Bioactive and total endotoxins in atmospheric aerosols in the Pearl River Delta region, China

Atmospheric Environment 47 (2012) 3e11

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Bioactive and total endotoxins in atmospheric aerosols in the Pearl River Delta region, China Jessica Y.W. Cheng a, Esther L.C. Hui b, Arthur P.S. Lau c, d, * a

Institute for the Environment, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China Environmental Engineering Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China c Division of Environment, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China d Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 June 2011 Received in revised form 18 November 2011 Accepted 22 November 2011

Endotoxin, a toxic and pyrogenic substance in gram-negative bacteria in atmospheric aerosols was measured over a period of one year at Nansha, Guangzhou and Hong Kong in the Pearl River Delta region, China. Atmospheric aerosols were collected by high-volume samplers. The bioactive endotoxin levels in the samples were determined using the Limulus Amebocyte Lysate (LAL) assay after extraction with pyrogen-free water while the total endotoxin levels were measured by quantifying the biomarker, 3hydroxy fatty acids (3-OHFAs) with GCeMS. Results showed that there was no significant difference (0.19 < p < 0.81) in the bioactive endotoxin level in PM10 among sites (average concentrations ranged from 0.34 to 0.39 EU m
3). However, Hong Kong showed a significantly lower (p < 0.05) total endotoxin level in PM10 (average of 17.4 ng m
3) compared with Nansha’s 29.4 ng m
3 and Guangzhou’s 32.7 ng m
3. The bioactive endotoxins were found to be associated with the coarse mode (PM2.5-10) of the particulates of natural origins while the total endotoxins were associated more with the fine mode (PM2.5) of the particulates of anthropogenic origins. When normalized with particulate mass, the endotoxin loading is much higher in summer as a result of the increased growth of the bacteria when climatic conditions are favorable. The chemically determined total endotoxins were 3e4 orders of magnitude higher than the bioactive endotoxins quantified using the LAL assay. Correlation analyses between the bioactive endotoxins and 3-OHFAs with different carbon length were analyzed. Results showed that the correlations detected vary among sites and particulate sizes. Although no generalization between the total and bioactive endotoxins can be drawn from the study, the levels reported in this study suggests that the discrepancies between the two measurement approaches, and the bioactive potential of 3-OHFAs with individual carbon chains deserve further investigation. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Airborne endotoxin Limulus Amebocyte Lysate (LAL) assay 3-Hydroxy fatty acid Biomarker Pearl River Delta

1. Introduction The Pearl River Delta (PRD), located in the southern part of China, is one of the most populated areas of China. Since the 1980s, it has become one of the leading economic regions and a major manufacturing center of China and the world. The heavy industrial activity has unavoidably resulted in the deterioration of the air quality in the region. In the past decade, many researchers have concentrated their efforts on understanding the air quality in this region (Guo et al., 2010, 2009; Zhang et al., 2010; Liu et al., 2008; Lang et al., 2007; Haggler et al., 2006). Most of these studies have * Corresponding author. Division of Environment, The Hong Kong University of Science and Technology, Clear Water Bay Road, Kowloon, Hong Kong, China. Tel.: þ852 2358 6915; fax: þ852 2358 1582. E-mail address: [email protected] (A.P.S. Lau). 1352-2310/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.atmosenv.2011.11.055

focused on the source identification, transportation or seasonal variation of different chemical pollutants. Relatively little is known about the airborne microbiological constituents in this region. Airborne endotoxin is of particular interest because of its strong chronic health effects (Wang et al., 2005). Endotoxins are amphiphilic lipopolysaccharide molecules composed of a unique structure of lipid A and a large polysaccharide part in the outer membrane of the gram-negative bacteria. Inhalation of endotoxins can cause fever, headache, and exacerbation of airway symptoms such as coughing, shortness of breath and airway inflammation (Liebers et al., 2008; Douwes et al., 2003; Rylander, 2002). Endotoxins as aeroallergens are commonly studied in indoor environment (Tager et al., 2010; Yao et al., 2009; Rao et al., 2005) as well as the occupational exposure (Saboli c Pipini c et al., 2010; Pomorska et al., 2007; Visser et al., 2006). Only a few studies have measured the atmospheric endotoxin concentrations systematically and most

