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Biotic and abiotic degradation of illicit drugs, their precursor, and by-products in soil
Chemosphere 85 (2011) 1002–1009
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Biotic and abiotic degradation of illicit drugs, their precursor, and by-products in soil Raktim Pal a,b, Mallavarapu Megharaj a,b,⇑, K. Paul Kirkbride c, Tunde Heinrich a,b, Ravi Naidu a,b,⇑ a
Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia CRC for Contamination Assessment and Remediation of the Environment, University of South Australia, Australia c Australian Federal Police Forensic and Data Centres, Canberra, Australia b
a r t i c l e
i n f o
Article history: Received 26 March 2011 Received in revised form 23 June 2011 Accepted 26 June 2011 Available online 22 July 2011 Keywords: Illicit drugs Methamphetamine MDMA Pseudoephedrine N-formylmethylamphetamine 1-Benzyl-3-methylnaphthalene
a b s t r a c t This study presents the first systematic information on the degradation patterns of clandestine drug laboratory chemicals in soil. The persistence of five compounds – parent drugs (methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA)), precursor (pseudoephedrine), and synthetic by-products Nformylmethylamphetamine and 1-benzyl-3-methylnaphthalene) – were investigated in laboratory scale for 1 year in three different South Australian soils both under non-sterile and sterile conditions. The results of the degradation study indicated that 1-benzyl-3-methylnaphthalene and methamphetamine persist for a long time in soil compared to MDMA and pseudoephedrine; N-formylmethylamphetamine exhibits intermediate persistence. The role of biotic versus abiotic soil processes on the degradation of target compounds was also varied significantly for different soils as well as with the progress in incubation period. The degradation of methamphetamine and 1-benzyl-3-methylnaphthalene can be considered as predominantly biotic as no measureable changes in concentrations were recorded in the sterile soils within a 1 year period. The results of the present work will help forensic and environmental scientists to precisely determine the environmental impact of chemicals associated with clandestine drug manufacturing laboratories. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of illicit drugs has gained worldwide concern due to their significant adverse impacts on human health and wellbeing of the society (Sloan, 2008; Rieckermann and Christakos, 2008). Illicit drugs are those whose nonmedical use is prohibited by the international law, and mainly belong to the classes of opiates, cocaine, cannabis, amphetamines and ecstasy-group substances (UNODC, 2007; Hall et al., 2008). Amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine currently demand the most attention of all the synthetic illicit drugs (EMCDDA, 2008). The amphetamines and ecstasy-group of illicit drugs (ATSs) are usually manufactured in clandestine laboratories. The chemicals associated with these illegal laboratories including precursors and by-products as well as the synthesized drugs are often illegally buried in soil or public waste management facilities, or disposed of into sinks or toilets after which they enter the sewerage system (Janusz et al., 2003; Scott et al., 2003). The degradation pattern of an illicit
⇑ Corresponding authors at: Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia. Tel.: +61 8 83025044; fax: +61 8 8302 3057. E-mail addresses: [email protected] (M. Megharaj), [email protected] (R. Naidu). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.06.102
drug and a related precursor and manufacturing by-product in soil was initially reported by our research group (Janusz et al., 2003). In this study, the persistence behavior of methylamphetamine sulfate (MAS) and phenyl-2-propanone (P2P, a key precursor and byproduct) in South Australian agricultural soils were presented. It was reported that P2P was rapidly degraded in all the test soils but the degradation of MAS was very slow with the level remaining practically constant over a period of 6 weeks. Recently, the potential impacts of these toxic chemicals are being recognized as a growing concern among environmental scientists and it is necessary to investigate the behavior of these compounds in the environment. The majority of work so far has focused on analytical detection techniques (Castiglioni et al., 2006; Sach and Woo, 2007), and chemical impurity profiling of the illicit drugs (Qi et al., 2006; Waddell-Smith, 2007). A series of reports have been published on the presence of illicit drugs in surface and waste waters from several countries (Jones-Lepp et al., 2004; Zuccato et al., 2005; Hummel et al., 2006; Castiglioni et al., 2006, 2007; Bones et al., 2007; Boleda et al., 2007; Huerta-Fontela et al., 2007, 2008; Kasprzyk-Hordern et al., 2008). Kaleta et al. (2006) reported the presence of amphetamine in the low ppb range in sewage sludge from Austria. There is a lack of information on the behavior of these compounds in the environment and there is no information available in the scientific literature on the fate of these compounds in soil.
Table 1 General information on the target compounds in the present study. Short name
IUPAC nomenclature
Molecular formula
Molecular weight
Methamphetamine
MAP
N-methyl-1 -phenyl-propan-2-amine
C10H15N
149.24
3,4-methylenedioxymethamphetamine
MDMA
1 -(benzo [ 1,3 ] dioxol-5 -yl-N-methylpropan-2amine
C11H15NO2
193.25
Pseudoephedrine
PSE
(1S,2S)-2-methylamino-1 -phenylpropan-1-ol
C10H15NO
165.23
Molecular structure
NHCH3
Solubility in H2O(mg L 1)
log Kow
pKa
1.33E + 04
2.07
9.87
1.06E + 05
0.89
10.3
NHCH 3
O O OH
NHCH 3
N-formylmethylamphetamine
FMA
N,N-dimethyl-1 -phenyl-propan-2-amine
C11H15NO
177.25
CHO NCH 3
l-benzyl-3-methylnaphthalene
BMN
l-benzyl-3-methylnaphthalene
C18H16
232.32
R. Pal et al. / Chemosphere 85 (2011) 1002–1009
Target compound full name
1003
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009
Table 2 Basic physico-chemical properties of the test soils. Soil
Mawson Lakes Sturt Gorge Waite campus
Short name
ML SG WC
pH (1:2.5 H2O)
8.91 5.98 5.64
Electrical conductivity (lS cm 1)
Cation Exchange capacity (cmol(p+) kg 1)
Organic carbon (%)
159 36 965
19.24 6.30 17.42
1.11 2.88 2.26
The research we present here was designed to understand the degradation pattern of ATS chemicals in soils with contrasting physico-chemical properties, and in particular, understand the soil factors controlling the degradation of those potential pollutants in soil. 2. Materials and methods 2.1. Target compounds The target compounds selected for study were the parent drugs (methamphetamine (MAP), and 3,4-methylenedioxymethamphetamine (MDMA)), a key precursor chemical (pseudoephedrine (PSE)) and two common synthetic by-products N-formylmethylamphetamine (FMA, encountered in the Leuckardt and other preparations of methamphetamine) and 1-benzyl-3-methylnaphthalene (BMN, encountered in the conversion of pseudoephedrine into methamphetamine using the Nagai method). The target compounds represent the chemicals associated with the most common methods for clandestine drug manufacture of MAP and MDMA in Australia. Basic information including IUPAC nomenclature, molecular formula, molecular weight, chemical structure of the target compounds are briefly summarized in Table 1. 2.2. Soils Three contrasting soils were used in this study: an alkaline sandy loam collected from the Mawson Lakes (ML) campus of the University of South Australia; slightly acidic sandy loam from Sturt Gorge (SG); and a slightly acidic loam from the Waite Campus (WC) of The University of Adelaide, South Australia. The soils were from an urban area (ML), native bush land (SG), and agricultural land (WC). Samples of surface soils (0–15 cm) were collected from each site, stored in polyethylene buckets, and brought to the laboratory. The soils were then screened to remove any plant parts or other artifacts, passed through 2 mm sieve, and then placed in refrigerator operated at 4 °C. The basic physico-chemical properties of the soils are summarized in Table 2. The physico-chemical properties of the soils were measured following the standard analytical procedures. The three soils varied in terms of pH, organic carbon, clay content, and soil texture. The pH (in 1:2.5 H2O) of the soils ranged between 5.64 and 5.98 (slightly acidic) for WC and SG soils, respectively to 8.91 (alkaline) for ML soil. The cation exchange capacity of the soils ranged between 6.30 and 19.24 cmol(p+) kg 1 soil. The organic carbon content (on dry weight of soil basis) varied between 1.11% (ML) and 2.88% (SG). The soils contained a moderate level of clay (15–20%). The ML and SG soils were sandy loam while WC soil was loam in texture. 2.3. Experimental approach The degradation of the compounds was studied both under non-sterile and sterile conditions. The moisture level of each soil was adjusted to 50% of maximum water holding capacity (MWHC) and then pre-incubated at 25 °C in a constant temperature room
