Effects Of Phenanthrene And Its Nitrogen Heterocyclic Analogues Biology Essay

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The effects of phenanthrene and its nitrogen-polycyclic aromatic hydrocarbon analogues on soil microbial respiration were evaluated to assess the toxicity of the chemicals in soil using substrate induced respiration (SIR). Soil samples were amended with phenanthrene and its nitrogen containing analogues and respiration rates were measured over time. Results showed that inhibition of PAH amended soil plateaued after 30 d, while the nitrogen containing analogues showed a pattern of respiration inhibition/stress with increased concentration and aging. These may be attributed to irreversible sorption (for PAH), solubility, low Kow and/or bio-transformed metabolites (for N-PAHs). Time dependent percentage inhibition/stress was more than 25% which portrays N-PAHs toxicity and bioavailability. Also, ANOVA results showed significant and strong positive correlation between mean CO2 evolved, concentration and incubation days (aging). The estimated LOEC was 10 mg/kg based on the concentrations used. This suggests that soil microorganisms might be more sensitive to N-PAHs in soil than the homocyclic analogues.

Introduction

Microbes are more susceptible to contaminant pollution than humans. High concentrations of chemicals in soils can affect microbial growth, density, viability and development. As a result of relative sensitivity of microbes to contaminants, toxicity data will be important in determining safe levels for some contaminants in soil. Eco-toxicological effects of chemicals are dependent on the exposure and bioavailability of contaminants, uptake and metabolism, intracellular concentration, mode of toxic action and balance between toxicity and protective cellular response (Fent, 2004). Toxic effects of chemicals on microbial communities are linked with a stress and/or reduction of microbial respiration activity.

Phenanthrene and its nitrogen-containing analogues consist of three aromatic rings (carbon and/or nitrogen). These chemicals have been produced since the 80's. They are semi-volatile, persistent, toxic and ubiquitously distributed. They are widely produced by petroleum and industrial activities. Due to the widespread /distribution in the environment, their log Kow . Koc and solubility, they exert high potencies for carcinogenicity, mutagenicity, teratogenicity and genotoxicity. These contaminants in soil can enter food chains through the ecological receptors.

Toxicological effects of PAHs have been studied. However, gaps in the knowledge on toxicity of its nitrogen-containing analogous on soil fauna have been highlighted. Nitrogen-polycyclic aromatic hydrocarbons (N-PAHs) are produced in conjunction with their homologous PAH compounds. It has been noted that not only homocyclic aromatic compounds but also heterocyclic compounds contribute to the biological activities encountered in many environmental samples. From both toxicological and epidemiological studies, many heterocyclic aromatics are reported to be highly toxic. Although the available literature data are limited, there are considerable proves indicating their toxicity to humans and ecological receptors. Also N-PAHs polarity, solubility and low Kows, increases their bioavailability in soil.

Soil is a complex microhabitat which has diverse microbial population. Microorganisms play an important role in breakdown and transformation of organic matter in fertile soils with many species contributing to different aspects of soil fertility. Any interference with these biochemical processes would potentially affect nutrient cycling and alter soil fertility status of the soil (OECD, 2000). It is widely known that numerous indigenous microorganisms that utilize organic contaminants in soil as carbon and energy sources are ubiquitous in the environment. Microbial uptake and conversion of compounds from the local environment is continuously taking place in any niche in the biosphere. This metabolic diversity of natural microbial communities has saved mankind from self-intoxication. The biological removal of a compound varies considerably depending on the period of time that soil has been exposed to the compound (Semple et al, 2003), as organic contaminants degrade slowly. Any alteration of soil microbial community can have significant effect on soil ecosystems.

In assessing the toxicity of organic contaminants in soil ecosystem, various levels must be taken into consideration. A number of bioassay has been developed to assess contaminant toxicity in soil environments. However, among these bioassays, those exploiting respirometry toxicity tests have shown good correlations in soil environments because respiration rate can be inhibited or stressed in the presence of contaminants. Microbial respiration is an effective measure of rate of carbon mineralization, since about 70% carbon added to soil is lost as carbon dioxide mainly as a product of microbial respiration (Usman et al, 2004). Although some bioassays use other methods for quantifying respiration rate, the most common criterion is carbon dioxide released (ug carbon dioxide/g dry soil/h) or oxygen consumed (ug oxygen/g soil/h). Toxicity assessments are necessary to determine the concentration ranges within which organic contaminants poses risk on the microbial populations involved in the clean-up process and to evaluate the effect of remediation of toxic discharges. Assessing the potential impact of a chemical in soil should not only focus on mortality and lethal dose but also evaluation of activity and respiration rates of microbial communities responsible for carbon transformation, since it subjects these communities to carbon starvation, chemical stress/inhibition and death.