4

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

of them were conducted in European cities (Nilsson et al., 2011; Morgenstern et al., 2005; Heinrich et al., 2003). Carty et al. (2003), in particular, reported that higher average temperatures yielded higher levels of endotoxin in their study conducted in Munich, Germany. The PRD is located in the subtropics with year-round warm and humid weather and high vegetation coverage. The region is also densely populated. It is therefore important to investigate the levels of atmospheric endotoxin in this region and their potential impact to human health. This study was therefore designed to assess the spatial and temporal variations in the atmospheric endotoxin levels in PM2.5 and PM10 in the PRD. Endotoxin is commonly quantified using the Limulus Amebocyte Lysate (LAL) assay. This assay is based on an enzymatic coagulation of the blood of a primitive marine arthropod, the horseshoe crab, in the presence of endotoxin. It provides an easy and sensitive detection method for endotoxin, which is bioactive (Douwes et al., 2003). However, large variations in the LAL assay results were commonly reported. Factors causing such large variations include the solution used for extraction, the extraction methods, and the interference of b-1,3-glucan (Liebers et al., 2007; Spaan et al., 2008). Great discrepancies in inter-laboratory analyses have also been reported (Reynolds et al., 2005). Experiences and findings from the occupational studies thus emphasize the importance of optimizing the extraction protocols for data validity. Despite its disadvantages, LAL assay is commonly used as its results are more biologically relevant to human health effects. A chemical analytical method utilizing gas chromatographyemass spectrometry (GCeMS) has also been developed to quantify 3-OHFAs, with carbon chain lengths from 9 to 18, as a biomarker for estimating the lipid A content and the mass concentration of the endotoxin (Lee et al., 2007). This chemical method is less sensitive, but more specific, in detection and can measure total (both biologically active and inactive) endotoxins regardless of their bioactive potencies. This method also enables the concomitant analysis of other chemical species to provide information on the source origin of aerosols. However, the relationship between total endotoxins and health effects has yet to be established. There is also no parallel study on bioactive and total endotoxin in the atmosphere with these two analytical approaches in the literature. The aim of this study is therefore to investigate the prevalence of the

atmospheric endotoxins in the Pearl River Delta region; and to compare the chemical and biological approach in assaying the endotoxins in atmospheric aerosols for cross-referencing between the bioactive and total endotoxins in the field collected samples. 2. Methods 2.1. Sample collection Atmospheric aerosol samples were collected in Nansha (NS), Guangzhou (GZ) and Hong Kong (HK) of the PRD region (Fig. 1). According to the Statistics Bureau of Guangdong Province (2010) and the Census and Statistics Department of Hong Kong (2010), the population densities of NS, GZ and HK were about 350 people km
2, 1300 people km
2 and 6400 people km
2, respectively. NS is a suburban district and is sparsely populated. GZ is an urban city with many different manufacturing industries while HK is an urban city with a high population density. The sampling site in NS was located on the rooftop of a 5-storey building in a rural area while the sampling sites in GZ and HK were located on the rooftop of a 23-storey building and a 5-storey building in residential areas, respectively. Samples were collected once every six days at the NS site and once every twelve days at the GZ and HK sites from February 2008 to March 2009. A total of 68, 34 and 32 sets of samples were collected at the NS, GZ and HK sites, respectively. High-volume samplers with a flow rate of 1.13 m3 min
1 (Tisch, TE6070V-BL), and equipped with impactors with a cut-off diameter of 2.5 mm (Tisch, TE-231), were used to collect aerosol samples on a 24-h basis. Both coarse (2.5 mm  aerodynamic diameter  10 mm) and fine (aerodynamic diameter  2.5 mm) aerosols were collected on quartz filters (PM2.5-10: Pall, TissuquartzÔ, 10  8 inch; PM2.5: Tisch, slotted) which had been thermally pretreated at 500  C for 4 h. After sampling, all filters were stored at
20  C to inhibit the growth of microorganisms prior to chemical analysis. 2.2. Limulus Amebocyte Lysate (LAL) assay for bioactive endotoxin Endotoxin in the filter samples were extracted with pure pyrogen-free water, LRW (Associates of Cape Cod, Inc., MA, USA) in

Fig. 1. Map showing the sampling locations e Guangzhou, Nansha and Hong Kong e in this study.