Dissolved organic carbon (lg mL 8.71 5.84 3.90
Particle size distribution
Textural class
1
) Sand (%)
Silt (%)
Clay (%)
55.0 60.0 42.5
25.0 25.0 42.5
20.0 15.0 15.0
Sandy loam Sandy loam Loam
for 1 week. The soils were incubated in the dark to avoid photodegradation. Sub-samples of the pre-incubated soils (5 g) were weighed into individual amber colored glass vials fitted with Teflon lined solid screw caps. For sterile degradation studies the soils in individual vials were autoclaved at 121 °C for 20 min on three consecutive days (Megharaj et al., 1997). The sterile conditions were maintained throughout the study period and affirmed periodically by a microbiological plating technique. The soils were spiked with 100 lg g 1 of each target compound in separate vials. The stock solutions (2 g L 1) for MAP, MDMA, and PSE were prepared in water; while FMA and BMN stock solutions (20 g L 1) were prepared in acetone and hexane, respectively. In the case of the soils for sterile degradation, the stock solutions were passed through sterile 0.45 lm filters and the soils spiked aseptically within a laminar airflow cabinet. The spiked soils were homogenized by vortexing at very low speed for 10 s. Such level of vortexing has no destructive effect on microbial activity as measured by dehydrogenase enzyme assay. The dehydrogenase is an intracellular enzyme linked to the respiratory activity of microorganisms and thus is an indication of microbial activity in soil (Megharaj et al., 2000). Dehydrogenase activity in soils was measured following the method of Casida et al. (1964). The lids of the vials containing FMA or BMN spiked soils were kept open for 15– 20 min to allow the solvents to evaporate. Three types of control soils were also maintained for both non-sterile and sterile conditions, one for each of the background solvents of the respective stock solutions without added compound. The vials for non-sterile degradation study were aerated aseptically within a laminar airflow every week. The moisture contents of the soils (both in nonsterile and sterile) were maintained by aseptic addition of sterile Milli-Q water. All the experimental treatments were conducted in duplicate. The concentrations of each compound were monitored at intervals for up to 1 year. 2.4. Extraction procedure Two extraction procedures were used to extract the compounds from the soil samples. For MAP, MDMA, and PSE the incubated soils were extracted with 40 mL of chloroform: acetonitrile: methanol: acetic acid (80:10:9:1) in two steps (20 mL each). The soils were vortexed and extracted twice on an electric shaker for the period of 1 h and 15 min, respectively, and each extraction was followed by ultrasonic vibration for 5 min at 30 °C. For each of the extraction steps the vials were centrifuged and the aliquots were filtered through 0.22 lm Teflon filters. The aliquots were combined, evaporated under nitrogen stream, and re-dissolved with HPLC grade methanol for direct HPLC analysis. For BMN three extraction steps were employed. To begin with, 10 mL of acetone was used followed by subsequent extractions with 10 mL of ethyl acetate. In each of the extraction steps the vials were vortexed for 1 min. followed by ultrasonic vibration for 15 min at 30 °C. The aliquots were filtered, combined, evaporated under nitrogen stream, and re-dissolved with chromatographic grade ethyl acetate for direct GC analysis. All analyses included appropriate positive and negative control samples.
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MDMA
120
Mawson Lakes
100
Sturt Gorge
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
MAP
Waite Campus
80 60 40 20 0
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0
0
100
200
300
400
0
50
Period ( Day)
PSE
150
FMA
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
100
Period (Day)
0
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0
0
50
100
150
0
100
Period (Day)
200
300
400
Period (Day)
Concentration (mg-kg -1 )
BMN 120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0 0
100
200
300
400
Period (Day) Fig. 1. Non-sterile degradation of target compounds in three experimental soils.
2.5. Determination of target compounds The determinations of MAP, MDMA, PSE and FMA were performed using HPLC (Agilent 1100 series) equipped with an autosampler, binary pump system, a mass selective detector (Agilent 1100), and Chemstation software for data integration. Chromatographic separation of the target compounds were made using a ZORBAX Eclipse XDB-C18 150 4.6 mm, 5 lm column operated at 25 °C. The mobile phase consisted of two combinations of solvent A (20% methanol + 0.1% acetic acid + 10 mM ammonium acetate) and solvent B (90% methanol + 0.1% acetic acid + 10 mM ammonium acetate) maintaining the flow-rate of 0.8 mL min 1. The timetable for the changes of the solvents of the mobile phase for the total run time (26 min) was 0–8 min (100% A), 8–12 min (90% A + 10% B), 12–25 min (100% B), and 25–26 min (100% A). The detector was operated in Electrospray ionization (ESI) mode with positive polarity. The scan parameters were fixed at the mass range of 100 (low) to 350 (high), Fragmentor 120, Gain EMV 3.0, Threshold 0.0, and step size 0.10. The nebulizer pressure in the spray chamber was 35 psig and drying gas 12.0 L min 1. Propranolol was used as the internal standard during the analysis. The determination of BMN was performed by GC (Agilent 6890 N system) equipped with an auto-sampler, a mass selective detector (Agilent 5973), and Chemstation software for data inte-
gration. The GC inlet was operated in splitless mode at 250 °C and helium was the carrier gas with constant flow mode. The inlet pressure was 96.46 kPa, purge flow 49 mL min 1, purge time 0.75 min, and total flow 52.8 mL min 1. The separation was achieved on a DB-5 column (30 m 0.25 mm 0.50 lm). The initial flow in column was 1 mL min 1. The oven temperature was started at 90 °C for 2.50 min and ramped at 45 °C min 1 to 300 °C, and held for 9.00 min. The mass spectra were collected after a 4 min solvent delay over the mass range of 50–550 m/z. Phenanthrene was used as the internal standard during the analysis. The detection limit of the analytical methods ranged between 0.05 ± 0.003 ng (MAP) and 0.11 ± 0.006 ng (FMA), and the limits of quantification were 127 ± 6.93 ng g 1 (MAP) to 704 ± 44.0 ng g 1 (BMN) when n = 3. The average recoveries of the analytical procedures were 51 ± 1.66 (PSE) to 86 ± 1.95% (FMA) when n = 3.
3. Results and discussion 3.1. Degradation patterns Figs. 1 and 2 show the changes in the concentrations of the compounds throughout the incubation period for all the three soils
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009 Mawson Lakes
PSE Mawson Lakes
120
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
MDMA Sturt Gorge
100
Waite Campus
80 60 40 20 0
Sturt Gorge
120
Waite Campus
100 80 60 40 20 0
0
20
40
60
80
0
20
Period (Day)
40
60
80
Period (Day)
Concentration (mg-kg -1 )
FMA
Mawson Lakes
120
Sturt Gorge
100
Waite Campus
80 60 40 20 0 0
50
100
150
200
Period (Day) Fig. 2. Sterile degradation of target compounds in three experimental soils.