Substrate induced respiration (SIR) using glucose is an indirect and simple method of estimating microbial activities to chemicals. Substrate-induced respiration is a measure of the CO2 evolved from a soil sample after administering an optimal concentration of an additional energy source and is directly proportional to the soil microbial biomass. With computerized respirometer, it is possible to obtain an hourly measurement of the CO2 production in many samples after addition of glucose.

Aging periods that have been used to assess effect of contaminant toxicity on micro-organisms cover a very wide range. Some studies have been for 7, 14, 21days, 12wks or 300days. Due to the diverse structure and physico-chemical properties of PAHs and N-PAHs, there is need for exploring new aspects of their toxicity.

Therefore, the aim of the present study was to obtain information on the impact of phenanthrene and its nitrogen-containing analogues on the microbial community/activity in a soil without contamination history.

Microbes are ecological receptor mentioned in recent reviews as requiring much research attention. For this purpose, a freshly sampled soil was spiked with phenanthrene and its nitrogen heterocyclic analogues, and incubated for 100days. At each time point, respiration measurements and biomass were carried out to determine the microbial activity and possible inhibition/stress in the microbial community-activities.

In addition, SIR using standard laboratory equipment the "Automated Columbus Instrument's Micro-Oxymax" was used. Description of the working of the Micro-Oxymass is on the Columbus Instrument Website. This is capable of providing rapid evaluation of hourly CO2 rate from soil microbes.

MATERIALS AND METHODS

Chemicals

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

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

http://www.chemspider.com/chemical-structures.8836.html

Soil preparation

Pristine 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). Soils with a known range of physico-chemical parameters were selected. 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 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, microbial respiration was analyzed for 12hours. Soils were analyzed at the beginning of the test with GC-MS.

Microbial test

Microbial test was performed according to OECD (2000) guideline draft 217 with little deviations. 10g of freshly sampled natural soil adjusted to 40% of water holding capacity was prepared and 3 replicates were designed for all treatment levels. Exposure concentrations (0, 10, 100, 250 and 500mg/kg) were prepared as described above. Water loss after evaporation was replenished and total water content was adjusted to 60% of water holding capacity. Soils were kept in amber bottles in the dark at 22 ± 2oC. Soil samples were analyzed after 0, 30, 60, and 90 days of incubation. The respiration was measured as CO2 production within the first 12 hours after the addition of glucose solution (5mgglucose/g dry wt soil). Microbial respiration rates were measured using an Automated Columbus Instrument's Micro-Oxymax. Sampling intervals was bihourly.

Data analysis

Statistical analyses were carried out with SPSS 19. The mean, standard deviation and standard error were calculated. Data was analyzed with general linear model of ANOVA using 12th hour cumulative mean respiration rates as dependent variables and time points (aging days) and concentrations as fixed factors. ANOVA was used to test the difference in mean respiration to determine the significance in respiration rates across concentrations and time points at p<0.05. Turkey's test was used to estimate the differences between concentrations and times. The percent deviation of the treated from the control was used to calculate percentage (%) inhibition/stress. Percentage inhibition/stress is a comparison of respiration in individual spiked soil to the control.

% inhibition = (1 - B/C) Ã- 100

Where B is the mean cumulative CO2 in spiked samples; C is the mean cumulative CO2 in control soil samples (Nwachukwu and Pulford, 2011).

Results

SIR has been recommended for interpretation of chemical inhibition (toxicity) on microbes (OECD, 2000). Availability of phenathrene and N-PAHs in amended samples was recorded in tablt 2. Table 3 showed significant inhibition and/or stress on microbial activities' at all the concentrations. Table 4 shows the mean cumulative CO2 evolved after 12 hours while table 5 showed mean biomass of amended soils. Results showed strong positive correlations between concentrations and aging times at p<0.05 in all the tested chemicals (Table 2). ANOVA results between the variables (mean respiration, aging times and concentration) were statistically significant in all compounds at p<0.05. A clear concentration-time effect was observed within each chemical which was significant at p<0.05. NOEC was not detected from the concentrations used since all concentration posed stress and/or inhibitions on the microbes. LOEC was 10mg/kg based on the concentrations used. Figure 1 shows the dose response curve of phenanthrene and its nitrogen-containing analogues.