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

pyrogen-free tubes by sonication for 30 min, followed by centrifugation at 2000g for 10 min. Experiences accumulated in the industrial or residential dusts studies indicated that LAL assay may subject to interferences from extraction methods, extraction matrices and filter paper types (Spaan et al., 2008). While it is better to optimize the conditions before LAL assay, it is not the primary aim of the present study. Extraction with Tween 20 had been reported to have higher endotoxin yields in industrial or residential dusts; however, the Tween also shifted the calibration curve to higher values (Spaan et al., 2008). To minimize the uncertainties of any additives in the extraction and reaction processes, pure LRW was thus adopted in the present study. The supernatant was collected for bioactive endotoxin analysis using the endpoint chromogenic method following the procedures suggested by the manufacturer of PyrochromeÒ (Associates of Cape Cod Inc., MA, USA). Control standard endotoxin or CSE from Escherichia coli O113:H10 (Associates of Cape Cod Inc., MA, USA) was used as a standard. The calibration curves were obtained using 5 different dilution points with the concentration ranged from 0.01 to 0.50 EU ml
1. Though the sensitivity of the assay can be as low as 0.001 EU ml
1, preliminary screening of the endotoxin in the coarse and fine aerosol samples (10 each) showed that its level was in the range of 0.01e0.50 EU ml
1 within 40 and 4 dilution, respectively. To minimize the systematic error due to too much dilution, the range of detection was set from 0.01 to 0.50 EU ml
1 and calibration curve was thus set within this range. A calibration curve was constructed with CSE for each batch of sample (40 in number) analysis. All calibration curves reported in this study had a correlation coefficients >0.98. Supernatant collected after sample extraction was then mixed with the PyrochromeÒ reagent according to the manufacturer’s instruction. The mixture was then incubated at 37  C for 30 min. After incubation, 50% acetic acid was added to stop the reaction and the optical density of the products was read by a plate reader (SunriseÔ, Tecan) at 405 nm. If the reading was larger than the highest calibration point (0.5 EU ml
1), sample was re-analyzed after dilutions with the LRW; 2e4 times for fine mode samples and 10e40 times for coarse mode samples. LRW and blank filter papers served as blank controls with readings well below 0.01 EU ml
1, the lowest detection limit, was also included in each batch of test. About 5% of the samples were duplicated for LAL analyses and the difference between the samples readings were all within 5% agreement. The concentrations of endotoxin in filter samples were calculated by comparing the optical density with the standard and expressed as endotoxin unit (EU) per m3 of sampled air. 2.3. Chemical analysis for total endotoxin The total endotoxin was calculated from the 3-OHFAs on the filter paper analyzed chemically using GCeMS. Filter samples (both fine and coarse modes) were analyzed following Lee et al.’s (2004) procedures. In short, one-eighth of each filter sample was first hydrolyzed in 4 M methanolic HCl at 100  C for 18 h. During the hydrolysis, the cell structure of bacterial cell membrane was broken

5

down and the dissociated 3-OHFAs reacted with the acidified methanol to form 3-hydroxy fatty acid methyl esters (3-OHFAMEs). The sample was then extracted with hexane twice and purified with a disposable silica gel column (100 mg/1.5 ml, Grace). Prior to GCeMS analysis, the sample was derivatized with 70 ml of N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA, Sigma) and 10 ml of pyridine at 80  C for 15 min. A gas chromatography (Agilent 7890A) with a fused-silica column (DB-5MS, 30 m length, 0.25 mm id, 0.25 mm film thickness, J&W Scientific) connected to a mass spectrometer (Agilent 5975C) was used to quantify the derivatized 3OHFAs with helium as the carrier gas. Methyl tridecanoate-D25 was used as an internal standard. Unlike the LAL bioassay which reflects only those bioactive portions leading to the coagulation of the enzyme, the chemical approach extracts all the endotoxins (total endotoxin hereafter) attached to the filter samples. The total endotoxin was calculated by assuming that each endotoxin molecule contained 4 3-OHFA molecules (Wilkinson, 1988) and the molecular mass of endotoxin was 8000 (Mielniczuk et al., 1993). The concentration of endotoxin in each sample was expressed as nanogram endotoxin per cubic meter of sampled air. 2.4. Statistical analysis The data collected were statistically analyzed using SPSS Forecasting 17.0 and R. As seasonal variation in airborne bacterial loading is expected, the “time series modeler” was used to remove outliers. To examine time trends in relation to endotoxin levels graphically, cubic spline smoothing was applied. Since the collected data were not normally distributed, ManneWhitney test was used when investigating the spatial variation in endotoxin. In all statistical testings, differences were considered significant when p-value was <0.05. Spearman’s partial correlation was used to study the relationships between bioactive endotoxin and individual 3-OHFAs yielded from the chemical analysis. 3. Results and discussion 3.1. Bioactive endotoxin from the LAL assay As there may be experimental error during the analysis, additive outliers were removed by the time series modeler in SPSS Forecasting 17.0. After removing, there were 63, 31 and 29 valid datasets for NS, GZ and HK correspondingly. The average bioactive endotoxin levels in PM10 were 0.345, 0.386 and 0.350 EU m
3 at the NS, GZ and HK sites respectively (Table 1). Among them, averages of 68%, 59% and 66% of endotoxin were found in PM2.5-10 at the NS, GZ and HK sites, respectively. The amounts of endotoxin reported in this study are comparable with those reported in other countries using similar LAL assay (Table 2). Table 2 shows that the bioactive endotoxin levels were low in Munich (Morgenstern et al., 2005) and Stockholm (Nilsson et al., 2011), the two cities with continental climate and a relatively lower average temperature. The bioactive endotoxin levels in PM10 in PRD cities were comparable with those

