Marine Pollution Bulletin 79 (2014) 34–38
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Growth, nutrient status, and photosynthetic response to diesel-contaminated soil of a cordgrass, Spartina argentinensis Susana Redondo-Gómez a,⇑, María C. Petenello b, Susana R. Feldman b a b
Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, Spain Biología, Facultad de Ciencias Agrarias y CIUNR, Universidad Nacional de Rosario, Argentina
a r t i c l e Keywords: Growth Diesel tolerance Mycorrhizal index Nutrient status Photosynthesis Soil contamination
i n f o
a b s t r a c t The present study was conduced to investigate the tolerance limits of Spartina argentinensis, which occurs in inland marshes of the Chaco-Pampean regions of Argentina, to diesel-contaminated soil. A glasshouse experiment was designed to investigate the effect of diesel fuel from 0% to 3% on growth and photosynthetic apparatus of S. densiflora by measuring gas exchange and photosynthetic pigments. We also performed chemical analysis of plant samples, and determined mycorrhizal index. Tiller and root biomasses declined with increasing diesel fuel concentration, as well as photosynthetic rate (A). Reductions in A could be accounted for by non-stomatal limitations. Mycorrhizal roots of S. argentinensis were reduced by the presence of diesel fuel, but did not affect its nutritional status; in fact, most element concentrations increased with diesel contamination. Despite the negative effect of diesel-contaminated soil, S. argentinensis continued growing, which could be useful management options for phytorremediation of diesel-contaminated soils. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Accidental oil spills represent a serious health risk to wetland systems throughout the world (Pezeshki et al., 2000). Diesel fuel is a complex mixture of petroleum hydrocarbons containing everything from alkanes to naphthalenes which may interfere with normal plant development. Furthermore, polycyclic aromatic hydrocarbons (PAHs) found in diesel spills are of particular concern as they are relatively persistent in the soil (Wang et al., 1990). There are urgent needs to find effective technologies to clean up contaminated soils. Phytoremediation, the use of plants to detoxify polluted environments, is under active investigation as a low-cost and environmentally friendly technique for restoration of contaminated soils and waters (Pilon-Smits, 2005). Plants have been shown to encourage organic contaminant reduction (Palmroth et al., 2002; Redondo-Gómez et al., 2011), principally by providing an optimal environment for microbial proliferation in the rhizosphere (Kruger et al., 1997). If plants can be successfully established on polluted soils, then the plant–microbial interaction in the rhizosphere may provide an economical method for enhancing microbial degradation of complex organic contaminants. But, plants are very sensitive and respond rapidly to diesel presence (Adam and ⇑ Corresponding author. Address: Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla, Spain. Tel.: +34 95 4557165; fax: +34 95 4615780. E-mail address: [email protected] (S. Redondo-Gómez). 0025-326X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2014.01.009
Duncan, 1999). Therefore, is very important to identify plants capable of growing in diesel-contaminated soils. Spartina argentinensis Parodi is the dominant species of temporally flooded inland marsh communities of Argentina, growing in hydro-halomorphic soils with high Na concentration in the upper layers. These communities cover approximately three millions hectares in the Chaco-Pampean regions of central Argentina (Cabrera and Willink, 1973). Furthermore, this species has shown to be useful for bioremediation; Redondo-Gómez et al. (2011) found that S. argentinensis is a chromium hyperaccumulator. Nonetheless, no studies have reported the physiological impact of diesel fuel. Considering that this species has high photosynthetic and growth rates, resprouts after disturbance even under severe drought conditions (Feldman et al., 2004; Feldman and Lewis, 2007), can germinate on diesel contaminated substrates (Petenello and Feldman, 2012), and that the closely related species, S. densiflora demonstrated high tolerance to phenantrene (represents a class of more persistent PAHs; Redondo-Gómez et al., 2011), it was hypothesized that S. argentinensis could be used for diesel phytoremediation. Consequently, the main objective of the present study was to evaluate the tolerance of S. argentinensis to diesel contaminated soil. The specific objectives were to: (1) analyze the growth of plants in experimental diesel treatments ranging from 0% to 3% (v/w) diesel fuel; (2) ascertain the extent to which effects on the photosynthetic apparatus, gas exchange characteristics and photosynthetic pigments determine plant performance with diesel increasing; and (3) examine possible role of mycorrhizal-colonization in roots of
