3.12 %) and root elongation inhibitions (0.68, 2.33, and 2.76 %) for the tested plants, it was obvious that the toxic influence of TPH-contaminated soil was more pronounced on root growth inhibition than seed germination inhibition. Most previous studies show that the root length is a more sensitive toxicity end point than seed germination (Oleszczuk and Hollert 2011). Thus, our results implied the suitability of root growth rather than seed germination as an end point in toxicity characterization of long-term TPH-contaminated soil.
https://static-content.springer.com/image/art%3A10.1007%2Fs11270-013-1553-x/MediaObjects/11270_2013_1553_Fig2_HTML.gif
Fig. 2

Inhibition of root biomass for Z. mays and C. sativus over the tested range of TPH-contaminated soils. Error bars represents the standard deviation of three independent measurements

The same trend as observed in germination and root growth inhibition was noticed in RBI of the tested species over the tested range of TPH-contaminated soil (Fig. 2). The recorded suppressions in root biomass of Z. mays at the lowest (1.0 %) and the highest TPH contamination levels (2.9 %) were 12 and 60 %, respectively. As a response to TPH contaminants, root biomass of C. sativus showed 27 % of inhibition at the lowest and the 70 % of inhibition at the highest level of TPH dose. All the measured values were significantly different from the control as well as from each other, and the IC50 for C. sativus and Z. mays were 2.0 and 2.6 %, respectively. According to the results, the root biomass of both tested plants showed the higher sensitivity as a toxicity end point compared to the calculated IC50 values on the basis of end points, seed germination, and root growth inhibition. Since root biomass of C. sativus displayed higher inhibition than that for Z. mays, it confirmed the applicability of root biomass of C. sativus as a toxicity end point in phytotoxicity evaluation of long-term TPH-contaminated soil.

As previously mentioned under seed germination inhibition, possible negative impact on radical cells due to toxicity of TPH and their metabolites may also be one of the major reasons for the inhibition in root growth and biomass as these two parameters are interrelated. Reynose-Cuevas et al. (2008) have pointed out that the possibility of root growth inhibition through blocking the water and nutrient uptake due to attachment of contaminants to the root surface. However, toxic contaminants such as petroleum hydrocarbon and their microbial metabolites may inhibit the root growth and subsequently inhibit the biomass due to their toxic influence on the apical meristem and elongation zone rather than block nutrient uptake through attachment to the root surface. Root elongation inhibition may arise from the presence of hydrogen ions in the medium (Pessarakli 1999). As the pH of the tested soil was not in an acidic range, this cannot be an acceptable reason to elucidate the results. The observed species-dependent susceptibility in both root growth and biomass inhibition could be due to differences in the responses of radical cell to toxic influence and/or differences in binding affinities of TPH contaminants with roots of different species.

3.3 Root Morphometric Parameters

Root morphology may also be changed due to the negative influence of hydrocarbon-contaminated soil on root growth (Reynose-Cuevas et al. 2008). However, the phytotoxicity data of the contaminated soil on the root morphological parameter are scarce (Merkl et al. 2005). Therefore, root morphometric studies were carried out to find out the toxic influence of TPH-contaminated soil on root meristematic and elongation zone of Z. mays and C. sativus. In this work, L. sativa L. was not employed due to its poor root growth in the contaminated soil.

Figure 3a, b shows the increase in inhibition of both cross-section areas of the MZ and EZ with the increase in TPH level in the contaminated soil. All the taken measurements over the tested range were significantly different from each other. An inhibition of the cross-section area of the MZ of Z. mays at the lowest TPH (1.0 %) and the highest tested TPH dose (2.9 %) were 16 and 60 %, respectively. The relevant data taken from C. sativus (71 %) were significantly higher than that of Z. mays (23 %). Furthermore, C. sativus showed higher decreases in the cross-section areas taken from both the MZ and EZ at almost all the tested TPH levels than Z. mays. The recorded inhibitions in the EZ of Z. mays and C. sativus at the lowest TPH dose were 13 and 16 %, respectively, and at the highest TPH level, the measured inhibitions 56 and 63 %, respectively. The calculated IC50 on the basis of inhibition in the MZ were 1.94 and 2.19 % for C. sativus and Z. mays, and the calculated IC50 on the basis of inhibition in the EZ were 2.4 and 2.7 %, respectively.
https://static-content.springer.com/image/art%3A10.1007%2Fs11270-013-1553-x/MediaObjects/11270_2013_1553_Fig3_HTML.gif
Fig. 3