Table 1 Bioactive endotoxin levels at different locations quantified using the LAL assay (unit: EU m
3). Fine Max Nansha (NS) Guangzhou (GZ) Hong Kong (HK) Significant difference a

Coarse Min

0.247 0.012 0.310 0.030 0.275 0.018 GZ > NS & HKa

>: p-value in ManneWhitney test <0.05.

PM10

Average

Max

Min

0.085 0.148 0.099

1.326 0.020 0.890 0.003 1.121 0.012 No sig. difference

Average

Max

0.266 0.261 0.248

1.479 0.033 1.017 0.106 1.245 0.080 No sig. difference

Min

Average 0.345 0.386 0.350

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

Table 2 Summary of atmospheric bioactive endotoxin measured in different studies.

0.2

01-Apr-09

01-Mar-09

01-Feb-09

01-Jan-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Aug-08

01-Jul-08

01-Jun-08

0.0 01-May-08

1.2

Guangzhou

1.0

0.8

0.6

0.4

0.2

01-Apr-09

01-Mar-09

01-Feb-09

01-Jan-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Aug-08

01-Jul-08

01-Jun-08

0.0

1.4 1.2

Hong Kong

1.0 0.8 0.6 0.4 0.2

01-Apr-09

01-Mar-09

01-Feb-09

01-Jan-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Aug-08

01-Jul-08

0.0 01-Jun-08

in California (Mueller-Anneling et al., 2004) and Turin (Traversi et al., 2010), the two cities that share a similar annual temperature range with the PRD. Despite lower endotoxin levels in Munich and Stockholm, both Morgenstern et al. (2005) and Nilsson et al. (2011) found that more bioactive endotoxins were associated with PM2.5-10 as reported in this study. ManneWhitney tests showed that there was no significant spatial variation in bioactive endotoxins in PM2.5-10 and PM10. However, GZ showed a significantly higher concentration of endotoxins in PM2.5 than NS and HK (p < 0.05). Although vegetation in rural areas is a main source of bacterial endotoxin, the results of this study show that the source strength of vegetation in NS may be much weaker than that of other sources in urban GZ. Potential sources of endotoxin in urban areas include kitchen emission (Morgenstern et al., 2005) and cigarette smoking (Sebastian et al., 2006), which are mainly PM2.5. Waste handling/ treatment is another source of endotoxin in urban areas (Liebers et al., 2006). Although these sources can also be found in NS, the amounts should be much lower than those in GZ due to the lower population. It is also interesting to note that even though HK is a much more populated city (at 6400 people km
2), it had a significantly lower bioactive endotoxin in PM2.5 than GZ. This is probably due to the different compositions of aerosols at two sites. LAL assay is a sensitive method and as there was no spike control in this study, inhibition or enhancement in the analysis could be occurred in the presence of some substances, such as divalent cations (Sullivan and Watson, 1974). Presence of beta-1,3-glucan in atmospheric fungal spores would also lead to overestimation of endotoxin. Further investigation is required. The atmospheric bioactive endotoxin in GZ also showed a different seasonal pattern compared with that in NS and HK (Fig. 2). It was found that the bioactive endotoxin concentration in GZ increased with the onset of spring through summer and decreased in fall, while the concentrations in NS and HK only started to increase in summer and remained high in winter. Carty et al. (2003) also found that endotoxin in PM2.5 increased with the temperature, which according to them was due to the increased growth of bacteria and plants. Although NS and HK share similar climate conditions to GZ, the increase in bioactive endotoxin concentration occurred later in these two cities than in GZ. Factors beyond the climatic (e.g. temperature) and the substrate (e.g. vegetation), such as differences in the natural assemblage of the bacterial fauna and possibly in the growth rates of the assemblages, lead to variations in the annual pattern of the airborne endotoxin level in the three cities. The Dutch Expert Committee on Occupational Safety and Health (DECOS) has recommended an occupational exposure limit of 50 EU m
3, based on the study of Castellan et al. (1987) which