Table 3 Regression equation, rate constant (k), and half-life (t½) values for the degradation of target compounds under non-sterile and sterile conditions.
Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus
Compound
MAP
MDMA
PSE
FMA
BMN
Non-sterile
Sterile
Regression equation
t½ (d)
Regression equation
t½ (d)
y= y= y= y= y= y= y= y= y= y= y= y= y= y= y=
274 131 502 59.0 15.4 26.6 3.70 30.1 5.44 35.0 43.6 57.9 10 034 151 602
y= y= y= y= y= y= y= y= y=
83.6 75.3 108 143 502 376 301 188 215
0.0011x + 1.9563 0.0023x + 2.0450 0.0006x + 1.9489 0.0051x + 2.9735 0.0195x + 2.7630 0.0113x + 2.9967 0.0814x + 3.1800 0.0100x + 2.9782 0.0553x + 3.1646 0.0086x + 3.2364 0.0069x + 3.1672 0.0052x + 3.1817 0.00003x + 1.9542 0.0020x + 2.0461 0.0005x + 2.0108
both under non-sterile and sterile conditions. The results showed MAP and BMN to be quite persistent even under non-sterile conditions; therefore Fig. 2 does not include graphs for these two compounds. In non-sterile soils, loss (in percent) of MAP over a period of 1 year were recorded at 68.4 ± 1.27, 89.6 ± 0.59, and 45.8 ± 1.24 for ML, SG, and WC soils, respectively, while that for the BMN were 15.3 ± 1.41, 84.1 ± 0.64, and 32.8 ± 1.41, respectively. The results of the present study corroborates well with our previous report on the biotic–abiotic degradation of methylamphetamine sulfate in South Australian agricultural soils (Janusz et al., 2003). In the previous study, the degradation of methylamphetamine sulfate (spiked at 500 lg g 1 soil) was investigated over a period of 6 weeks in soils from Tailem Bend (pH 6.95, electrical conductivity 86 lS cm 2, organic carbon 2.60%, and clay, 13.0%). Although some initial degradation of the compound was shown in both the nonsterile and sterile soils over 12 d, the concentration level remained nearly constant after this period and two-thirds of the initial concentration was still recorded after 6 weeks. Although there is a difference in basic physico-chemical properties of the soils, spiking
Degradation rate constant (k-1)
Soil
0.0036x + 1.9753 0.0040x + 1.9471 0.0028x + 1.9751 0.0021x + 1.9921 0.0006x + 1.9816 0.0008x + 1.9861 0.0010x + 1.9426 0.0016x + 1.9290 0.0014x + 1.9324
0.09 ML
0.08 0.07
SG
0.06
WC
0.05 0.04 0.03 0.02 0.01 0.00 MAP MDMA
PSE
FMA
BMN MDMA
Non-sterile
PSE
FMA
Sterile
Experimental condition Fig. 3. A plot for the rate constant (k 1) values to compare the degradation potential of the target compounds under different experimental conditions.
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3.2. Degradation potential The regression equations, regression coefficient (r2), rate constant (k 1), and half-life (t½) values to describe the degradation of MAP, MDMA, PSE, FMA, and BMN in both non-sterile and sterile conditions are presented in Table 3. It should however be noted that the regression equations for MAP and BMN in sterile soils were not calculated due to non-measurable changes in concentration over the 1 year incubation period as mentioned earlier. The experimental data were fitted to simple regression equations considering first order reaction, where y is concentration and x is time. The half-life values were calculated from the best fit lines of the logarithm of residual concentrations vs. time elapsed in the incubation period. The results show that the degradation behavior of the target compounds, when compared in terms of the half-life values, generally followed the descending order of MAP BMN > MDMA FMA > PSE in non-sterile soils. Using the regression equation, the half-life (d) values for the non-sterile soils were predicted and these ranged from
Percent degradation
MDMA 100
Day 7
80
Day 15 Day 60
60 40 20 0
ML
SG
WC
ML
Total
SG
WC
ML
Abiotic
SG
WC
Biotic
Nature of degradation
PSE Percent degradation
level of the compound, and the overall incubation period, a close similarity in the degradation behavior between the two studies is noticeable. MAP is fairly stable in soils and little affected by the physico-chemical and biological soil processes. The relatively small decrease in the concentration of BMN especially in ML and WC soils over 1 year incubation period under non-sterile condition might be ascribed to its molecular nature (i.e., benzyl group in the a-position of naphthalene may cause steric hindrance) making it less susceptible to biotic and abiotic soil processes. However, in SG soil BMN recorded a fairly steady degradation pattern over 1 year period. The significantly higher degradation in SG soil compared to ML and WC soils might be ascribed to the soil microbiological processes. The soil microbes in SG soil might have been adapted between the initial to mid phase of the incubation period and thereafter became efficient in utilizing BMN as one of the carbon source. Therefore, the persistence pattern of BMN seems to be closely related to the basic soil characters in addition to its own molecular properties. Recent studies by Abouseoud et al. (2010) demonstrate the marked effect of alkaline pH or high salinity on the naphthalene solubility of a biosurfactant. These findings suggest that the high pH of ML soil compared to the SG and WC soils and extremely high electrical conductivity of WC soil followed by ML soil might be the dominating factor for the high persistency of BMN in ML and WC soils. Thus, no specific comment can be made on degradation behavior of BMN before any thorough study employing a series of soils varying in their physico-chemical properties. Under non-sterile conditions, FMA was also found to be relatively stable for about 4 months in all the soils irrespective of experimental conditions. Thereafter the concentration falls to practically zero after 9 months in ML and SG soils, and to practically zero after 12 months in WC soil. This delay could be related to adaptation of the microbes so that they acquire ability to decompose a synthetic compound over time. The result for FMA might be attributed to the presence of tertiary amine group in the molecule, which has been reported to inhibit biodegradation (Hiromatsu et al., 2000). On the other hand, FMA concentration under sterile conditions declined moderately over 1 month and showed nearly a stable pattern in all the three soils after this period until 6 months incubation. The results for MDMA showed a steady decline over the 4 month incubation period in non-sterile soils recording residual concentrations up to the levels of 17.9 ± 0.93, 0.21 ± 0.03, and 2.27 ± 0.17% for ML, SG, and WC soils, respectively. PSE concentrations declined rapidly in non-sterile ML and WC soils to 0.69 ± 0.06 and 3.18 ± 0.11% of their original concentrations within 4 weeks, while the concentration in SG soil declined to 4.81 ± 0.13% after 4 months.
100
Day 7
80
Day 15
60 40 20 0
ML
SG Total
WC
ML
SG
WC
ML
Abiotic
SG
WC
Biotic
Nature of degradation Fig. 4. A plot for the comparative role of biotic and abiotic factors on degradation of parent drug and precursor compound at different periods of incubation.
