Phytotoxicity of Long-Term Total Petroleum Hydrocarbon-Contaminated Soil—A Comparative and Combined Approach
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- Masakorala, K., Yao, J., Guo, H. et al. Water Air Soil Pollut (2013) 224: 1553. doi:10.1007/s11270-013-1553-x
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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.
KeywordsTotal petroleum hydrocarbonPhytotoxicityMorphometric parametersMetabolic heat
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
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 properties of uncontaminated control soil and TPH-contaminated soil
TPH (mg kg−1)
N-NO3− (mg kg−1)
P (mg kg−1)
Cd (mg kg−1)
Pb (mg kg−1)
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
2.6 Root Growth and Root Biomass
2.7 Root Morphometric Parameters
2.8 Microcalorimetric Analysis
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
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 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.
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
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.
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.
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.