0.4

01-May-08

Hong Kong, China

0.6

01-May-08

Guangzhou, China

0.8

01-Apr-08

Turin, Italy Nansha, China

1.0

01-Apr-08

California, USA

1.2

01-Mar-08

0.42 0.085 0.345 0.148 0.386 0.099 0.350

Munich, Germany

Nansha

1.4

01-Apr-08

PM10 PM2.5 PM10 PM2.5 PM10 PM2.5 PM10

Nilsson et al. (2011) Morgenstern et al. (2005) Mueller-Anneling et al. (2004) Traversi et al. (2010) This study

01-Mar-08

0.015 0.05 0.019 0.081 0.44

01-Feb-08

PM2.5 PM10 PM2.5 PM10 PM10

01-Feb-08

Stockholm, Sweden

1.6

01-Feb-08

Reference

Biologically active endotoxin (EU/m 3)

Endotoxin level (EU m
3)

Biologically active endotoxin (EU/m 3)

Size fraction

Biologically active endotoxin (EU/m 3 )

Location

01-Mar-08

6

Fig. 2. Smoothed plots of the atmospheric bioactive endotoxin in PM10 at the three sampling sites over the sampling period. Dotted lines represent the 95% confidence intervals of the smoothing spline.

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

showed a no-effect-level for healthy subjects in a 6-h work exposure. When an exposure limit for the general population is to be determined, it should take susceptible individuals and longer exposure periods into account. For PM10, the exposure limit suggested by the Occupational Safety and Health Administration in the United States is 5000 mg m
3. This is 100 times the guideline for exposure for the general population suggested by the World Health Organization. If the guideline for exposure to endotoxin for the general population is to be 100 times more stringent, a level of 0.5 EU m
3 is expected based on the DECOS limit. The measured bioactive endotoxin levels reported in this work (Table 2) and some other studies are in fact close to 0.5 EU m
3. The general exposure to ambient endotoxins is thus worth further in-depth investigations from a public health perspective. 3.2. Total endotoxins resulting from chemical analysis Fig. 3 shows the relative abundance of different 3-OHFAs in PM2.5-10 and PM2.5. It was found that the 3-OHFA with 16 carbon atoms (C16 3-OHFA) was the most abundant 3-OHFA species in the atmospheric endotoxin (w25%), followed by C9 3-OHFA. It was also

7

found that the compositions of different 3-OHFAs were similar at all sites despite large variations in the relative abundance of C9, C10 and C16 3-OHFAs. The total endotoxin was calculated from the amounts of individual 3-OHFAs and is reported in Table 3. Although chemical analysis had been applied previously to quantify the amount of airborne endotoxin, most of these studies focused on the indoor environments, such as animal houses (Pomorska et al., 2007) and aircraft cabins (Hines et al., 2003). Only a limited number of studies reported the atmospheric concentration for comparison (Lee et al., 2004). Fox et al. (2005) and Liu et al. (2000) measured the airborne 3-OHFAs in total suspended particulates in schools and found that the outdoor airborne endotoxin concentration, by summing up the amounts of endotoxin from all the 3-OHFAs with an even number of carbon atoms, was 20e30 ng m
3. This is highly comparable with the mean concentrations measured in the current study (NS: 29.4 ng m
3; GZ: 32.7 ng m
3; HK: 17.4 ng m
3) with all 3-OHFAs (i.e. those with an odd number of carbon atoms and those with an even number of carbon atoms) included. It was also found that GZ and NS showed no significant difference in total endotoxin concentration in PM10, but both showed

Fine particles

40

Nansha

relative abundance (%)

35

Guangzhou

30

Hong Kong

25 20 15 10 5 0

C9

C10

C11

C12

C13 C14 3-OHFAs

C15

C16

C18

Coarse particles

40

Nansha

35 relative abundance (%)

C17

Guangzhou

30

Hong Kong

25 20 15 10 5 0

C9

C10

C11

C12

C13 C14 3-OHFAs

C15

C16

C17

C18

Fig. 3. Relative distribution of different 3-OHFAs in atmospheric aerosols collected at Nansha, Guangzhou and Hong Kong. No significant differences were found among the three sites with respect to individual carbon chain 3-OHFAs (ANOVA, p > 0.05). C9eC18 denotes the carbon chain length of the 3-OHFAs.