131 (SG) to 502 (WC) for MAP, 15.4 (SG) to 59.0 (ML) for MDMA, 3.70 (ML) to 30.1 (SG) for PSE, 35.0 (ML) to 57.9 (WC) for FMA, and 151 (SG) to 10034 (ML) for BMN. However, the half-lives (d) for the sterile soils ranged from 75.3 (SG) to 108 (WC) for MDMA, 143 (ML) to 502 (SG) for PSE, and 188 (SG) to 301 (ML) for FMA. The results thus showed that BMN was quite stable in ML soil and almost completely stable in WC soils. The high persistency of BMN in ML and WC soils might be ascribed to their relatively high pH and salinity as discussed earlier. It can also be noted that MAP remained fairly stable in WC soil. To examine the degradation potential of the target compounds under diverse experimental conditions, the results were expressed in terms of degradation rate constant (k 1: degradation of target compound per unit time). In Fig. 3, the rate constant values are compared for degradation of all the compounds in non-sterile and sterile soils. The results were initially compared within soils under nonsterile condition. The highest k values for PSE (0.0814) and FMA (0.0086) were recorded in ML soil, while that for MAP (0.0023), MDMA (0.0195), and BMN (0.0020) were in SG soil. Alternatively, the lowest k values were recorded for MDMA (0.0051) and BMN (0.00003) in ML soil, PSE (0.0100) in SG soil, and MAP (0.0006) and FMA (0.0052) in WC soil. The results thus indicate that PSE and FMA has highest degradation potential in ML soil as indicated by the respective k values, while that for MAP, MDMA, and BMN was in SG soil. Interestingly, some matching pattern was also apparent between non-sterile and sterile conditions. In accord with the non-sterile soils, MDMA and PSE recorded their maximum k values in SG and ML soil, respectively under sterile condition. On the basis of the above results the potential for degradation of target compounds among soils follow the descending order of SG > ML > WC. However, the results from all the three non-sterile soils showed
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009
that parent drug (MAP and MDMA) and by-products (FMA and BMN) recorded much lower k values compared to the precursor chemical (PSE). 3.3. Role of biotic–abiotic soil processes in degradation Biodegradation is considered to be one of the most important factors in risk assessment of many chemical substances due to influence on their removal after release into to the environment (Hiromatsu et al., 2000; Jaworska et al., 2003). In the present study, the aim was directed to determine the role of the biotic and/or abiotic soil process in the degradation of the target compounds. The present experiment was thus designed to eliminate any chances of photodegradation of the target compounds. To accomplish this, soils were incubated in amber colored glass vials in the dark. Thus, the degradation of the compounds in sterile soils can be attributed to abiotic factors such as oxidation or hydrolysis. In addition, it is also reasonable to assume that the higher degradation in non-sterile compared to sterile soils is indicative of the role of soil microbes. To investigate the possible role of the biotic–abiotic soil processes in the degradation of target compounds, the percent degradation data for the two least stable compounds (e.g., MDMA and PSE) were compared at different periodic intervals (Fig. 4). The abiotic losses of MDMA showed an increase over time irrespective of soils and its biotic degradation in SG and WC soils followed a similar pattern. However, in ML soil a decrease in biotic degradation of MDMA was evident after 7 d, while in SG soil the percent degradation appeared virtually stable over time. The results thus revealed that in SG and WC soils, a similar contribution of biotic and abiotic soil processes are maintained at least at the later stages of incubation, while in ML soil the abiotic processes are more dominant. For PSE an increase over time is apparent for biotic losses especially in ML and WC soils and abiotic losses in all the soils. However, the magnitude of losses due to biotic soil processes significantly exceeds losses due to abiotic processes for PSE. Compared to MDMA therefore, abiotic degradation for PSE was more prominent. In contrast, biotic degradation in ML and WC soils was higher for PSE than MDMA, while the reverse was apparent for SG soil. The higher biotic degradation of PSE might be ascribed to the presence of OH substituent in the molecule, which has been reported to enhance the biodegradability of compounds regardless of the skeleton structure (Hiromatsu et al., 2000). 4. Conclusion The present study clearly indicates that the environmental behavior of clandestine laboratory chemicals is a fairly complex process and depends on several factors. The overall degradation pattern of the test compounds depended mostly on the role of biotic and abiotic properties of individual soil. The nature of the test compounds (e.g., chemical structure, nature of functional groups, etc.) was also a determining factor, as they have the potential to strongly influence the degradation pattern (Hiromatsu et al., 2000). MAP and BMN recorded highest half life values followed by FMA, MDMA, and PSE in non-sterile soils and in most cases the half life values were higher in ML and WC soils compared to SG soil. Caution must be exercised in extrapolation of the results described herein to actual field conditions. Prior to any comment being made on the ‘‘real-life’’ degradation pattern of chemicals involved with illicit drug manufacture, the overall result of this study suggest that there are number of factors to be considered for further research in particular: (i) the affect of different soil
physico-chemical properties on the degradation of these compounds, (ii) the influence of soil moisture regime (e.g., field capacity of soil, waterlogged condition, etc.), and (iii) degradation under anaerobic conditions. The results under diverse environmental conditions may reflect the degradation pattern, metabolite profile, and distribution potential of these chemicals in soil. The results can then be extrapolated to determine the potential hazard arising from the release of these compounds into environment and an assessment be made on the environmental impact of clandestine drug laboratories.
Acknowledgment This work was supported by a National Drug Law Enforcement Research Fund (NDLERF) Grant. References Abouseoud, M., Yataghene, A., Amrane, A., Maachi, R., 2010. Effect of pH and salinity on the emulsifying capacity and naphthalene solubility of a biosurfactant produced by Pseudomonas fluorescens. J. Hazard. Mater. 180, 131–136. Boleda, M.A., Galceran, M.T., Ventura, F., 2007. Trace determination of cannabinoids and opiates in wastewater and surface waters by ultra-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 1175, 38–48. Bones, J., Thomas, K.V., Paul, B., 2007. Using environmental analytical data to estimate levels of community consumption of illicit drugs and abused pharmaceuticals. J. Environ. Monit. 9, 701–707. Casida Jr., L.E., Klein, D., Santoro, T., 1964. Soil dehydrogenase activity. Soil Sci. 98, 371–376. Castiglioni, S., Zuccato, E., Crisci, E., Chiabrando, C., Fanelli, R., Bagnati, R., 2006. Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry. Anal. Chem. 78, 8421–8429. Castiglioni, S., Zuccato, E., Chiabrando, C., Faneli, R., Bagnati, R., 2007. Detecting illicit drugs and metabolites in wastewater using high performance liquid chromatography-tandem mass spectrometry. Spectroscopy Europe 19, 7–9. EMCDDA (European Monitoring Centre for Drugs and Drug Addiction), 2008. Annual Report 2007: The state of the drugs problem in Europe., (accessed 24.09.08). Hall, W., Degenhardt,L., Sindicich, N., 2008. In: Heggenhougen, K., Quah, S., (Eds.), Illicit Drug use and the Burden of Disease: International Encyclopedia of Public Health, Elsevier, pp. 523–530. Hiromatsu, K., Yakabe, Y., Katagiri, K., Nishihara, T., 2000. Prediction for biodegradability of chemicals by an empirical flowchart. Chemosphere 41, 1749–1754. Huerta-Fontela, M., Galceran, M.T., Ventura, F., 2007. Ultraperformance liquid chromatography-tandem mass spectrometry analysis of stimulatory drugs of abuse in wastewater and surface water. Anal. Chem. 79, 3821–3829. Huerta-Fontela, M., Galceran, M.T., Martin-Alonso, J., Ventura, F., 2008. Occurrence of psychoactive stimulatory drugs in wastewaters in north-eastern Spain. Sci. Total Environ. 397, 31–40. Hummel, D., Löffler, D., Fink, G., Ternes, T.A., 2006. Simultaneous determination of psychoactive drugs and their metabolites in aqueous matrices by liquid chromatography mass spectrometry. Environ. Sci. Technol. 40, 7321–7328. Janusz, A., Kirkbride, K.P., Scott, T.L., Naidu, R., Perkins, M.V., Megharaj, M., 2003. Microbial degradation of illicit drugs, their precursors, and manufacturing byproducts: implications for clandestine drug laboratory investigation and environmental assessment. Forensic Sci. Int. 134, 62–71. Jaworska, J.S., Boethling, R.S., Howard, P.H., 2003. Recent development in broadly applicable structure-biodegradability relationships. Environ. Toxicol. Chem. 22, 1710–1723. Jones-Lepp, T.L., Alvarez, D.A., Petty, J.D., Huckins, J.N., 2004. Polar organic chemical integrative sampling and liquid chromatography-electrospray/ion trap mass spectrometry for assessing selected prescription and illicit drugs in treated sewage effluents. Arch. Environ. Contam. Toxicol. 47, 427–439. Kaleta, A., Ferdig, M., Buchberger, W., 2006. Semiquantitative determination of residues of amphetamine in sewage sludge samples. J. Sep. Sci. 29, 1662–1666. Kasprzyk-Hordern, B., Dinsdale, R.M., Guwy, A.J., 2008. Multiresidue methods for the analysis of pharmaceuticals, personal care products and illicit drugs in surface water and wastewater by solid-phase extraction and ultra performance liquid chromatography-electrospray tandem mass spectrometry. Anal. Bioanal. Chem. 391, 1293–1308. Megharaj, M., Singleton, I., McClure, N.C., Naidu, R., 2000. Influence of petroleum hydrocarbon contamination on microalgae and microbial activities in a longterm contaminated soil. Arch. Environ. Contam. Toxicol. 38, 439–445. Megharaj, M., Wittich, R.-M., Blasco, R., Pieper, D.H., Timmis, K.N., 1997. Superior survival and degradation of dibenzo-p-dioxin and bibenzofuran in soil by soiladapted and non-adapted Sphingomonas sp. strain RW1. Appl. Microbiol. Biot 48, 109–114.