S. Redondo-Gómez et al. / Marine Pollution Bulletin 79 (2014) 34–38
S. argentinensis and nutrient status in response to increasing external diesel contamination in explaining effects on growth. A number of studies (Adam and Duncan, 1999, 2003; Kechavarzi et al., 2007; Lin and Mendelssohn, 2009; Lam, 2012; Petenello and Feldman, 2012) have investigated the effect of diesel on plant growth. However, to our knowledge, little is known about the extent to which effects of diesel on photosynthetic apparatus and nutrient status determine plant performance. 2. Materials and methods 2.1. Plant material and stress treatments Ripe spikes of S. argentinensis were collected in February 2012 from the glen of Ludueña stream (15 km SW Rosario, 32°450 S; 60°350 W, Santa Fe, Argentina). Caryopses were stripped from the spikes and those containing seeds were selected and stored at 25 °C (in the dark). Commercial diesel fuel (density, 0.875–0.900 g cm3 at 15 °C; 500 ppm S; Cetane index, 46; flashpoint, 48 °C; and viscosity, 3.5–5.0 cSt at 37.8 °C) was mixed with Vertic Argiudol soil (Roldán series; clay 24.1%, silt 71.4% and sand 4.4%; total organic carbon 3.68%; total nitrogen 0.28%; assimilable phosphorous 44.1 ppm; Cation Exchange Capacity 17.67; pH 6.65) from Faculty of Agricultural Sciences (33°010 S; 60°530 W, Zavalla, Argentina) at 0%, 1%, 2% and 3% (v/w), and field-moist soil was placed into polyethylene bags. The soil–diesel mixture was allowed to stabilise for 72 h before planting. Because preliminary experiments indicated that the use of high soil–diesel concentrations inhibited the growth of S. argentinensis from seeds, the higher concentration of diesel fuel was set to 3%. In April 2012, seeds were placed in individual plastic pots (15 cm of diameter and 20 of height) filled with 800 g of the appropriate soil–diesel mixture. The pots were allocated in shallow trays; fifteen pots per tray, with one tray per diesel fuel treatment. Trays were placed in a glasshouse (Rosario, Santa Fe, Argentina) with controlled temperature of 21–25 °C, 40–60% relative humidity and natural daylight. Pots were irrigated with tap water as necessary. The possibility of adding NaCl to the culture medium was disregarded because salt does not affect either the photosynthetic functions or the growth of S. argentinensis. Plants were grown for 250 days under the previously described conditions, long enough to assess the tolerance in diesel-fuel-treated plants.
35
0.05). In addition, tiller and root Cu, Mn and P contents were correlated (Cu, r = 0.64, P < 0.05; Mn, r = 0.88, P < 0.0001; P, r = 0.73, P < 0.01). 3.3. Gas exchange Net photosynthetic rate (A) declined with increasing diesel concentration (r = 0.37, P < 0.01); and the highest A value was recorded at 0% diesel (ANOVA, P < 0.0001; Fig. 2A). Furthermore, there was a significant relationship between total biomass and A (r = 0.99, P < 0.01). In contrast, there were not relationships between stomatal conductance (Gs) and A, or Gs and diesel concentration (Fig. 2B). Moreover, intercellular CO2 concentration (Ci) increased with increasing diesel concentration (r = 0.40, P < 0.01; Fig. 2C). 3.4. Photosynthetic pigment concentration There was no effect of diesel treatment on chlorophyll a content (Chl a; Fig. 3A), but chlorophyll b content (Chl b) diminished with
Fig. 1. Tiller (A) and root (B) biomasses of Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 15. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
increasing external diesel concentration after 250 d of treatment (r = 0.61, P < 0.01; Fig 3B). Carotenoids (Cx + c) also showed no relationship with diesel concentration (Fig. 3C). 3.5. Mycorrhizal index The percentage of mycorrhizal roots declined with increasing diesel fuel concentration (r = 0.94, P < 0.001), ranging between 77% and 7% for the control and the highest diesel concentration, respectively (Fig. 4). 4. Discussion Spartina argentinensis demonstrated low tolerance to diesel fuel in the soil because its grown was lower than 50% of the control at all diesel dosages (total biomass decreased by 65% at 1% diesel fuel) after 250 days of exposure; the higher the level of pollution, the more the effect. Hernández-Ortega et al. (2012) obtained similar result for Melilotus albus; treatment of 0.75% diesel fuel reduced its total biomass by 75% with respect to the control. Nevertheless, residual oil concentrations in the sediment of up to 5% did not adversely affect Spartina alterniflora (DeLaune et al., 1979), suggesting the great difference in tolerance between plant species. However, Lam (2012) recorded 60% and 15% survival of Spartina foliosa for the crude oil and diesel treatments, respectively, possibly because diesel refined nature and low-molecular weight make it more bioavailable to the plant. Otherwise, Lin and Mendelssohn (2009) found that belowground biomass in the 8–16 cm subsurface sediment was affected at all diesel dosages, while live stem density was significantly affected at oil dosages P16%. In