8

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

Table 3 Total endotoxin levels at different locations quantified using chemical analysis (unit: ng m
3). Fine Max Nansha (NS) Guangzhou (GZ) Hong Kong (HK) Significant difference a

Coarse Min

59.22 1.01 67.55 9.27 35.33 0.16 GZ > NS > HKa

PM10

Average

Max

Min

22.17 26.78 14.90

31.82 0.84 12.60 2.67 6.31 0.19 NS > GZ > HKa

Average

Max

6.76 5.89 2.80

91.04 1.85 75.66 12.18 41.64 0.35 NS & GZ > HKa

Min

Average 29.38 32.68 17.39

>: p-value in ManneWhitney test <0.05.

significantly higher concentration than HK (ManneWhitney tests, p < 0.05, Table 3). Similar to the results of bioactive endotoxin in PM2.5, GZ showed significantly higher total endotoxin concentration in fine aerosols than NS and HK (ManneWhitney tests, p < 0.05). The average total endotoxin concentration in PM2.5 in GZ was 26.8 ng m
3 compared with 22.2 and 14.9 ng m
3 in NS and HK respectively (Table 3). Fine mode endotoxins were mainly originated from combustion, such as cigarette smoke (Sebastian et al., 2006) and kitchen emission (Lee et al., 2007). Most of these endotoxins were biologically inactive (compare Table 3 with Table 1) as the high temperature during combustion inactivated the endotoxins (Tsuji and Harrison, 1978). Hasday et al. (1999) found that about 99% of endotoxins in cigarettes become inactive after burning. On the contrary, NS showed significantly higher total endotoxin concentration in PM2.5-10 than GZ, which in turn showed significantly higher concentration than HK. This indicates that in NS, more of the endotoxins are from natural sources. This is in agreement with the larger vegetation (and soil) coverage in NS compared with the other two sites. The significantly lower total endotoxin levels of all size fractions in HK may be due to the better hygiene practice and the fewer natural sources. Unlike bioactive endotoxin, no clear seasonal variation in total endotoxin was found at the NS and GZ sites. However, it was observed (Fig. 4) that the total endotoxin concentrations were lower from June to August at all sites. This concurred with the lower ambient concentration of particulate matter in the PRD region during the same period (Guangdong Provincial Environmental Monitoring Centre, 2009). The lower level of particulate matter was due to heavier rainfall and higher mixing layer height in these summer months, which favored the deposition and dispersion of pollutants. The relatively clean maritime air stream that prevailed in the region during summer time under the influence of the southern monsoon also accounted for the lower level of particulate matter. Hence, the total and the bioactive endotoxin adhered to the particulate matters will be deposited or dispersed along with other particulates during the summer months. It is interesting then to note that the lower summer trend in the total endotoxin (Fig. 4) is in contrast with the higher summer trend in the bioactive endotoxin (Fig. 2). This apparent discrepancy can be accounted for by the higher loading of endotoxin per unit mass of particulates in the summer time. Although the mass concentration of PM10 was not measured in this study as quartz filters were used for the bioassay or chemical analysis, PM2.5 was collected on Teflon filters using mid-volume samplers during the same sampling periods, and the mass concentrations of PM2.5 were measured (data not shown). The bioactive endotoxin contents in PM2.5 were calculated (Table 4) and the results showed that the average bioactive endotoxin content in PM2.5 from June to August was about four times that during the rest of the year in NS and GZ. This agrees with a study conducted by Traversi et al. (2010) who reported that the endotoxin content in PM10 in summer was triple that in winter. It has been reported that the bioactive endotoxin per mass of particulate matter was higher in summer (Carty et al., 2003; Heinrich et al., 2003). The ManneWhitney tests showed that the bioactive endotoxin content

