Nitrogen Polycyclic Aromatic Hydrocarbon Analogues Biology Essay

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The effects of phenanthrene and its nitrogen-containing polycyclic aromatic hydrocarbon analogues on seedling emergence and biomass of two terrestrial plants species, Latuca sativa (lettuce) and Loliun perenne (rye grass) were assessed. The effects were studied over a 21d exposure period. ANOVA results revealed that seedling emergence and biomass (growth) were significantly affected by the tested chemicals even at the lowest exposure concentration. The greatest % inhibition was 33.3 (lettuce) and 46.5 (rye) for the PAH and 53.3 (lettuce) and 93.3 (rye) for N-PAHs. N-PAHs showed greater inhibition effects on seedling emergence and biomass than the homologues PAH. The results demonstrate greater toxicity and bioavailability of N-PAHs to plants.

Introduction

Phytotoxicity can be referred to as any alteration / abnormality in the normal development of plant species due to the presence of a pollutant in soil. Soil, which provides rooting material for plants, is a major settlement for organic pollutants in the environment. Industrial activities lead to the discharge of these pollutants in soils, which possess adverse ecological and toxicological effects. Soils consist of living and non-living components which exists in complex and heterogeneous mixtures. It is structured, heterogeneous and discontinuous. The nature of organic matter, pH, and particle size distribution etc, in connection with physico-chemical properties determines the bioavailability of contaminants to plants (Semple et al, 2007). It is impossible to measure all chemical contaminants in soil, including their bioavailability and bio-transformed products which may occur. Therefore risk assessment can only consider those pollutants and concentration ranges which can actually be detected by chemical and biological methods.

Phenanthrene and its nitrogen-containing analogues have three carbon, hydrogen and/or nitrogen aromatic rings. Bioavailability and toxicity of these contaminants in the environment are of great concern. Phenanthrene-nitrogen containing analogues are polar, more soluble with low Kows. Concentrations of nitrogen-containing polycyclic aromatics found in the environment are reported to be one or two order magnitude lower than its PAH analogues (Blumer et al, 1977) but their biological effects can be of similar in magnitude. Their sources are similar to those of PAHs, including incomplete combustion of fossil fuels, petroleum derivatives, coal production and some industrial processes (i.e. petrogenic, pyrogenci and biogenic). They can easily be photo-modified. This contaminant raises a number of environmental concerns because it appears to be more toxic than PAHs.

Green plants are primary producers and form the foundation of all ecosystem and animals, bacteria, fungi etc rely on plants for energy. Plants as primary producers are essential components of the terrestrial ecosystem, integral part of agricultural and non-crop sites, and should be included in the study of the fate of contaminants. They have also been used in literature for phyto-remediation of contaminated lands pollution (Smith et al, 2006; Liste and Prutz, 2006). Effective pathways for uptake of contaminants into plants includes: root uptake and translocation; uptake from vapour; uptake of contaminated soil or dust by shoots and uptake and transport in oil cells (for oil-containing plants). Effects on germination, growth and/or reproduction of plants may reflect the eco-toxicological potential of contaminated soils. Biological monitoring of these contaminants is a useful approach for estimating toxicity and should not be seen as an alternative to physical and chemical method.

Risk assessment is a systematic process for identifying and analysing the hazards attributed in a contaminated lands. It helps make logical and justifiable decisions. In chemical risk assessment, effect concentrations are related to exposure data (soil concentration of contaminants). The use of biological assays allows the effects on ecological receptors originating from existing contamination to be evaluated more accurately than is possible by only chemical assays. The commonly used chemical analysis could not identify all the potential toxic substances or predict their toxicity on plants. Bioavailability remains the key point in the fate of pollutants in soils and their effects on plants. The need to evaluate bioavailability arises from the fact that phenanthrene and its n.heterocyclic analogues vary in their polarity, solubility, Kows, and capacity to irreversibly-sorb to soil organic matter, which may influence their uptake by plants hence bioavailability.

Aging was assumed as a limiting factor for bioavailability due to sequestration of contaminants. Once the contaminant is sorbed by the soil, aging (interaction of chemical with soil over time) may lead to development of inner sphere complexes, surface diffusion within micro-pores or surface precipitates (Aharoni and Sparks, 1991), resulting to decrease in bioavailability. However the effects of such on plants have not been measured using phenanthrene and its nitrogen-containing analogues. Research works has reported the adverse effects of PAHs and N-PAHs on germination but many of these trials involve freshly spikes soils or subjection of seed to PAHs solutions using filter paper (Chung et al, 2007; Paskova et al, 2006; Sverdrup et al, 2003). Phyto-toxicity test such as seedling emergence and growth have been recommended as part of a battery test in order to develop a comprehensive toxicity profile for hazardous sites. Data such as toxicity of nitrogen-containing polycyclic aromatic compounds to plants in aged soil are scarce in literature. Therefore, the aim of this study was to investigate the effects of phenanthrene and their nitrogen-containing analogues on plants, focusing on;

Using spiked soil to generate phyto-toxicity data of phenanathrene and its nitrogen-containing analogues in aging soil.