contrast, the effect of diesel fuel in the soil on aboveground biomass of S. argentinensis was more severe compared to the belowground component (root biomass diminished by 65%, 88% and 91% and tiller biomass by 71%, 90% and 92% at 1%, 2% and 3% diesel fuel, respectively). Similar result was recorded for perennial ryegrass in 2.5% diesel contaminated subsurface soil layers (Kechavarzi et al., 2007). The declining growth of S. argentinensis in presence of diesel contamination may be attributed to a decrease in A. Pajevic´ et al. (2009) also found that photosynthetic rates of willow genotypes diminished in soil polluted with 1% diesel fuel, and explained that inhibition of Gs in plants grown under conditions of soil contamination indicated that depression of photosynthesis occurred to a great extent at the stomatal level. In the present experiment, the limitation to net photosynthetic rate was not because of Gs; there is no correlation between A and Gs. Furthermore, we recorded an increase of Ci with increasing diesel concentration, which could be linked to a decrease in RuBisCO activity (Monnet et al., 2001; Mateos Naranjo et al., 2008). On the other hand, diesel dosages higher than 2% enhanced tiller Mn concentrations and decreased chlorophyll b contents of S. argentinensis. Related to this, Mn (55 mg kg1) has been described to have a toxic effect on chlorophyll content, especially on concentrations of chlorophyll b (Macfie and Taylor, 1992). Additionally, decreased chlorophyll b contents could be partially attributed to lower tiller copper concentration in presence of diesel fuel, as noted Peng et al. (2013). The decrease in pigment concentration or an increase in pigment degradation could also lead to a decline in photosynthetic functioning of S. argentinensis. Mycorrhizal-colonization in roots of S. argentinensis was significantly reduced by the presence of diesel fuel, although mycorrhizal associations were still developed. Kirk et al. (2005) noted that, in the presence of plant roots, diesel reduced fungal colonization, germ tube and hyphal growth; they hypothesized that diesel interfered with communication between the plant and fungus as well as having toxic effects on fungal growth. However, it did not affect the
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Table 1 Total Ca, Cu, Fe, K, Mg, Mn, N, P and Zn concentrations for tillers and roots of Spartina alterniflora grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 3. Means within a diesel treatment that have different letter are significantly different from each other (LSD test, P < 0.05). Treatment
Tiller concentration
Diesel fuel (%)
Ca (mg g1)
Cu (mg kg1) a
0 0.09 ± 0.006 1 0.09 ± 0.004a 2 0.10 ± 0.007a 3 0.10 ± 0.010a Root concentration 0 0.17 ± 0.018a 1 0.18 ± 0.024a 2 0.28 ± 0.014b 3 0.28 ± 0.020b
c
2.1 ± 0.08 1.8 ± 0.06bc 1.7 ± 0.16ab 1.4 ± 0.11a 5.7 ± 0.62a 7.2 ± 0.66ab 11.1 ± 0.76c 9.3 ± 0.71bc
Fe (mg kg1) 76 ± 10.8 86 ± 5.5a 81 ± 2.4a 93 ± 3.4a
a
836 ± 58.5a 1707 ± 222.2b 4933 ± 319.7c 5164 ± 229.6c
K (mg g1)
Mg (mg g1) a
Mn (mg kg1) a
N (mg g1)
1.51 ± 0.133 1.68 ± 0.007a 1.74 ± 0.039a 1.71 ± 0.164a
a
b
0.09 ± 0.008 0.10 ± 0.002ab 0.13 ± 0.004b 0.12 ± 0.011ab
37 ± 2.8 37 ± 0.7a 124 ± 5.4b 122 ± 15.4b
11.1 ± 0.16 9.9 ± 0.04a 12.9 ± 0.41c 12.5 ± 0.38c
0.53 ± 0.016b 0.57 ± 0.007b 0.46 ± 0.029a 0.71 ± 0.016c
0.07 ± 0.04a 0.09 ± 0.007a 0.11 ± 0.005b 0.13 ± 0.006b
206 ± 15.4a 453 ± 61.5b 1047 ± 73.6c 937 ± 66.5c
7.5 ± 0.30a 8.9 ± 0.79a 9.1 ± 0.73a 9.4 ± 0.92a
P (mg g1)
Zn (mg kg1) a
0.07 ± 0.006 0.07 ± 0.001a 0.10 ± 0.003b 0.08 ± 0.008ab
7.5 ± 0.43a 5.6 ± 0.14a 6.9 ± 0.86a 6.7 ± 0.76a
0.04 ± 0.002a 0.07 ± 0.003b 0.13 ± 0.006c 0.14 ± 0.005c
19.5 ± 2.05a 22.1 ± 2.22a 32.5 ± 1.74b 33.0 ± 2.10b
Fig. 2. (A) Net photosynthetic rate (A), (B) stomatal conductance (Gs), (C) intercellular CO2 concentration (Ci) in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 12. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
Fig. 3. (A) Chlorophyll a (Chl a), (B) chlorophyll b (Chl b) and (C) carotenoid (Cx + c) concentrations in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 5. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
nutrient uptake of S. argentinensis, since its element concentrations increased with diesel contamination; except tiller Cu and K concentrations, which diminished and remained unchanged, respectively. Thus, the role of mycorrhizal associations in nutrient uptake of S. argentinensis was insignificant in the current study. Hernández-Ortega et al. (2012) found that plants of Melilotus albus treated with 0.75% diesel had reduced content of elements compared to plants without the contaminant, although arbuscular
mycorrhizal fungi (AMF)-plants had greater content of microelements than non-AMF plants. When petroleum hydrocarbons contaminate soil, the added carbon stimulates microbial numbers and causes an imbalance in the C:N ratio which may result in immobilization of soil nitrogen by the microbial biomass, leaving none available for plant growth (Xu and Johnson, 1997). Thus, studies on phytoremediation pay particular attention to the performance of legumes versus other