R. Pal et al. / Chemosphere 85 (2011) 1002–1009 Qi, Y., Evans, I.D., McCluskey, A., 2006. Australian federal police seizure of illicit crystalline methamphetamine (‘ice’) 1998–2002: impurity analysis. Forensic Sci. Int. 164, 201–210. Rieckermann, J., Christakos, G., 2008. Sewage epidemiology – assessing community health factors by wastewater monitoring. Geophys. Res. Abst. 10. EGU2008-A11908. Sach, S.B., Woo, M.S., 2007. A detailed mechanistic fragmentation analysis of methamphetamine and selected regioisomers by GC/MS. J. Forensic Sci. 52, 308–319. Scott, T.L., Janusz, A., Perkins, M.V., Megharaj, M., Naidu, R., Kirkbride, K.P., 2003. Effect of amphetamine precursors and by-products on soil enzymes of two urban soils. Bull. Environ. Contam. Toxicol. 70, 824–831.
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Biotic and abiotic degradation of illicit drugs, their precursor, and by-products in soil Raktim Pal a,b, Mallavarapu Megharaj a,b,⇑, K. Paul Kirkbride c, Tunde Heinrich a,b, Ravi Naidu a,b,⇑ a
Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia CRC for Contamination Assessment and Remediation of the Environment, University of South Australia, Australia c Australian Federal Police Forensic and Data Centres, Canberra, Australia b
a r t i c l e
i n f o
Article history: Received 26 March 2011 Received in revised form 23 June 2011 Accepted 26 June 2011 Available online 22 July 2011 Keywords: Illicit drugs Methamphetamine MDMA Pseudoephedrine N-formylmethylamphetamine 1-Benzyl-3-methylnaphthalene
a b s t r a c t This study presents the first systematic information on the degradation patterns of clandestine drug laboratory chemicals in soil. The persistence of five compounds – parent drugs (methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA)), precursor (pseudoephedrine), and synthetic by-products Nformylmethylamphetamine and 1-benzyl-3-methylnaphthalene) – were investigated in laboratory scale for 1 year in three different South Australian soils both under non-sterile and sterile conditions. The results of the degradation study indicated that 1-benzyl-3-methylnaphthalene and methamphetamine persist for a long time in soil compared to MDMA and pseudoephedrine; N-formylmethylamphetamine exhibits intermediate persistence. The role of biotic versus abiotic soil processes on the degradation of target compounds was also varied significantly for different soils as well as with the progress in incubation period. The degradation of methamphetamine and 1-benzyl-3-methylnaphthalene can be considered as predominantly biotic as no measureable changes in concentrations were recorded in the sterile soils within a 1 year period. The results of the present work will help forensic and environmental scientists to precisely determine the environmental impact of chemicals associated with clandestine drug manufacturing laboratories. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of illicit drugs has gained worldwide concern due to their significant adverse impacts on human health and wellbeing of the society (Sloan, 2008; Rieckermann and Christakos, 2008). Illicit drugs are those whose nonmedical use is prohibited by the international law, and mainly belong to the classes of opiates, cocaine, cannabis, amphetamines and ecstasy-group substances (UNODC, 2007; Hall et al., 2008). Amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine currently demand the most attention of all the synthetic illicit drugs (EMCDDA, 2008). The amphetamines and ecstasy-group of illicit drugs (ATSs) are usually manufactured in clandestine laboratories. The chemicals associated with these illegal laboratories including precursors and by-products as well as the synthesized drugs are often illegally buried in soil or public waste management facilities, or disposed of into sinks or toilets after which they enter the sewerage system (Janusz et al., 2003; Scott et al., 2003). The degradation pattern of an illicit
⇑ Corresponding authors at: Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, Adelaide, South Australia 5095, Australia. Tel.: +61 8 83025044; fax: +61 8 8302 3057. E-mail addresses: [email protected] (M. Megharaj), [email protected] (R. Naidu). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.06.102
drug and a related precursor and manufacturing by-product in soil was initially reported by our research group (Janusz et al., 2003). In this study, the persistence behavior of methylamphetamine sulfate (MAS) and phenyl-2-propanone (P2P, a key precursor and byproduct) in South Australian agricultural soils were presented. It was reported that P2P was rapidly degraded in all the test soils but the degradation of MAS was very slow with the level remaining practically constant over a period of 6 weeks. Recently, the potential impacts of these toxic chemicals are being recognized as a growing concern among environmental scientists and it is necessary to investigate the behavior of these compounds in the environment. The majority of work so far has focused on analytical detection techniques (Castiglioni et al., 2006; Sach and Woo, 2007), and chemical impurity profiling of the illicit drugs (Qi et al., 2006; Waddell-Smith, 2007). A series of reports have been published on the presence of illicit drugs in surface and waste waters from several countries (Jones-Lepp et al., 2004; Zuccato et al., 2005; Hummel et al., 2006; Castiglioni et al., 2006, 2007; Bones et al., 2007; Boleda et al., 2007; Huerta-Fontela et al., 2007, 2008; Kasprzyk-Hordern et al., 2008). Kaleta et al. (2006) reported the presence of amphetamine in the low ppb range in sewage sludge from Austria. There is a lack of information on the behavior of these compounds in the environment and there is no information available in the scientific literature on the fate of these compounds in soil.