Bioavailability of chemicals to plants over time, in a laboratory setting.

Materials and methods

Chemicals

Phenanthrene, 1,7-phenanthroline, 4,7-phenanthroline, 1,10-phenanthroline, benzo(h)quinoline and phenanthrene were purchased from Sigma-Aldrich, company UK. Chemicals were dissolved in acetone.

The test chemicals and their physico-chemical properties are shown in table 1

Table 1: The tested chemicals and their physico-chemical properties

Chemical

Chemical formula

Chemical structure

Molecular mass

Boiling point

Log Kow

Solubility mg/L 25oC

% purity

Phenanthrene

C14H10

CAS-NO 85-01-8http://www.chemspider.com/ImagesHandler.ashx?id=970&w=200&h=200

178.2

340

4.46

0.677

96

1,10 phenenthroline

C12H8N2

CAS-NO 66-71-7InChI=1/C12H8N2/c1-3-9-5-6-10-4-2-8-14-12(10)11(9)13-7-1/h1-8H

180.21

365.1

2.51

30.64

99

1,7 phenanthroline

C12H8N2

CAS-NO 230-46-6InChI=1/C12H8N2/c1-3-9-10-4-2-8-14-12(10)6-5-11(9)13-7-1/h1-8H

180.21

365.1

2.51

30.64

99

4,7 phenanthroline

C12H8N2

CAS-NO 230-07-9 InChI=1/C12H8N2/c1-3-7-11-9(5-1)10-6-2-4-8-12(10)14-13-11/h1-8H

180.21

361.2

2.40

38.04

98

Benzo(h) quinoline

C12H9N

CAS-NO 230-27-3InChI=1/C13H9N/c1-2-6-12-10(4-1)7-8-11-5-3-9-14-13(11)12/h1-9H

179.2

339

3.43

78.7

97

Source: http://www.chemspider.com/chemical-structures.8836.htm

Test seeds: Lolium perenne (Rye grass) Lactuca sativa (Lettuce).

Rye grass and lettuce are important agricultural crops and widely recommended for plant toxicity test (OECD, 2006, USEPA, 1996). Rye grass is important for both pasture for grazing and hay for livestock. It is a highly nutritious stock feed. It is also useful in soil erosion control programs. In Britain, it is used as an indicator of non-species rich grassland. Rye grass has demonstrated resistance to PAH pollution (Smith et al, 2006; Liste and Prutz, 2006). The seeds were purchased from B&T World Seeds United States and B&Q Seeds United Kingdom respectively.

Soil preparation

Soil from Myerscough Agricultural College in Lancashire was prepared for the test. The soil was collected from the top layer of field about 0-20cm. The soil was clay-loam with an organic matter content of 2.7% and pH 6.5. The soil was air dried at room temperature and then sieved with 2mm mesh size. Spiking procedure followed those described in (Doick et al, 2003). Triplicate of each soil(g) were placed in bowls and 1/3 of the soil were spiked with dissolved chemical to give final concentrations of 10, 100, 250 and 500mg/kg. The soil was left to evaporate for 2-4 hours after which soil was mixed with the remaining 2/3 soil. Analytical blanks were prepared using acetone to serve as control. The soils were then put in amber glass jar and aged for 0, 30, 60 and 90 days. After each aging period, plant toxicity was measured for 21days.

Plant Bioassay

The tests were performed according to OECD Guidelines 208, 2006 and EPA, 1996 with small changes. 40g soil (with known concentration) with 60% water holding capacity (WHC) was weighed into a plastic flower pot. The bioassay was performed in the dark to prevent photo-modification of compounds. There were three replicates for each concentration of contaminant and 5 healthy seeds each were introduced. The pots were arranged under a randomized design in the laboratory under room temperature. The pots were covered with glass plates until seedlings emerged. After removal of glass plates, the water contents was checked daily and replenished as required. Leached water accumulated at the bottom of the pots was used for watering to avoid chemical loss. Solvent controls were prepared for each concentration to provide reference values. Seedling emergence and plant biomass were selected endpoints to reflect the growth and health status of the plant.

Seedling emergence percentage was determined when 90% of seeds in controls germinated (5 days for lettuce and 7 days for rye grass). The number of germinated seeds and plant biomass were recorded. Biomass involves the measurement of the fresh and dry weights (drying for 20hrs at 70OC) of the plants. Only seeds defined as germinated were measured for biomass, dead/withered plants were not used. Prior to measurements, plants were carefully harvested, washed and wrapped with blue roll (to aid draining of water and reduce drying of the plants). Other criteria were visible abnormal changes on different parts of the plants.