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S. Redondo-Gómez et al. / Marine Pollution Bulletin 79 (2014) 34–38
Fig. 4. Percentage of mycorrhizal roots in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 12. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
plants species (Adam and Duncan, 2003). Surprisingly, tissue nitrogen content of S. argentinensis increased with diesel pollution. Otherwise, nitrate reductase (NRA) is an important enzymatic activity in plants exposed to different environmental stresses (Foyer et al., 1998; Taiz and Zeiger, 2002). In this regard, Hernández-Ortega et al. (2012) noted that diesel induced greater root NRA in Melilotus albus, suggesting that nitrogen assimilation is a crucial process for plant establishment at contaminated soils. In conclusion, diesel contamination resulted in negative effects on the growth of S. argentinensis, which could be consequence of reduced photosynthesis, but did not affect its nutritional status. Despite the negative impact of the presence of diesel fuel, the plants continued growing and did not exhibit chlorosis after 250 d of treatment; therefore, this species can be useful management options for phytorremediation of diesel-contaminated soils. However, the continuous exposition to diesel-contaminated soils might affect in acute manner photosynthesis, with consequences at the mineral balance. Thus, long-term studies will be needed in the future. Acknowledgements We are grateful to the Council of the National University of Rosario (Argentina; AGR-164) and Spanish Science and Technology Ministry and Andalusian Council for their supports (projects CTM2008-04453 and P11-RNM-7274, respectively). References Adam, G., Duncan, H.J., 1999. Effect of diesel fuel on growth of selected plant species. Environ. Geochem. Health 21, 353–357. Adam, G., Duncan, H., 2003. The effect of diesel fuel on common vetch (Vicia sativa L.) plants. Environ. Geochem. Health 25, 123–130. Cabrera, A.L., Willink, A., 1973. Biogeografía de América Latina, Serie de Biología, Monografía No. 13. OEA, Washington, D.C.. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Figueroa, M.E., 2008. Comparison of the role of two Spartina species in terms of phytostabilization and bioaccumulation of metals in the estuarine sediment. Mar. Pollut. Bull. 56, 2037–2042. DeLaune, R.D., Patrick Jr., W.H., Buresh, R.J., 1979. Effect of crude oil on a Louisiana Spartina alterniflora salt marsh. Environ. Pollut. 20, 21–30. Feldman, S.R., Bisaro, V., Lewis, J.P., 2004. Photosynthetic and growth responses to fire of the subtropical-temperate grass Spartina argentinensis Parodi. Flora 199, 491–499.
Feldman, S.R., Lewis, J.P., 2007. Effect of fire on Spartina argentinensis Parodi demographic characteristics. Wetlands 27, 785–793. Foyer, C.H., Valadier, M.H., Migge, A., Becker, T.W., 1998. Drought/induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves. Plant Physiol. 117, 283–292. Hernández-Ortega, H.A., Alarcón, A., Ferrera-Cerrato, R., Zavaleta-Mancera, H.A., López-Delgado, H.A., Mendoza-López, M.R., 2012. Arbuscular mycorrhizal fungi on growth, nutrient status, and total antioxidant activity of Melilotus albus during phytoremediation of a diesel-contaminated substrate. J. Environ. Manage. 95, S319–S324. Kechavarzi, C., Pettersson, K., Leeds-Harrison, P., Ritchie, L., Ledin, S., 2007. Root establishment of perennial ryegrass (L. perenne) in diesel contaminated subsurface soil layers. Environ. Pollut. 145, 68–74. Kirk, J.L., Moutoglis, P., Klironomos, J., Lee, H., Trevors, J.T., 2005. Toxicity of diesel fuel to germination, growth and colonization of Glomus intraradices in soil and in vitro transformed carrot root cultures. Plant Soil 270, 23–30. Kruger, E.L., Anderson, T.A., Coats, J.R., 1997. Phytoremediation of contaminated water and soil. In: Kruger, E.L., Anderson, T.A., Coats, J.R. (Eds.), Phytoremediation of soil and water contaminants. ACS Symposium Series 664. Americam Chemical Society, Washintong D.C., pp. 2–17. Lam, K., 2012. Alaska crude oil and diesel effects on Californian Spartina foliosa and Spartina foliosa alterniflora hybrid cordgrass. Oil Effects on marsh cordgrass. Environmental Sciences Senior Thesis Symposium, UC Berkeley Environmental Sciences. University of California at Berkeley, Berkeley, California, pp. 1–16. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Method Enzymol. 148, 350–382. Lin, Q., Mendelssohn, I.A., 2009. Potential of restoration and phytoremediation with Juncus roemerianus for diesel-contaminated coastal wetlands. Ecol. Eng. 35, 85– 91. Macfie, S.M., Taylor, G.J., 1992. The effects of excess manganese on photosynthetic rate and concentration of chlorophyll in Triticum aestivum in solution culture. Physiol. Plant. 