Table 1 General information on the target compounds in the present study. Short name
IUPAC nomenclature
Molecular formula
Molecular weight
Methamphetamine
MAP
N-methyl-1 -phenyl-propan-2-amine
C10H15N
149.24
3,4-methylenedioxymethamphetamine
MDMA
1 -(benzo [ 1,3 ] dioxol-5 -yl-N-methylpropan-2amine
C11H15NO2
193.25
Pseudoephedrine
PSE
(1S,2S)-2-methylamino-1 -phenylpropan-1-ol
C10H15NO
165.23
Molecular structure
NHCH3
Solubility in H2O(mg L 1)
log Kow
pKa
1.33E + 04
2.07
9.87
1.06E + 05
0.89
10.3
NHCH 3
O O OH
NHCH 3
N-formylmethylamphetamine
FMA
N,N-dimethyl-1 -phenyl-propan-2-amine
C11H15NO
177.25
CHO NCH 3
l-benzyl-3-methylnaphthalene
BMN
l-benzyl-3-methylnaphthalene
C18H16
232.32
R. Pal et al. / Chemosphere 85 (2011) 1002–1009
Target compound full name
1003
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009
Table 2 Basic physico-chemical properties of the test soils. Soil
Mawson Lakes Sturt Gorge Waite campus
Short name
ML SG WC
pH (1:2.5 H2O)
8.91 5.98 5.64
Electrical conductivity (lS cm 1)
Cation Exchange capacity (cmol(p+) kg 1)
Organic carbon (%)
159 36 965
19.24 6.30 17.42
1.11 2.88 2.26
The research we present here was designed to understand the degradation pattern of ATS chemicals in soils with contrasting physico-chemical properties, and in particular, understand the soil factors controlling the degradation of those potential pollutants in soil. 2. Materials and methods 2.1. Target compounds The target compounds selected for study were the parent drugs (methamphetamine (MAP), and 3,4-methylenedioxymethamphetamine (MDMA)), a key precursor chemical (pseudoephedrine (PSE)) and two common synthetic by-products N-formylmethylamphetamine (FMA, encountered in the Leuckardt and other preparations of methamphetamine) and 1-benzyl-3-methylnaphthalene (BMN, encountered in the conversion of pseudoephedrine into methamphetamine using the Nagai method). The target compounds represent the chemicals associated with the most common methods for clandestine drug manufacture of MAP and MDMA in Australia. Basic information including IUPAC nomenclature, molecular formula, molecular weight, chemical structure of the target compounds are briefly summarized in Table 1. 2.2. Soils Three contrasting soils were used in this study: an alkaline sandy loam collected from the Mawson Lakes (ML) campus of the University of South Australia; slightly acidic sandy loam from Sturt Gorge (SG); and a slightly acidic loam from the Waite Campus (WC) of The University of Adelaide, South Australia. The soils were from an urban area (ML), native bush land (SG), and agricultural land (WC). Samples of surface soils (0–15 cm) were collected from each site, stored in polyethylene buckets, and brought to the laboratory. The soils were then screened to remove any plant parts or other artifacts, passed through 2 mm sieve, and then placed in refrigerator operated at 4 °C. The basic physico-chemical properties of the soils are summarized in Table 2. The physico-chemical properties of the soils were measured following the standard analytical procedures. The three soils varied in terms of pH, organic carbon, clay content, and soil texture. The pH (in 1:2.5 H2O) of the soils ranged between 5.64 and 5.98 (slightly acidic) for WC and SG soils, respectively to 8.91 (alkaline) for ML soil. The cation exchange capacity of the soils ranged between 6.30 and 19.24 cmol(p+) kg 1 soil. The organic carbon content (on dry weight of soil basis) varied between 1.11% (ML) and 2.88% (SG). The soils contained a moderate level of clay (15–20%). The ML and SG soils were sandy loam while WC soil was loam in texture. 2.3. Experimental approach The degradation of the compounds was studied both under non-sterile and sterile conditions. The moisture level of each soil was adjusted to 50% of maximum water holding capacity (MWHC) and then pre-incubated at 25 °C in a constant temperature room
Dissolved organic carbon (lg mL 8.71 5.84 3.90
Particle size distribution
Textural class
1
) Sand (%)
Silt (%)
Clay (%)
55.0 60.0 42.5
25.0 25.0 42.5
20.0 15.0 15.0
Sandy loam Sandy loam Loam
for 1 week. The soils were incubated in the dark to avoid photodegradation. Sub-samples of the pre-incubated soils (5 g) were weighed into individual amber colored glass vials fitted with Teflon lined solid screw caps. For sterile degradation studies the soils in individual vials were autoclaved at 121 °C for 20 min on three consecutive days (Megharaj et al., 1997). The sterile conditions were maintained throughout the study period and affirmed periodically by a microbiological plating technique. The soils were spiked with 100 lg g 1 of each target compound in separate vials. The stock solutions (2 g L 1) for MAP, MDMA, and PSE were prepared in water; while FMA and BMN stock solutions (20 g L 1) were prepared in acetone and hexane, respectively. In the case of the soils for sterile degradation, the stock solutions were passed through sterile 0.45 lm filters and the soils spiked aseptically within a laminar airflow cabinet. The spiked soils were homogenized by vortexing at very low speed for 10 s. Such level of vortexing has no destructive effect on microbial activity as measured by dehydrogenase enzyme assay. The dehydrogenase is an intracellular enzyme linked to the respiratory activity of microorganisms and thus is an indication of microbial activity in soil (Megharaj et al., 2000). Dehydrogenase activity in soils was measured following the method of Casida et al. (1964). The lids of the vials containing FMA or BMN spiked soils were kept open for 15– 20 min to allow the solvents to evaporate. Three types of control soils were also maintained for both non-sterile and sterile conditions, one for each of the background solvents of the respective stock solutions without added compound. The vials for non-sterile degradation study were aerated aseptically within a laminar airflow every week. The moisture contents of the soils (both in nonsterile and sterile) were maintained by aseptic addition of sterile Milli-Q water. All the experimental treatments were conducted in duplicate. The concentrations of each compound were monitored at intervals for up to 1 year. 2.4. Extraction procedure Two extraction procedures were used to extract the compounds from the soil samples. For MAP, MDMA, and PSE the incubated soils were extracted with 40 mL of chloroform: acetonitrile: methanol: acetic acid (80:10:9:1) in two steps (20 mL each). The soils were vortexed and extracted twice on an electric shaker for the period of 1 h and 15 min, respectively, and each extraction was followed by ultrasonic vibration for 5 min at 30 °C. For each of the extraction steps the vials were centrifuged and the aliquots were filtered through 0.22 lm Teflon filters. The aliquots were combined, evaporated under nitrogen stream, and re-dissolved with HPLC grade methanol for direct HPLC analysis. For BMN three extraction steps were employed. To begin with, 10 mL of acetone was used followed by subsequent extractions with 10 mL of ethyl acetate. In each of the extraction steps the vials were vortexed for 1 min. followed by ultrasonic vibration for 15 min at 30 °C. The aliquots were filtered, combined, evaporated under nitrogen stream, and re-dissolved with chromatographic grade ethyl acetate for direct GC analysis. All analyses included appropriate positive and negative control samples.
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MDMA
120
Mawson Lakes
100
Sturt Gorge
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
MAP
Waite Campus
80 60 40 20 0
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0
0
100
200
300
400
0
50
Period ( Day)
PSE
150
FMA
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
100
Period (Day)
0
120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0
0
50
100
150
0
100
Period (Day)
200
300
400
Period (Day)
Concentration (mg-kg -1 )
BMN 120
Mawson Lakes
100
Sturt Gorge Waite Campus
80 60 40 20 0 0
100
200
300
400
Period (Day) Fig. 1. Non-sterile degradation of target compounds in three experimental soils.