Statistical analysis

Data analysis was carried out using SPSS 19. Seedling emergence (SE) was compared with control and % effect was determined using: SE = emergence (treated)-emergence (control) Ã-100

emergence (control)

The test was conducted in triplicates and results were presented as mean ± SD and SEM (standard error of mean). Generalized linear model was used to determine the significant effect between weight, concentrations, time and concentrations * time at p<0.05. Using linear model: Weightijk = Concentrationi + Timej + ijk + (Concentration*Time)ij. Where i = concentrations, j = time points (days) and k = replicates within each concentration. One way ANOVA was used to test effects of the chemicals on seedling emergence at = p<0.05. Dose-response curves were plotted using Sigma Plot 10 software.

RESULTS

Effects of phenanthrene and its nitrogen analogues on seedling emergence are summarized in Table 2. Exposure of the plants to NHAs resulted in significant inhibited germination. Also biomass (determined as growth-weight) of lettuce and rye grass exposed to different concentrations of PAH and NHAs were plotted as dose response effects (Figs.1 & 2). Chemical analysis on availability of chemicals over time was reported on Anyanwu and Semple, 2012a &b. The result shows that NHAs are more toxic than PAHs. This depends greatly on their physico-chemical properties such as solubility and log Kow (table 1).

Phenanthrene and its nitrogen containing analogues had more effect on plant biomass than seedling emergence. Literatures have recorded that seed germinations may be insensitive to toxic chemicals (Smith el al, 2009; Eom et al, 2007; Sverdrup et al, 2003; Hulzebos et al, 1993; Chung et al, 2007). This may be because many seedlings derive its food from internal materials. Although seedling emergence was slightly above 25% in most of the chemicals, ANOVA results proved that it was significant at p<0.05 except for 4,7-phenanthroline which showed no significant inhibition to lettuce at p<0.05 level of significance (table 2). ANOVA also revealed that benzo(h)quinoline mostly affected seedling emergence.

Table2. % seedling emergence (SE) of L. perenne and L. sativa after exposed to phenanthrene and its nitrogen containing analogues.

% SE

Lettuce

% SE Rye

Conc (mg)

0

10

100

250

500

0

10

100

250

500

Chem.

Days

0

100

80

100

100

93.4

100

73.4

73.4

100

66.7

Phen

30

100

80

100

93.4

86.7

100

67

53.5

73.4

80

60

100

80

93.4

93.4

73.4

100

73.4

73.4

73.4

86.7

90

100

80

100

100

93.4

100

100

60

60

73.4

BhQ

0

100

66.7

80

73.4

66.7

100

86.7

93.4

66.7

60

30

100

73.4

80

93.4

73.4

100

6.7

33.4

60

53.4

60

100

93.4

86.7

86.7

100

100

66.7

73.4

60

86.7

90

100

10

93.4

86.7

100

100

80

93.4

73.6

73.6

4,7-phen

0

100

73.6

60

86.7

100

100

86.7

80

53.4

73.6

30

100

86.7

93.4

86.7

93.4

100

60

40

66.7

86.7

60

100

100

93.4

93.4

86.7

100

73.4

73.4

80

86.7

90

100

86.7

100

93.4

80

100

66.7

60

86.7

60

1,7-phen

0

100

93.4

73.4

86.7

46.7

100

93.4

73.4

73.4

33.4

30

100

86.7

80

100

86.7

100

86.7

60

93.4

73.4

60

100

93.4

86.7

86.7

86.7

100

86.7

73.4

93.4

80

90

100

100

100

86.7

86.7

100

86.7

93.4

60

60

1,10-phen

0

100

86.7

93.4

53.4

86.7

100

80

80

66.7

60

30

100

93.4

80

86.7

100

100

66.7

80

86.7

86.7

60

100

93.4

100

73.4

93.4

100

80

73.4

80

73.4

90

100

86.7

80

86.7

100

100

86.7

80

66.7

73.4

Table3. Correlations of phenanthrnene and its N-containing analogues on L. Sativa & L. perenne

Compound

Plant

R2 (fresh biomass)

R2 (dry biomass)

phenanthrene

L.sativa

0.745

0.670

L. perenne

0.809

0.766

Benzo(h)quinoline

L.sativa

0.714

0.629

L. perenne

0.854

0.770

4,7-phenanthroline

L.sativa

0.636

0.578

L. perenne

0.824

0.759

1,7-phenanthroline

L.sativa

0.766

0.725

L. perenne

0.860

0.683

1,10-phenanthroline

L.sativa

0.669

0.507

L. perenne

0.736

0.680

Fig 1: Effects of phenanthrene and its N-containing analogues on biomass of lettuce sativa