in PM2.5 was significantly higher from June to August in NS and GZ (p < 0.05) in this study as well. The higher bioactive endotoxin loading can be explained by the increased growth of bacteria and plants during the warmer months, augmenting the source strength of the bioactive endotoxin enough to offset the wind dispersion and deposition or washing effects due to rains. 3.3. Comparing between LAL assay and chemical analysis Large differences were found between the results of endotoxin concentration measured using the LAL assay and chemical analysis, or between the bioactive and total endotoxin. Benchmarking with the reference standard endotoxin from E. coli O113:H10 and the equivalence of 1 ng of endotoxin to 10 EU, the average endotoxin level measured using the LAL assay was about 0.04 ng m
3 in PM10. This is about three to four orders of magnitude smaller than the 20e30 ng m
3 in PM10 measured using the chemical approach. Large discrepancies between LAL assay and chemical analysis have also been reported in the literature. Sonesson et al. (1990) found 10e50 times more 3-OHFAs in dust from slaughterhouses than could be accounted for using LAL assay. Similar differences were also found in settled house dust by Park et al. (2004) if the molecular weight of 8000 was assumed for endotoxin. The discrepancy is partly due to the fact that the LAL assay does not measure all endotoxins. Most of the endotoxins, including the bioactive ones, may not be exposed at the bacterial surface due to their intact configuration and therefore unavailable to react with the Limulus enzymes. Hence, the shed and loose endotoxins are more sensitive to the LAL assay than the intact cells (MattsbyBaltzer et al., 1991). The chemical analysis, on the other hand, dissociates all endotoxins, regardless of whether they are cell-wall-associated, shed or attached to other organic molecules, through the hydrolysis reactions. This obviously yields much higher levels of total endotoxins. Enzymatic reactions however usually require complementary configuration; the complete dissociation of the endotoxins from the cell membrane in the chemical hydrolysis will also destroy the spatial configuration of the endotoxin associated with the intact membrane, rendering a lower bioactive potency to the LAL enzymatic reactions. A deeper understanding of these factors to generalize the relationship between total and bioactive endotoxin should be sought. The bioactive and total endotoxin measured also showed different size distribution patterns. About 60% of bioactive endotoxins quantified using the LAL assay were associated with PM2.5-10, while about 80% of total endotoxins resulted from the chemical approach were found in PM2.5 (Table 3). PM2.5-10 originates mainly from natural sources (Seinfeld and Pandis, 1998), such as soil dust and vegetation. The results indicate that bioactive endotoxins were more associated with natural sources, which are composed of intact or fragmented bacterial cells with the endotoxin moieties on their membranes well preserved to give their bioactive potency. On the contrary, large amounts of the total endotoxins were associated with the PM2.5, which may likely originate from anthropogenic

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

Table 4 The bioactive endotoxin content in PM2.5 at different sampling sites (unit: EU mg
1 PM2.5).

100

Nansha

Average bioactive endotoxin content in PM2.5 (maxemin)

Total endotoxin (ng/m3)

80 60

NS GZ HK

20

01-Apr-09

01-Feb-09

01-Mar-09

01-Jan-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Aug-08

01-Jul-08

01-Jun-08

01-May-08

01-Apr-08

01-Mar-08

01-Feb-08 80 70 Total endotoxin (ng/m3)

JuneeAugust

Rest of the year

7.03 (1.25e12.93) 9.18 (3.50e15.01) 3.18 (1.95e5.15)

1.59 (0.26e10.32) 2.05 (0.39e6.17) 3.40 (0.56e12.55)

40

0

Guangzhou

60 50 40 30 20 10

01-Apr-09

01-Mar-09

01-Jan-09

01-Feb-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Aug-08

01-Jul-08

01-Jun-08

01-Apr-08

01-May-08

01-Mar-08

01-Feb-08

0

50

Hong Kong 40 Total endotoxin (ng/m3 )