Table 2. Availability of PAH and N-PAHs in soil samples

Compound

Conc mg/kg

0d

30d

60d

90d

Phenanthrene

500

250

100

10

1,7-phenanthroline

500

250

100

10

4,7-phenantholine

500

250

100

10

1,10-phenanthroline

500

250

100

10

Benzo(h)quinoline

500

250

100

10

Table 2 Percentage inhibition and/or stress effects of phenanthrene and its nitrogen analogues on CO2 evolved

Compound

Conc mg/kg

0d

30d

60d

90d

LOEC mg/kg

R2

Phenanthrene

500

250

100

10

15.7

12.1

12.5

5.0

29.5

20.7

3.2

3.9

31.7

22.5

11.4

12.6

26.6

17.1

8.8

8.4

10

.926

1,7-phenanthroline

500

250

100

10

27.7

29.3

31.0

37.6

9.4

25.9

41.7

89.3

66.8

14.9

9.3

4.8

69.9

1.8

3.7

9.8

10

.932

4,7-phenantholine

500

250

100

10

8.5

5.6

4.0

1.4

157.7

121.3

118.8

152.9

159.4

120.8

102.8

142.0

103.9

101.4

78.4

102.0

10

.989

1,10-phenanthroline

500

250

100

10

18.6

11.7

10.3

4.7

52.6

76.0

78.0

85.8

52.2

68.5

66.8

46.3

44.6

60.2

55.4

33.4

10

.988

Benzo(h)quinoline

500

250

100

10

8.7

6.0

2.6

0.4

49.5

61.0

56.5

8.4

40.5

9.4

4.6

45.6

45.8

20.6

23.4

22.3

10

.942

Table 4: Mean cumulative (± S.E) of CO2 evolved by PAH and its NHAs amended soil samples

Chem.