85, 467–475. Mateos Naranjo, E., Redondo-Gómez, S., Cambrollé, J., Luque, T., Figueroa, M.E., 2008. Growth and photosynthetic responses to zinc stress of an invasive cordgrass, Spartina densiflora. Plant Biol. 10, 754–762. McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L., Swan, J.A., 1990. A new method which gives an objective measure of colonization of roots by vesiculararbuscular mycorrhizal fungi. New Phytol. 115, 495–501. Monnet, F., Vaillant, N., Vernay, P., Coudret, A., Sallanon, H., Hitmi, A., 2001. Relationship between PSII activity, CO2 fixation, and Zn, Mn and Mg contents of Lolium perenne under zinc stress. J. Plant Physiol. 158, 1137–1144. Pajevic´, S., Borišev, M., Nataša, N., Krstic´, B., Pilipovic´, A., Orlovic´, S., 2009. Phytoremediation capacity of poplar (Populus spp.) and willow (Salix spp.) clones in relation to photosynthesis. Arch. Biol. Sci., Belgrade 61, 239–247. Palmroth, M.R.T., Pichtel, J., Puhakka, J.A., 2002. Phytoremediation of sudarctic soil contaminated with diesel fuel. Bioresour. Technol. 84, 221–228. Peng, H., Kroneck, P.M.H., Küpper, H., 2013. Toxicity and deficiency of copper in Elsholtzia splenden affect photosynthesis biophysics, Pigments and metal accumulation. Environ. Sci. Technol. 47, 6120–6128. Petenello, M.C., Feldman, S.R., 2012. Evaluation of tolerance to soils contaminated with diesel oil in plant species with bioremediation potencial. Acta Biológica Colombiana 17, 589–598 (in Spanish). Pezeshki, S.R., Hester, M.W., Lin, Q., Nyman, J.A., 2000. The effects of oil spill and clean-up on dominant US Gulf Coast marsh macrophytes: a review. Environ. Pollut. 108, 129–139. Pilon-Smits, E., 2005. Phytoremediation. Annu. Rev. Plant Biol. 56, 15–39. Redondo-Gómez, S., Mateos-Naranjo, E., Davy, A.J., Fernández-Muñoz, F., Castellanos, E., Luque, T., Figueroa, M.E., 2007. Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides. Ann. Bot. 100, 555–563. Redondo-Gómez, S., Andrades-Moreno, L., Parra, R., Valera-Burgos, J., Real, M., Mateos-Naranjo, E., Cox, L., Cornejo, J., 2011. Spartina densiflora demonstrates high tolerante to phenanthrene in soils and reduces it concentration. Mar. Pollut. Bull. 62, 1800–1808. Vannette, R.L., Rasmann, S., 2012. Arbuscular mycorrhizal fungi mediate belowground plant-herbivore interactions: a phylogenetic study. Funct. Ecol. 26, 1033–1042. Wang, X., Yu, X., Bartha, R., 1990. Effect of bioremediation of PAH residues in soil. Environ. Sci. Technol. 24, 1086–1089. Xu, J.G., Johnson, R.L., 1997. Nitrogen dynamics in soils with different hydrocarbon contents planted to barley and field pea. 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Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Growth, nutrient status, and photosynthetic response to diesel-contaminated soil of a cordgrass, Spartina argentinensis Susana Redondo-Gómez a,⇑, María C. Petenello b, Susana R. Feldman b a b
Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Apartado 1095, 41080 Sevilla, Spain Biología, Facultad de Ciencias Agrarias y CIUNR, Universidad Nacional de Rosario, Argentina
a r t i c l e Keywords: Growth Diesel tolerance Mycorrhizal index Nutrient status Photosynthesis Soil contamination
i n f o
a b s t r a c t The present study was conduced to investigate the tolerance limits of Spartina argentinensis, which occurs in inland marshes of the Chaco-Pampean regions of Argentina, to diesel-contaminated soil. A glasshouse experiment was designed to investigate the effect of diesel fuel from 0% to 3% on growth and photosynthetic apparatus of S. densiflora by measuring gas exchange and photosynthetic pigments. We also performed chemical analysis of plant samples, and determined mycorrhizal index. Tiller and root biomasses declined with increasing diesel fuel concentration, as well as photosynthetic rate (A). Reductions in A could be accounted for by non-stomatal limitations. Mycorrhizal roots of S. argentinensis were reduced by the presence of diesel fuel, but did not affect its nutritional status; in fact, most element concentrations increased with diesel contamination. Despite the negative effect of diesel-contaminated soil, S. argentinensis continued growing, which could be useful management options for phytorremediation of diesel-contaminated soils. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Accidental oil spills represent a serious health risk to wetland systems throughout the world (Pezeshki et al., 2000). Diesel fuel is a complex mixture of petroleum hydrocarbons containing everything from alkanes to naphthalenes which may interfere with normal plant development. Furthermore, polycyclic aromatic hydrocarbons (PAHs) found in diesel spills are of particular concern as they are relatively persistent in the soil (Wang et al., 1990). There are urgent needs to find effective technologies to clean up contaminated soils. Phytoremediation, the use of plants to detoxify polluted environments, is under active investigation as a low-cost and environmentally friendly technique for restoration of contaminated soils and waters (Pilon-Smits, 2005). Plants have been shown to encourage organic contaminant reduction (Palmroth et al., 2002; Redondo-Gómez et al., 2011), principally by providing an optimal environment for microbial proliferation in the rhizosphere (Kruger et al., 1997). If plants can be successfully established on polluted soils, then the plant–microbial interaction in the rhizosphere may provide an economical method for enhancing microbial degradation of complex organic contaminants. But, plants are very sensitive and respond rapidly to diesel presence (Adam and ⇑ Corresponding author. Address: Dpto. Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla, Spain. Tel.: +34 95 4557165; fax: +34 95 4615780. E-mail address: [email protected] (S. Redondo-Gómez). 0025-326X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2014.01.009
Duncan, 1999). Therefore, is very important to identify plants capable of growing in diesel-contaminated soils. Spartina argentinensis Parodi is the dominant species of temporally flooded inland marsh communities of Argentina, growing in hydro-halomorphic soils with high Na concentration in the upper layers. These communities cover approximately three millions hectares in the Chaco-Pampean regions of central Argentina (Cabrera and Willink, 1973). Furthermore, this species has shown to be useful for bioremediation; Redondo-Gómez et al. (2011) found that S. argentinensis is a chromium hyperaccumulator. Nonetheless, no studies have reported the physiological impact of diesel fuel. Considering that this species has high photosynthetic and growth rates, resprouts after disturbance even under severe drought conditions (Feldman et al., 2004; Feldman and Lewis, 2007), can germinate on diesel contaminated substrates (Petenello and Feldman, 2012), and that the closely related species, S. densiflora demonstrated high tolerance to phenantrene (represents a class of more persistent PAHs; Redondo-Gómez et al., 2011), it was hypothesized that S. argentinensis could be used for diesel phytoremediation. Consequently, the main objective of the present study was to evaluate the tolerance of S. argentinensis to diesel contaminated soil. The specific objectives were to: (1) analyze the growth of plants in experimental diesel treatments ranging from 0% to 3% (v/w) diesel fuel; (2) ascertain the extent to which effects on the photosynthetic apparatus, gas exchange characteristics and photosynthetic pigments determine plant performance with diesel increasing; and (3) examine possible role of mycorrhizal-colonization in roots of
S. Redondo-Gómez et al. / Marine Pollution Bulletin 79 (2014) 34–38
S. argentinensis and nutrient status in response to increasing external diesel contamination in explaining effects on growth. A number of studies (Adam and Duncan, 1999, 2003; Kechavarzi et al., 2007; Lin and Mendelssohn, 2009; Lam, 2012; Petenello and Feldman, 2012) have investigated the effect of diesel on plant growth. However, to our knowledge, little is known about the extent to which effects of diesel on photosynthetic apparatus and nutrient status determine plant performance. 2. Materials and methods 2.1. Plant material and stress treatments Ripe spikes of S. argentinensis were collected in February 2012 from the glen of Ludueña stream (15 km SW Rosario, 32°450 S; 60°350 W, Santa Fe, Argentina). Caryopses were stripped from the spikes and those containing seeds were selected and stored at 25 °C (in the dark). Commercial diesel fuel (density, 0.875–0.900 g cm3 at 15 °C; 500 ppm S; Cetane index, 46; flashpoint, 48 °C; and viscosity, 3.5–5.0 cSt at 37.8 °C) was mixed with Vertic Argiudol soil (Roldán series; clay 24.1%, silt 71.4% and sand 4.4%; total organic carbon 3.68%; total nitrogen 0.28%; assimilable phosphorous 44.1 ppm; Cation Exchange Capacity 17.67; pH 6.65) from Faculty of Agricultural Sciences (33°010 S; 60°530 W, Zavalla, Argentina) at 0%, 1%, 2% and 3% (v/w), and field-moist soil was placed into polyethylene bags. The soil–diesel mixture was allowed to stabilise for 72 h before planting. Because preliminary experiments indicated that the use of high soil–diesel concentrations inhibited the growth of S. argentinensis from seeds, the higher concentration of diesel fuel was set to 3%. In April 2012, seeds were placed in individual plastic pots (15 cm of diameter and 20 of height) filled with 800 g of the appropriate soil–diesel mixture. The pots were allocated in shallow trays; fifteen pots per tray, with one tray per diesel fuel treatment. Trays were placed in a glasshouse (Rosario, Santa Fe, Argentina) with controlled temperature of 21–25 °C, 40–60% relative humidity and natural daylight. Pots were irrigated with tap water as necessary. The possibility of adding NaCl to the culture medium was disregarded because salt does not affect either the photosynthetic functions or the growth of S. argentinensis. Plants were grown for 250 days under the previously described conditions, long enough to assess the tolerance in diesel-fuel-treated plants.