2.5. Determination of target compounds The determinations of MAP, MDMA, PSE and FMA were performed using HPLC (Agilent 1100 series) equipped with an autosampler, binary pump system, a mass selective detector (Agilent 1100), and Chemstation software for data integration. Chromatographic separation of the target compounds were made using a ZORBAX Eclipse XDB-C18 150 4.6 mm, 5 lm column operated at 25 °C. The mobile phase consisted of two combinations of solvent A (20% methanol + 0.1% acetic acid + 10 mM ammonium acetate) and solvent B (90% methanol + 0.1% acetic acid + 10 mM ammonium acetate) maintaining the flow-rate of 0.8 mL min 1. The timetable for the changes of the solvents of the mobile phase for the total run time (26 min) was 0–8 min (100% A), 8–12 min (90% A + 10% B), 12–25 min (100% B), and 25–26 min (100% A). The detector was operated in Electrospray ionization (ESI) mode with positive polarity. The scan parameters were fixed at the mass range of 100 (low) to 350 (high), Fragmentor 120, Gain EMV 3.0, Threshold 0.0, and step size 0.10. The nebulizer pressure in the spray chamber was 35 psig and drying gas 12.0 L min 1. Propranolol was used as the internal standard during the analysis. The determination of BMN was performed by GC (Agilent 6890 N system) equipped with an auto-sampler, a mass selective detector (Agilent 5973), and Chemstation software for data inte-
gration. The GC inlet was operated in splitless mode at 250 °C and helium was the carrier gas with constant flow mode. The inlet pressure was 96.46 kPa, purge flow 49 mL min 1, purge time 0.75 min, and total flow 52.8 mL min 1. The separation was achieved on a DB-5 column (30 m 0.25 mm 0.50 lm). The initial flow in column was 1 mL min 1. The oven temperature was started at 90 °C for 2.50 min and ramped at 45 °C min 1 to 300 °C, and held for 9.00 min. The mass spectra were collected after a 4 min solvent delay over the mass range of 50–550 m/z. Phenanthrene was used as the internal standard during the analysis. The detection limit of the analytical methods ranged between 0.05 ± 0.003 ng (MAP) and 0.11 ± 0.006 ng (FMA), and the limits of quantification were 127 ± 6.93 ng g 1 (MAP) to 704 ± 44.0 ng g 1 (BMN) when n = 3. The average recoveries of the analytical procedures were 51 ± 1.66 (PSE) to 86 ± 1.95% (FMA) when n = 3.
3. Results and discussion 3.1. Degradation patterns Figs. 1 and 2 show the changes in the concentrations of the compounds throughout the incubation period for all the three soils
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009 Mawson Lakes
PSE Mawson Lakes
120
Concentration (mg-kg -1 )
Concentration (mg-kg -1 )
MDMA Sturt Gorge
100
Waite Campus
80 60 40 20 0
Sturt Gorge
120
Waite Campus
100 80 60 40 20 0
0
20
40
60
80
0
20
Period (Day)
40
60
80
Period (Day)
Concentration (mg-kg -1 )
FMA
Mawson Lakes
120
Sturt Gorge
100
Waite Campus
80 60 40 20 0 0
50
100
150
200
Period (Day) Fig. 2. Sterile degradation of target compounds in three experimental soils.
Table 3 Regression equation, rate constant (k), and half-life (t½) values for the degradation of target compounds under non-sterile and sterile conditions.
Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus Mawson Lakes Sturt Gorge Waite campus
Compound
MAP
MDMA
PSE
FMA
BMN
Non-sterile
Sterile
Regression equation
t½ (d)
Regression equation
t½ (d)
y= y= y= y= y= y= y= y= y= y= y= y= y= y= y=
274 131 502 59.0 15.4 26.6 3.70 30.1 5.44 35.0 43.6 57.9 10 034 151 602
y= y= y= y= y= y= y= y= y=
83.6 75.3 108 143 502 376 301 188 215
0.0011x + 1.9563 0.0023x + 2.0450 0.0006x + 1.9489 0.0051x + 2.9735 0.0195x + 2.7630 0.0113x + 2.9967 0.0814x + 3.1800 0.0100x + 2.9782 0.0553x + 3.1646 0.0086x + 3.2364 0.0069x + 3.1672 0.0052x + 3.1817 0.00003x + 1.9542 0.0020x + 2.0461 0.0005x + 2.0108
both under non-sterile and sterile conditions. The results showed MAP and BMN to be quite persistent even under non-sterile conditions; therefore Fig. 2 does not include graphs for these two compounds. In non-sterile soils, loss (in percent) of MAP over a period of 1 year were recorded at 68.4 ± 1.27, 89.6 ± 0.59, and 45.8 ± 1.24 for ML, SG, and WC soils, respectively, while that for the BMN were 15.3 ± 1.41, 84.1 ± 0.64, and 32.8 ± 1.41, respectively. The results of the present study corroborates well with our previous report on the biotic–abiotic degradation of methylamphetamine sulfate in South Australian agricultural soils (Janusz et al., 2003). In the previous study, the degradation of methylamphetamine sulfate (spiked at 500 lg g 1 soil) was investigated over a period of 6 weeks in soils from Tailem Bend (pH 6.95, electrical conductivity 86 lS cm 2, organic carbon 2.60%, and clay, 13.0%). Although some initial degradation of the compound was shown in both the nonsterile and sterile soils over 12 d, the concentration level remained nearly constant after this period and two-thirds of the initial concentration was still recorded after 6 weeks. Although there is a difference in basic physico-chemical properties of the soils, spiking
Degradation rate constant (k-1)
Soil
0.0036x + 1.9753 0.0040x + 1.9471 0.0028x + 1.9751 0.0021x + 1.9921 0.0006x + 1.9816 0.0008x + 1.9861 0.0010x + 1.9426 0.0016x + 1.9290 0.0014x + 1.9324
0.09 ML
0.08 0.07
SG
0.06
WC
0.05 0.04 0.03 0.02 0.01 0.00 MAP MDMA
PSE
FMA
BMN MDMA
Non-sterile
PSE
FMA
Sterile
Experimental condition Fig. 3. A plot for the rate constant (k 1) values to compare the degradation potential of the target compounds under different experimental conditions.