Days

500mg

250mg

100mg

10mg

Control

Phen

0

170.328 ± 4.2

177.6073 ± 1.4

176.7837 ± 7.3

212.3843 ± 5.4

202.1207 ± 2.4

30

216.0003 ± 8.4

243.1403 ± 11.8

296.818 ± 16.1

319.0633 ± 20.4

306.7983 ± 12.3

60

187.3123 ± 4.1

212.484 ± 9.2

242.7743 ± 8.4

309.083 ± 13.2

274.2857 ± 11.3

90

176.216 ± 7.0

199.7943 ± 1.4

219.6743 ± 6.4

261.562 ± 5.6

241.1313 ± 3.2

1,7-phen

0

258.164 ± 2.0

261.3653 ± 5.2

264.8627 ± 3.6

278.1867 ± 7.4

202.1207 ± 2.4

30

352.43 ± 4.5

405.5523 ± 5.1

456.684 ± 2.6

609.9153 ± 5.9

322.067 ± 6.9

60

188.8707 ± 6.9

655.6243 ± 45.1

623.395 ± 23.1

597.9153 ± 66.7

570.2633 ± 61.2

90

168.6727 ± 3.0

566 ± 36.5

534.5447 ± 18.9

500.8543 ± 32.2

555.4773 ± 72.1

BhQ

0

184.4807 ± 2.7

189.896 ± 1.0

196.8633 ± 1.9

201.3057 ± 3.3

202.1207 ± 2.4

30

481.7953 ± 8.4

518.7637 ± 17.4

504.2363 ± 4.8

349.1703 ± 6.2

322.067 ± 6.9

60

338.828 ± 10.4

516.258 ± 70.4

543.4747 ± 3.1

830.44 ± 46.0

570.2633 ± 61.2

90

300.78 ± 12.0

440.5437 ± 33.6

425.0213 ± 16.5

679.7877 ± 22.7

555.4773 ± 72.1

1,10-phen

0

202.0697 ± 2.2

219.3237 ± 4.6

222.6447 ± 4.2

236.5613 ± 2.7

248.412 ± 8.0

30

296.8137 ± 2.6

342.1187 ± 4.0

346.068 ± 6.0

361.2153 ± 4.9

194.3807 ± 3.3

60

241.0413 ± 1.2

266.8757 ± 2.9

264.1787 ± 6.6

231.8103 ± 4.5

158.352 ± 6.9

90

221.9677 ± 3.6

245.9113 ± 2.0

238.682 ± 2.3

204.8977 ± 0.2

153.497 ± 2.8

4,7-phen

0

227.0877 ± 7.7

234.3493 ± 2.0

238.4223 ± 3.8

252.1217 ± 3.8

248.412 ± 8.0

30

501.1123 ± 4.2

430.2453 ± 8.0

425.3057 ± 16.4

491.727 ± 2.7

194.3807 ± 3.3

60

410.9127 ± 3.9

349.705 ± 5.5

321.152 ± 5.2

383.3263 ± 9.4

158.352 ± 6.9

90

313.1107 ± 4.9

309.235 ± 2.9

273.947 ± 16.2

310.1123 ± 4.8

153.497 ± 4.5

Figure 1: Impact of phenanthrene and its nitrogen heterocyclic analogues on soil microbial activity.

Table 5: Mean microbial biomass (± S.E) of soil amended with phenanthrene and its nitrogen analogues

Chem.

Days

500mg

250mg

100mg

10mg

Control

Phen

0

156.6667±23.3

150±28.8

90±5.7

53.33333±17.6

40±5.7

30

143.3333±23.3

136.6667±8.8

223.3333±14.5

123.3333±14.5

160±20.8

60

106.6667±6.6

133.3333±33.3

123.3333±14.5

125±43.3

85±37.5

90

175±43.3

200±28.8

165±2.8

150±28.8

116.6667±8.8

1,7

0

86±8.0

73.66667±0.8

101±0.5

110±5.7

115±20.2

30

90±5.7

117.6667±1.4

155±2.8

175±2.8

195±2.8

60

61±0.5

76±13.8

87.33333±18.7

52.33333±1.4

31.66667±10.6

90

50±5.7

40.66667±10.2

50±5.7

55±2.8

38.33333±6.0

BhQ

0

35±8.6

41±12.1

42.66667±10.1

40±2.8

55±2.8

30

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

60

32.33333±10.1

19±2.3

35±8.6

21.33333±0.8

19±0.5

90

55±8.6

75±14.4

130±5.7

75±14.4

45±2.8

1,10

0

47.33333±1.4

47.33333±12.9

72.33333±4.3

90±5.7

110±5.7

30

50±2.8

50±17.3

65±8.6

60±5.7

100±1.1

60

75±8.6

75±2.8

99.33333±0.6

100±11.5

100.6667±0.6

90

106±7.0

106±7.0

90±17.3

115±20.2

75±14.4

4,7

0

35±8.6

31.33333±7.7

80±17.3

101±1.4

101±0.5

30

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

#DIV/0! ±

60

55±2.8

52.33333±1.4

65±5.7

35±8.6

35±8.6

90

40.66667±0.6

75±14.4

60±5.7

25±2.8

25±2.8

Discussion

The mean cumulative CO2 evolved by microbes was affected by chemical concentrations and aging period (Table 3).

The presence of contaminants changed the response of the microbial community (fig. 1). Effect of phenanthrene and its nitrogen analogues on microbial activity followed a predictable pattern. At 0d inhibition was mild for the N-PAHs, but the pattern changed after 30d (fig 1). Studies have shown that the nitrogen-polycyclic aromatic hydrocarbons often show no effect until they are metabolically activated (Warshawky et al, 1992; Kumar et al, 1989). From the result, % inhibition increased with increased aging time for NHAs (benzo(h)quinoline and 1,7-phenanthroline) while its homocyclic analogue plateaued after 30d, this can be attributed to their polarity and log Kow, hence bioavailability. Chemicals are known to undergo irreversible sorption which renders them unavailable to microbes. This may be the case of the homocyclic analogue.