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0.05). In addition, tiller and root Cu, Mn and P contents were correlated (Cu, r = 0.64, P < 0.05; Mn, r = 0.88, P < 0.0001; P, r = 0.73, P < 0.01). 3.3. Gas exchange Net photosynthetic rate (A) declined with increasing diesel concentration (r = 0.37, P < 0.01); and the highest A value was recorded at 0% diesel (ANOVA, P < 0.0001; Fig. 2A). Furthermore, there was a significant relationship between total biomass and A (r = 0.99, P < 0.01). In contrast, there were not relationships between stomatal conductance (Gs) and A, or Gs and diesel concentration (Fig. 2B). Moreover, intercellular CO2 concentration (Ci) increased with increasing diesel concentration (r = 0.40, P < 0.01; Fig. 2C). 3.4. Photosynthetic pigment concentration There was no effect of diesel treatment on chlorophyll a content (Chl a; Fig. 3A), but chlorophyll b content (Chl b) diminished with
Fig. 1. Tiller (A) and root (B) biomasses of Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 15. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
increasing external diesel concentration after 250 d of treatment (r = 0.61, P < 0.01; Fig 3B). Carotenoids (Cx + c) also showed no relationship with diesel concentration (Fig. 3C). 3.5. Mycorrhizal index The percentage of mycorrhizal roots declined with increasing diesel fuel concentration (r = 0.94, P < 0.001), ranging between 77% and 7% for the control and the highest diesel concentration, respectively (Fig. 4). 4. Discussion Spartina argentinensis demonstrated low tolerance to diesel fuel in the soil because its grown was lower than 50% of the control at all diesel dosages (total biomass decreased by 65% at 1% diesel fuel) after 250 days of exposure; the higher the level of pollution, the more the effect. Hernández-Ortega et al. (2012) obtained similar result for Melilotus albus; treatment of 0.75% diesel fuel reduced its total biomass by 75% with respect to the control. Nevertheless, residual oil concentrations in the sediment of up to 5% did not adversely affect Spartina alterniflora (DeLaune et al., 1979), suggesting the great difference in tolerance between plant species. However, Lam (2012) recorded 60% and 15% survival of Spartina foliosa for the crude oil and diesel treatments, respectively, possibly because diesel refined nature and low-molecular weight make it more bioavailable to the plant. Otherwise, Lin and Mendelssohn (2009) found that belowground biomass in the 8–16 cm subsurface sediment was affected at all diesel dosages, while live stem density was significantly affected at oil dosages P16%. In contrast, the effect of diesel fuel in the soil on aboveground biomass of S. argentinensis was more severe compared to the belowground component (root biomass diminished by 65%, 88% and 91% and tiller biomass by 71%, 90% and 92% at 1%, 2% and 3% diesel fuel, respectively). Similar result was recorded for perennial ryegrass in 2.5% diesel contaminated subsurface soil layers (Kechavarzi et al., 2007). The declining growth of S. argentinensis in presence of diesel contamination may be attributed to a decrease in A. Pajevic´ et al. (2009) also found that photosynthetic rates of willow genotypes diminished in soil polluted with 1% diesel fuel, and explained that inhibition of Gs in plants grown under conditions of soil contamination indicated that depression of photosynthesis occurred to a great extent at the stomatal level. In the present experiment, the limitation to net photosynthetic rate was not because of Gs; there is no correlation between A and Gs. Furthermore, we recorded an increase of Ci with increasing diesel concentration, which could be linked to a decrease in RuBisCO activity (Monnet et al., 2001; Mateos Naranjo et al., 2008). On the other hand, diesel dosages higher than 2% enhanced tiller Mn concentrations and decreased chlorophyll b contents of S. argentinensis. Related to this, Mn (55 mg kg1) has been described to have a toxic effect on chlorophyll content, especially on concentrations of chlorophyll b (Macfie and Taylor, 1992). Additionally, decreased chlorophyll b contents could be partially attributed to lower tiller copper concentration in presence of diesel fuel, as noted Peng et al. (2013). The decrease in pigment concentration or an increase in pigment degradation could also lead to a decline in photosynthetic functioning of S. argentinensis. Mycorrhizal-colonization in roots of S. argentinensis was significantly reduced by the presence of diesel fuel, although mycorrhizal associations were still developed. Kirk et al. (2005) noted that, in the presence of plant roots, diesel reduced fungal colonization, germ tube and hyphal growth; they hypothesized that diesel interfered with communication between the plant and fungus as well as having toxic effects on fungal growth. However, it did not affect the
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Table 1 Total Ca, Cu, Fe, K, Mg, Mn, N, P and Zn concentrations for tillers and roots of Spartina alterniflora grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 3. Means within a diesel treatment that have different letter are significantly different from each other (LSD test, P < 0.05). Treatment
Tiller concentration
Diesel fuel (%)