Inhibition in cross-section area of a root meristematic and b root elongation zone of Z. mays and C. sativus over the tested range of TPH-contaminated soils. Error bars represents the standard deviation of three independent measurements

The results imply the differential responses of two different root zones marking comparatively high sensitivity of both tested end points. The MZ of both tested species show higher sensitivity than the EZ for TPH contaminants, suggesting the applicability of root morphometric parameters specially inhibition in the cross-section area of the MZ as a toxicity end point. Since the highest sensitivity exhibited the MZ of C. sativus, root morphometric parameter also confirm the suitability of C. sativus as a test plant.

Merkl et al. (2005) found that the decrease in root length and increase in root diameter in tested graminoides (Brachiaria brizantha, Cyperus aggregatus) as a response to laboratory-spiked crude oil-contaminated soil. Although Z. mays is also a graminoid, we observed the inhibition in root length and root cross-section area, implying the species specificity as well as specificity of toxicity end point in response to petroleum-contaminated soil. The meristematic zone of a root is involved in cell division, and the elongation zone is involved in cell expansion. Thus, the contribution of both zones is important for root growth. The suppressions in the both the MZ and the EZ reflect the toxic influence of TPH-contaminated soil on both processes. Cell division in the MZ was highly impacted than the cell elongation in the MZ due to the higher sensitivity of cells in the MZ for contaminants than to that of cells in the EZ.

Inckot et al. (2011) reported the reduced size in apical meristem due to toxicity of petroleum-contaminated soil, which further strengthens our findings. However, subsequent molecular mechanism of TPH contaminant toxicity on root MZ and EZ is still not clearly understood.

3.4 Microcalorimetric Analysis

Heat emission is the result of biochemical reactions of energy metabolism in living cells. Since microcalorimetric technique has been applied to evaluate metabolic activities, responses for stress condition can be characterized (Smith et al. 2000) as the amount of heat emission that reflects how they react with stressors such as toxins (Edelstein et al. 2001). In the present study, the microcalorimetric technique was employed to characterize the influence of TPH-contaminated soil on the early seedling growth. The metabolic heat emission rate–time curves of the tested seedlings (Z .mays, C. sativus, and L. sativa L.), which were obtained after incubation of seeds at 25 °C in the uncontaminated control soil and in the TPH-contaminated soils with different doses, are shown in Fig. 4. The control experiments of all the tested species displayed an increasing trend in the MHER up to a maximum point and a tendency to stabilize at the end of the experimental time without showing considerable changes. However, the MHER of the tested seedlings which were germinated in soil containing different doses of TPH showed the deviation from the control and the tendency of decreasing with the rising TPH-contaminated level. Thus, the seed germination in TPH-contaminated soil caused a reduction in the metabolism of seedlings due to the imposed toxic effect during the germination, and the extent of the impact increased with the rising TPH dose. The seedlings of different species over the tested TPH doses reached to maximum MHER at different periods with obvious differences in the obtained MHER–time curves. Therefore, our findings infer that the susceptibility to the phytotoxic effect of TPH-contaminated soil is dose-dependent and species-specific.
https://static-content.springer.com/image/art%3A10.1007%2Fs11270-013-1553-x/MediaObjects/11270_2013_1553_Fig4_HTML.gif
Fig. 4

The metabolic heat emission rate-time curves for Z. mays, C. sativus, and L. sativa L. Seedlings were germinated in uncontaminated (control) and contaminated soils with (a) 1.0, (b) 1.5, (c) 2.0, (d) 2.5, and (e) 2.9 % w/w TPH