Microbial biomass in highly contaminated soils has been found to be consistently lower than in uncontaminated or low conditions (Barajas-Aceves, 2005) (table 4). Low organic content of the soil would offer limited energy source to the microbes. There was significant increase in CO2 evolved once substrate was added. The mean CO2 evolved was highest at 10mg/kg, for all chemicals except 4,7-phenanthroline. The ability of the contaminants to sustain microbial respiration in the presence of toxicity was significant at low concentrations. This may be attributed to the fact that microorganisms differ in their sensitivity to contaminant toxicity. It is possible that at low concentrations, the microbes may compensate by a higher C turnover and so lead to a gradual change in viability (Giller et al, 1998). The reverse was the case at much higher concentration; the contaminants cause inhibitions and/or much stress in the microbial community. This is due to a community shift in which case the tolerance of the dominant microbial group determines the respiration (Giller et al, 1998).

Various authors have made numerous suggestions on respiration rates of micro-organisms. Domsch et al, (1983) documented that any alteration caused by either natural agents or pollutants which returns to normal microbiology values within 30 days should be considered normal fluctuations; alterations lasting for 60 days can be regarded as tolerable but those persisting for over 90 days are stress agents. The result shows that, N-PAHs can be regarded as stress agents to microbial community. This implies that the contaminants probably may have a long term impact on soil microbial communities especially benzo(h)quinoline and 1,7-phenanthroline.

Estimating the impact of contaminants on microbial community may be more complex than imagined. Since different chemicals cause different toxic effects, comparing the toxicity of one with another is hard. Our result portrays benzo(h)quinoline to be more toxic since there is a gradual but continuous inhibition at higher concentrations with increased aging. 4,7- phenanthroline is regarded as the least toxic. With the high %CO2 evolved, it is envisaged that the microbes were utilizing it as a carbon and energy source which can enter their central metabolic pathways.

Comparison of respiration rates with incubation days

Aging is toxicologically significant because the assimilation, acute and chronic toxicity of harmful contaminants reduce as they persist and become increasingly sequestered with time (Alexander, 2000, Semple et al, 2007). It also reduces the effectiveness of some toxic and geno-toxic compounds in soil. This was observed in the case of phenanthrene. It has been reported that prolonged incubation (aging) may lead to the depletion of available C and N sources in soil and consequently cause remarkable changes in soil microbial biomass and respiration (Brohon et al. 1999). This was also observed in the lowest concentrations. Nutrient deficiencies may have caused microbes to become vulnerable to the contaminants. In spite of the fact that aging declines exposure, toxicity and risk of various contaminants, it does not eliminate exposure and risk. Also a time dependent reduction in bioavailability does not occur always (Alexander, 2000). This was significant in the case of the tested nitrogen- containing compounds. It may be attributed to their polarity, solubility, low Kow, and/or bio-transformed metabolites. The results further depict the significance of metabolites in the fate and eco-toxicology studies of chemicals. The cumulative CO2 released per time point depicts that some of the contaminants are toxic and able to persist in the soil.

Biomass inhibition of microorganisms correlates with the increased persistence of some NHAs in soil in spite the fact that microbes lowered the effect of metabolic poisoning of some chemicals. In this study, persistence is attributed to the amended soil due to the inhibition and/or stress on microbial community. The results allow an estimation to be made of the time required for all the carbon in the amended soil to be utilized. However, the values suggest that the contaminants can remain in the soil for a considerable time. Little has been done on the microbial activities on these chemicals so the discovery cannot be compared.

Conclusions

Relatively, high toxicity could be expected for organic compounds soluble in aqueous phase with low sorption in soils. Effect of PAH and N-PAHs on soil microbial community were studied. N-PAHs were shown to stress/inhibit respiration than its homocyclic analogues in aging soil. The results reveal that extent of respiration inhibition/stress portrays contaminant's bioavailability and toxicity. However, the effects of bio-transformed metabolites needs further investigation since our results reveals that microbes may become more susceptible to N-PAHs as they persist in soil.

The present risk assessment for PAHs is solely based on homocyclic PAHs. Yet, from the present review it becomes clear that this approach fails to protect against a vast number of heterocyclic compounds and biotransformation products that may exhibit stronger or other toxic effects than their homocyclic analogues. Therefore, incorporating the role of heterocyclic compounds and their metabolism appears to be a necessity for a reliable risk assessment for polycyclic aromatic compounds. In addition, reliable long-term protection against PAHs demands data on chronic toxicity, including teratogenicity, both for homocyclic as for heterocyclic compounds.

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