Ca (mg g1)
Cu (mg kg1) a
0 0.09 ± 0.006 1 0.09 ± 0.004a 2 0.10 ± 0.007a 3 0.10 ± 0.010a Root concentration 0 0.17 ± 0.018a 1 0.18 ± 0.024a 2 0.28 ± 0.014b 3 0.28 ± 0.020b
c
2.1 ± 0.08 1.8 ± 0.06bc 1.7 ± 0.16ab 1.4 ± 0.11a 5.7 ± 0.62a 7.2 ± 0.66ab 11.1 ± 0.76c 9.3 ± 0.71bc
Fe (mg kg1) 76 ± 10.8 86 ± 5.5a 81 ± 2.4a 93 ± 3.4a
a
836 ± 58.5a 1707 ± 222.2b 4933 ± 319.7c 5164 ± 229.6c
K (mg g1)
Mg (mg g1) a
Mn (mg kg1) a
N (mg g1)
1.51 ± 0.133 1.68 ± 0.007a 1.74 ± 0.039a 1.71 ± 0.164a
a
b
0.09 ± 0.008 0.10 ± 0.002ab 0.13 ± 0.004b 0.12 ± 0.011ab
37 ± 2.8 37 ± 0.7a 124 ± 5.4b 122 ± 15.4b
11.1 ± 0.16 9.9 ± 0.04a 12.9 ± 0.41c 12.5 ± 0.38c
0.53 ± 0.016b 0.57 ± 0.007b 0.46 ± 0.029a 0.71 ± 0.016c
0.07 ± 0.04a 0.09 ± 0.007a 0.11 ± 0.005b 0.13 ± 0.006b
206 ± 15.4a 453 ± 61.5b 1047 ± 73.6c 937 ± 66.5c
7.5 ± 0.30a 8.9 ± 0.79a 9.1 ± 0.73a 9.4 ± 0.92a
P (mg g1)
Zn (mg kg1) a
0.07 ± 0.006 0.07 ± 0.001a 0.10 ± 0.003b 0.08 ± 0.008ab
7.5 ± 0.43a 5.6 ± 0.14a 6.9 ± 0.86a 6.7 ± 0.76a
0.04 ± 0.002a 0.07 ± 0.003b 0.13 ± 0.006c 0.14 ± 0.005c
19.5 ± 2.05a 22.1 ± 2.22a 32.5 ± 1.74b 33.0 ± 2.10b
Fig. 2. (A) Net photosynthetic rate (A), (B) stomatal conductance (Gs), (C) intercellular CO2 concentration (Ci) in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 12. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
Fig. 3. (A) Chlorophyll a (Chl a), (B) chlorophyll b (Chl b) and (C) carotenoid (Cx + c) concentrations in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 5. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
nutrient uptake of S. argentinensis, since its element concentrations increased with diesel contamination; except tiller Cu and K concentrations, which diminished and remained unchanged, respectively. Thus, the role of mycorrhizal associations in nutrient uptake of S. argentinensis was insignificant in the current study. Hernández-Ortega et al. (2012) found that plants of Melilotus albus treated with 0.75% diesel had reduced content of elements compared to plants without the contaminant, although arbuscular
mycorrhizal fungi (AMF)-plants had greater content of microelements than non-AMF plants. When petroleum hydrocarbons contaminate soil, the added carbon stimulates microbial numbers and causes an imbalance in the C:N ratio which may result in immobilization of soil nitrogen by the microbial biomass, leaving none available for plant growth (Xu and Johnson, 1997). Thus, studies on phytoremediation pay particular attention to the performance of legumes versus other
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Fig. 4. Percentage of mycorrhizal roots in Spartina argentinensis grown at 0%, 1%, 2% and 3% diesel fuel over 250 days. Values represent mean ± SE, n = 12. Different letters indicate means that are significantly different from each other (LSD test, P < 0.05).
plants species (Adam and Duncan, 2003). Surprisingly, tissue nitrogen content of S. argentinensis increased with diesel pollution. Otherwise, nitrate reductase (NRA) is an important enzymatic activity in plants exposed to different environmental stresses (Foyer et al., 1998; Taiz and Zeiger, 2002). In this regard, Hernández-Ortega et al. (2012) noted that diesel induced greater root NRA in Melilotus albus, suggesting that nitrogen assimilation is a crucial process for plant establishment at contaminated soils. In conclusion, diesel contamination resulted in negative effects on the growth of S. argentinensis, which could be consequence of reduced photosynthesis, but did not affect its nutritional status. Despite the negative impact of the presence of diesel fuel, the plants continued growing and did not exhibit chlorosis after 250 d of treatment; therefore, this species can be useful management options for phytorremediation of diesel-contaminated soils. However, the continuous exposition to diesel-contaminated soils might affect in acute manner photosynthesis, with consequences at the mineral balance. Thus, long-term studies will be needed in the future. Acknowledgements We are grateful to the Council of the National University of Rosario (Argentina; AGR-164) and Spanish Science and Technology Ministry and Andalusian Council for their supports (projects CTM2008-04453 and P11-RNM-7274, respectively). References Adam, G., Duncan, H.J., 1999. Effect of diesel fuel on growth of selected plant species. Environ. Geochem. Health 21, 353–357. Adam, G., Duncan, H., 2003. The effect of diesel fuel on common vetch (Vicia sativa L.) plants. Environ. Geochem. Health 25, 123–130. Cabrera, A.L., Willink, A., 1973. Biogeografía de América Latina, Serie de Biología, Monografía No. 13. OEA, Washington, D.C.. Cambrollé, J., Redondo-Gómez, S., Mateos-Naranjo, E., Figueroa, M.E., 2008. Comparison of the role of two Spartina species in terms of phytostabilization and bioaccumulation of metals in the estuarine sediment. Mar. Pollut. Bull. 56, 2037–2042. DeLaune, R.D., Patrick Jr., W.H., Buresh, R.J., 1979. Effect of crude oil on a Louisiana Spartina alterniflora salt marsh. Environ. Pollut. 20, 21–30. Feldman, S.R., Bisaro, V., Lewis, J.P., 2004. Photosynthetic and growth responses to fire of the subtropical-temperate grass Spartina argentinensis Parodi. Flora 199, 491–499.
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