Fig 2: Effects of phenanthrene and its N-containing analogues on biomass of lolium perenne

Dose-response curves showed significant effects even at the lowest concentration of 10mg/kg (Fig.1).. ANOVA results showed strong positive correlations (Table 3) and significant effects between biomass, concentrations and time (days) except for 4,7-phenanatroline which showed no significant time effect on lettuce (fresh biomass) and rye (fresh & dry biomass), 1,7-phenanthroline on rye (dry biomass) and 1,10-phenanthroline on rye (fresh & dry biomass) at p<0.05. Also ANOVA results further depicts significant concentration effects on both plants at p<0.05.

DISCUSSION

In this study, both phenanthrene and its N-containing analogues slightly inhibited seedling emergence of the tested plants but ANOVA reveals that the inhibition was significant at p<0.05. Rye grass germination was the most affected by the tested compounds. The observation with N-PAHs indicates the greater toxicity of these chemicals and this can be attributed to their polarity, solubility and low Kow (table 1), hence bioavailability. This observation is in accordance with the findings of Hanner et al, 1999 who reported that volatile, water soluble, low molecular weight hydrocarbons and other toxic compounds strongly inhibit plant germination and growth. Chaineau et al, (1997) which reported inhibited seed germination and reduced plant growth while Paskova et al, (2006) reported N-PAHs as chemicals with strongest inhibition effect on germination and growth of plants. Although the chemicals exhibit effects on the tested plant biomass, nitrogen-PAHs showed higher toxicity (table 3). Unlike the eco-toxicity test with soil organisms (Anyanwu and Semple, 2012 a & b), phenanthrene effects was evident throughout the time points. This maybe because the roots hairs were able to penetrate the soil pores, hence bioavailability.

All N-PAHs in this study, exhibits effects to lolium perenne and latuca sativa. In contrast Sverdrup et al, 2003 recorded no toxicity with acridine. In spite the fact that effect values were lower on dry biomass than fresh biomass as recorded by Eon et al, 2007 and Sverdrup et al, 2003, we observed that this depends largely on the chemical (table 3).

Wang et al, 2007, reported fresh weight of oilseed rape seedlings reduced at 10µg/g compared to 1µg/g and control. Our results also revealed significant effects even at the lowest concentration of 10mg/kg (especially benzo(h)quinoline). This was also reported by Gissel-Niesen and Nielsen (1996) whose observations reveal that acridine inhibits the seed production of navew and the growth of Italian rye-grass. Inhibition was noted at 1 ppm level. At 100 ppm nawev seeds either did not germinate or the sprouts did not survive.

Although our findings are in agreement with the findings of other studies on plant toxicity in literature and some documented greater toxicity of N-PAHs (Paskova et al, 2006; Van Vlaardigen et al, 1996; Eon et al, 2007, Sverdrup et al, 2003, Gissel-nielsen and Nielsen (1996), Henner et al, 1999, Chaineau et al, 1997, Smith et al, 2006).

Documented literatures on phytotoxicity of N-PAHs in aging soil are limited, so comparing our results with literature will be limited. However, our results correspond with the findings of Gissel-nielsen and Nielsen 1996 which reported that acridine affected seed germination and growth (survival), Henner et al, 1999, Paskova et al, 2006; Van Vlaardigen et al, 1996; Eon et al, 2007, Sverdrup et al, 2003, Wang et al, 2007, Chaineau et al, 1997. The effects of the chemicals on plant biomass (seedling growth) and higher toxicities of N-PAHs is also in accordance with the findings of Gissel-nielsen and Nielsen 1996, Hulzebos et al, 1993 who reported higher toxicities of N compounds and Sverdrup et al, 2003 which recorded seedling growth to be far more sensitive endpoint than seed emergence but in contrast, found no toxicity in acridine. Other visible abnormalities observed were death, chlorosis, narcosis, wilting, withering and stunted growth (especially in the nitrogen containing compounds). We also observed that although rye grass was mostly affected during germination, it can survive chemical concentrations even at 500mg/kg. This was also reported by Korade and Fulekar, 2009.

CONCLUSIONS

Phenanthrene and its nitrogen-containing analogues significantly inhibited seedling emergence and plant growth (biomass) of the two plant species (lettuce and rye grass). Nitrogen-polycyclic aromatic hydrocarbons demonstrated significantly higher toxicity than their PAH analogue. This confirms the higher bioavailability and greater toxicity of N-PAHs than PAHs. There is need to further investigate the factors that affect bioavailability of N-PAHs and their uptake by plants.

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