The MHER of Z. mays seedling of control reached to about 225 μW 0.1 g−1 DW at the peak of the heat emission, and it decreased to about 210 μW 0.1 g−1 DW at the end of the incubation period. The suppression in MHER at the maximum point as a percentage of control varied nearly from 2.0 to 35 % over the tested range of TPH doses. However, at the end of the experimental time, inhibition in MHER at the lowest and the highest doses were around 9.5 and 52 %, respectively. Although the metabolism of Z. mays seedling is negatively affected due to exposure to contaminants, over 50 % inhibition (about 52 %) compared to the control was found at the end of the experimental time only in seedling which were exposed to the highest dose of TPH during the germination. These results infer that the considerable impact on the metabolism of Z. mays seedlings can be expected only in seedlings which are germinated in the highly TPH-contaminated soil. Both C. sativus and L. sativa L. exhibited higher reduction in MHER than Z. mays over the tested range of TPH doses. An inhibition in MHER as a percentage of control at the highest recorded point of C. sativus changed from about 12 to 66 %. At the end of the experiment time, the recorded values varied from 49 to 97 % over the tested range, displaying higher decreasing tendency than at the peak.

L. sativa L. showed higher inhibition in MHER than Z. mays and C. sativus at the peak and end of the experimental time. At the lowest and the highest tested TPH doses, the recorded decreases in MHER at the peak were about 39 and 71 %, respectively, as a percentage of control. The values at the end of the experiment were 65 and 98 %. When comparing with the inhibitions in MHER, the germination of L .sativa L. is the most susceptible to the toxic effect of TPH contaminants.

Seed germination consists of different phases including imbibition, catabolism, anabolism, and finally radicle projection marking the end of the germination and starting the seedling growth (Cosgrove 1997). Phytotoxic compounds inhibit the seed germination or have negative influence, resulting abnormalities in seedling development (Kupidlowska et al. 2006). The abnormalities in seedling development due to exposure to TPH contaminants during the germination could have been the reason for the observed inhibition in MHER of the tested seedlings.

The microcalorimetric analysis shows that the impact on seedlings due to the imposed phytotoxicity of TPH-contaminated soil is Z. mays < C. sativus < L. sativa L. This is in complete agreement with the order of susceptibility of the tested species to the phytotoxicity of the long-term TPH-contaminated soil. In essence, microcalorimetry can be a suitable tool to characterize the phytotoxicity of the long-term TPH-contaminated soil.

4 Conclusion

The tested long-term TPH-contaminated soils show contamination level-dependent inhibition in all the examined parameters of tested species, implying phytotoxicity with a dose-dependent relationship. The tested soils elicit highly toxic effect on both seed germination and root elongation of L. sativa L., and the root elongation is the most susceptible end point. IC50 values show that for both Z. mays and C. sativus, an inhibition in the cross-section area of the MZ is the most sensitive toxicity end point, and the inhibition in seed germination is the least sensitive toxicity end point. The IC50 values for all other tested end points imply their appropriateness to be applied in toxicity assessment for TPH-contaminated soils. Since inhibition in the cross-section area of root in the EZ also shows considerable sensitivity for phytotoxic impact, the tested root morphometric parameters of Z. mays and C. sativus can be applied as novel end points in toxicity characterization of long-term TPH-contaminated soil. Microcalorimetric analysis confirms the applicability of MHER as a comprehensive toxicity end point, and it is a useful approach to investigate the phototoxic effect of long-term TPH-contaminated soil. All the IC50 of C. sativus prove the applicability of C. sativus as a novel test plant. Considering all the IC50 in the present combined approach, it can be concluded that the phytotoxicity of the tested long-term TPH-contaminated soil is species-specific and the susceptibility of the tested species is Z. mays < C. sativus < L. sativa L. The findings of this work may not only provide useful information on phytotoxicity assessments in long-term TPH-contaminated soil but also assist in bioremediated soil or species screening for phytoremediation of hydrocarbon-contaminated soil.

Acknowledgments

This work is supported in part by grants from the National Outstanding Youth Research Foundation of China (40925010), International Joint Key Project from the National Natural Science Foundation of China (40920134003), International Joint Key Project from the Chinese Ministry of Science and Technology (2010DFA12780 and 2009DFA92830), and the National Natural Science Foundation of China (41273092). KM acknowledges the receipt of a Chinese Government Scholarship from the Chinese Scholarship Council.

Supplementary material

11270_2013_1553_MOESM1_ESM.doc (30 kb)
ESM 1(DOC 30 kb)

Copyright information

© Springer Science+Business Media Dordrecht 2013