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R. Pal et al. / Chemosphere 85 (2011) 1002–1009
3.2. Degradation potential The regression equations, regression coefficient (r2), rate constant (k 1), and half-life (t½) values to describe the degradation of MAP, MDMA, PSE, FMA, and BMN in both non-sterile and sterile conditions are presented in Table 3. It should however be noted that the regression equations for MAP and BMN in sterile soils were not calculated due to non-measurable changes in concentration over the 1 year incubation period as mentioned earlier. The experimental data were fitted to simple regression equations considering first order reaction, where y is concentration and x is time. The half-life values were calculated from the best fit lines of the logarithm of residual concentrations vs. time elapsed in the incubation period. The results show that the degradation behavior of the target compounds, when compared in terms of the half-life values, generally followed the descending order of MAP BMN > MDMA FMA > PSE in non-sterile soils. Using the regression equation, the half-life (d) values for the non-sterile soils were predicted and these ranged from
Percent degradation
MDMA 100
Day 7
80
Day 15 Day 60
60 40 20 0
ML
SG
WC
ML
Total
SG
WC
ML
Abiotic
SG
WC
Biotic
Nature of degradation
PSE Percent degradation
level of the compound, and the overall incubation period, a close similarity in the degradation behavior between the two studies is noticeable. MAP is fairly stable in soils and little affected by the physico-chemical and biological soil processes. The relatively small decrease in the concentration of BMN especially in ML and WC soils over 1 year incubation period under non-sterile condition might be ascribed to its molecular nature (i.e., benzyl group in the a-position of naphthalene may cause steric hindrance) making it less susceptible to biotic and abiotic soil processes. However, in SG soil BMN recorded a fairly steady degradation pattern over 1 year period. The significantly higher degradation in SG soil compared to ML and WC soils might be ascribed to the soil microbiological processes. The soil microbes in SG soil might have been adapted between the initial to mid phase of the incubation period and thereafter became efficient in utilizing BMN as one of the carbon source. Therefore, the persistence pattern of BMN seems to be closely related to the basic soil characters in addition to its own molecular properties. Recent studies by Abouseoud et al. (2010) demonstrate the marked effect of alkaline pH or high salinity on the naphthalene solubility of a biosurfactant. These findings suggest that the high pH of ML soil compared to the SG and WC soils and extremely high electrical conductivity of WC soil followed by ML soil might be the dominating factor for the high persistency of BMN in ML and WC soils. Thus, no specific comment can be made on degradation behavior of BMN before any thorough study employing a series of soils varying in their physico-chemical properties. Under non-sterile conditions, FMA was also found to be relatively stable for about 4 months in all the soils irrespective of experimental conditions. Thereafter the concentration falls to practically zero after 9 months in ML and SG soils, and to practically zero after 12 months in WC soil. This delay could be related to adaptation of the microbes so that they acquire ability to decompose a synthetic compound over time. The result for FMA might be attributed to the presence of tertiary amine group in the molecule, which has been reported to inhibit biodegradation (Hiromatsu et al., 2000). On the other hand, FMA concentration under sterile conditions declined moderately over 1 month and showed nearly a stable pattern in all the three soils after this period until 6 months incubation. The results for MDMA showed a steady decline over the 4 month incubation period in non-sterile soils recording residual concentrations up to the levels of 17.9 ± 0.93, 0.21 ± 0.03, and 2.27 ± 0.17% for ML, SG, and WC soils, respectively. PSE concentrations declined rapidly in non-sterile ML and WC soils to 0.69 ± 0.06 and 3.18 ± 0.11% of their original concentrations within 4 weeks, while the concentration in SG soil declined to 4.81 ± 0.13% after 4 months.
100
Day 7
80
Day 15
60 40 20 0
ML
SG Total
WC
ML
SG
WC
ML
Abiotic
SG
WC
Biotic
Nature of degradation Fig. 4. A plot for the comparative role of biotic and abiotic factors on degradation of parent drug and precursor compound at different periods of incubation.
131 (SG) to 502 (WC) for MAP, 15.4 (SG) to 59.0 (ML) for MDMA, 3.70 (ML) to 30.1 (SG) for PSE, 35.0 (ML) to 57.9 (WC) for FMA, and 151 (SG) to 10034 (ML) for BMN. However, the half-lives (d) for the sterile soils ranged from 75.3 (SG) to 108 (WC) for MDMA, 143 (ML) to 502 (SG) for PSE, and 188 (SG) to 301 (ML) for FMA. The results thus showed that BMN was quite stable in ML soil and almost completely stable in WC soils. The high persistency of BMN in ML and WC soils might be ascribed to their relatively high pH and salinity as discussed earlier. It can also be noted that MAP remained fairly stable in WC soil. To examine the degradation potential of the target compounds under diverse experimental conditions, the results were expressed in terms of degradation rate constant (k 1: degradation of target compound per unit time). In Fig. 3, the rate constant values are compared for degradation of all the compounds in non-sterile and sterile soils. The results were initially compared within soils under nonsterile condition. The highest k values for PSE (0.0814) and FMA (0.0086) were recorded in ML soil, while that for MAP (0.0023), MDMA (0.0195), and BMN (0.0020) were in SG soil. Alternatively, the lowest k values were recorded for MDMA (0.0051) and BMN (0.00003) in ML soil, PSE (0.0100) in SG soil, and MAP (0.0006) and FMA (0.0052) in WC soil. The results thus indicate that PSE and FMA has highest degradation potential in ML soil as indicated by the respective k values, while that for MAP, MDMA, and BMN was in SG soil. Interestingly, some matching pattern was also apparent between non-sterile and sterile conditions. In accord with the non-sterile soils, MDMA and PSE recorded their maximum k values in SG and ML soil, respectively under sterile condition. On the basis of the above results the potential for degradation of target compounds among soils follow the descending order of SG > ML > WC. However, the results from all the three non-sterile soils showed
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that parent drug (MAP and MDMA) and by-products (FMA and BMN) recorded much lower k values compared to the precursor chemical (PSE). 3.3. Role of biotic–abiotic soil processes in degradation Biodegradation is considered to be one of the most important factors in risk assessment of many chemical substances due to influence on their removal after release into to the environment (Hiromatsu et al., 2000; Jaworska et al., 2003). In the present study, the aim was directed to determine the role of the biotic and/or abiotic soil process in the degradation of the target compounds. The present experiment was thus designed to eliminate any chances of photodegradation of the target compounds. To accomplish this, soils were incubated in amber colored glass vials in the dark. Thus, the degradation of the compounds in sterile soils can be attributed to abiotic factors such as oxidation or hydrolysis. In addition, it is also reasonable to assume that the higher degradation in non-sterile compared to sterile soils is indicative of the role of soil microbes. To investigate the possible role of the biotic–abiotic soil processes in the degradation of target compounds, the percent degradation data for the two least stable compounds (e.g., MDMA and PSE) were compared at different periodic intervals (Fig. 4). The abiotic losses of MDMA showed an increase over time irrespective of soils and its biotic degradation in SG and WC soils followed a similar pattern. However, in ML soil a decrease in biotic degradation of MDMA was evident after 7 d, while in SG soil the percent degradation appeared virtually stable over time. The results thus revealed that in SG and WC soils, a similar contribution of biotic and abiotic soil processes are maintained at least at the later stages of incubation, while in ML soil the abiotic processes are more dominant. For PSE an increase over time is apparent for biotic losses especially in ML and WC soils and abiotic losses in all the soils. However, the magnitude of losses due to biotic soil processes significantly exceeds losses due to abiotic processes for PSE. Compared to MDMA therefore, abiotic degradation for PSE was more prominent. In contrast, biotic degradation in ML and WC soils was higher for PSE than MDMA, while the reverse was apparent for SG soil. The higher biotic degradation of PSE might be ascribed to the presence of OH substituent in the molecule, which has been reported to enhance the biodegradability of compounds regardless of the skeleton structure (Hiromatsu et al., 2000). 4. Conclusion The present study clearly indicates that the environmental behavior of clandestine laboratory chemicals is a fairly complex process and depends on several factors. The overall degradation pattern of the test compounds depended mostly on the role of biotic and abiotic properties of individual soil. The nature of the test compounds (e.g., chemical structure, nature of functional groups, etc.) was also a determining factor, as they have the potential to strongly influence the degradation pattern (Hiromatsu et al., 2000). MAP and BMN recorded highest half life values followed by FMA, MDMA, and PSE in non-sterile soils and in most cases the half life values were higher in ML and WC soils compared to SG soil. Caution must be exercised in extrapolation of the results described herein to actual field conditions. Prior to any comment being made on the ‘‘real-life’’ degradation pattern of chemicals involved with illicit drug manufacture, the overall result of this study suggest that there are number of factors to be considered for further research in particular: (i) the affect of different soil
physico-chemical properties on the degradation of these compounds, (ii) the influence of soil moisture regime (e.g., field capacity of soil, waterlogged condition, etc.), and (iii) degradation under anaerobic conditions. The results under diverse environmental conditions may reflect the degradation pattern, metabolite profile, and distribution potential of these chemicals in soil. The results can then be extrapolated to determine the potential hazard arising from the release of these compounds into environment and an assessment be made on the environmental impact of clandestine drug laboratories.
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