9

30

20

10

combustion processes. Although the combustion processes may help break down or release the endotoxin moieties on the bacterial membranes, the heat may inactivate the bioactive potency of the endotoxins, as discussed in the previous section, giving a low bioactive response in PM2.5 as reported in the LAL assay. The low bioactive endotoxin in PM2.5 may also be due to their strong adsorption on the particle surfaces (Sebastian et al., 2006), so that they are unavailable for the enzymatic reaction in the LAL assay. Although endotoxins in both PM2.5 and PM2.5-10 can be adsorbed, PM2.5 provides a larger surface-area-to-volume ratio for adsorption. Hence less endotoxin was found in PM2.5 when the LAL assay was used. Non-parametric correlation analysis between total endotoxin and bioactive endotoxin were conducted in this study. However, results showed that bioactive potential cannot be assessed simply from the total endotoxin levels as significant correlation could not be found at any of the sites. The poor correlation may be due to the fact that endotoxins with different molecular structures or carbon chain lengths have different LAL activation capability (Kabanov and Prokhorenko, 2010). Lentschat et al. (1999) found that lipopolysaccharides with more heptose, hexose and phosphate residues were more biologically active. Rietschel et al. (1994, 1993) also reported that differences in the substituent groups and the composition of fatty acids in lipids A led to different bioactivities. Therefore correlation analyses between the bioactive endotoxin and individual 3-OHFAs were further conducted in this study. Results of Spearman’s partial correlation showed that the bioactive endotoxin in PM2.5 at the NS site was correlated with none of its 3-OHFA content, while the bioactive endotoxins in PM2.5 at the GZ and HK sites were correlated with the C18 and C15 3OHFAs, respectively (Table 5). For the PM2.5-10, bioactive endotoxin was positively correlated with the C10, C14 and C16 3-OHFAs at the NS site, and with the C12 3-OHFA at the GZ site. It was also negatively correlated with the C9 and C18 3-OHFAs at the NS site, with the C15 3-OHFA at the GZ site and with the C10 3-OHFA at the HK site. Previous studies showed that the correlation between the bioactive endotoxin level and the 3-OHFA concentrations varies with the nature of the sample. Endotoxin in house dust correlated best with the C10, C12 and C14 3-OHFAs (Saraf et al., 1999), while

Table 5 Different 3-OHFAs significantly correlated with the bioactive endotoxin at different sampling locations.

0

01-Apr-09

01-Mar-09

01-Feb-09

01-Jan-09

01-Dec-08

01-Nov-08

01-Oct-08

01-Sep-08

01-Jul-08

01-Aug-08

01-Jun-08

01-May-08

01-Apr-08

01-Mar-08

01-Feb-08

Fine

Fig. 4. Smoothed plots of the total atmospheric endotoxin in PM10 at the three sampling sites over the sampling period. Dotted lines represent the 95% confidence intervals of the smoothing spline.

Coarse

Location

3-OHFA

Correlation coefficient

p-value

GZ HK NS

C18 C15 C9 C10 C14 C16 C18 C12 C15 C10

0.388 0.395
0.560 0.504 0.521 0.487
0.439 0.424
0.650
0.463

<0.05 <0.05 <0.0001 <0.0001 <0.0001 <0.0001 <0.0005 <0.05 <0.0001 <0.05

GZ HK

10

J.Y.W. Cheng et al. / Atmospheric Environment 47 (2012) 3e11

endotoxin in aircraft seat dust correlated best with the C16 3-OHFA followed by the C14 3-OHFA (Hines et al., 2003). In agricultural dust, endotoxin correlated with the C12 and C14 3-OHFAs but not with the C16 3-OHFA (Reynolds et al., 2005). Results of the current study showed that the correlations between bioactive potential and different 3-OHFAs also varied with the sampling sites. This may be due to the different assemblages of bacteria with different compositions of 3-OHFAs associated with different localities. The results reported in this work and a few other studies demonstrate that there is as yet no general trend. More studies are needed, particularly on the bioactive potencies of different endotoxins, in addressing their impact on public health. 4. Conclusions The quantities, size distribution and seasonal variations of endotoxin in the PRD measured using the LAL assay were much lower than those measured using 3-OHFAs as biomarkers. While the LAL bioassay provided direct information on the bioactive potential of the endotoxins, it did not account for the presence of biologically active yet unexposed endotoxins. These endotoxins are not exposed to the Limulus enzymes at the surface of the outer membrane but can still cause endotoxic effects when inhaled. Therefore, any assessment using the LAL assay would underestimate the potential health effects caused by airborne endotoxins. The alternative approach is to analyze chemically the biomarker, 3-OHFAs. This approach provides the total endotoxin level which is 3e4 orders of magnitude higher than that available from the biological LAL assay as a result of the complete dissociation of the 3-OHFAs from the membrane through the hydrolysis reactions. Data from this study show that the composition of 3-OHFAs with respect to different carbon chains varies with localities, and 3-OHFAs with different carbon chain lengths may have different endotoxic potencies. Although the bioactive endotoxins were correlated with some of the 3-OHFAs in this study, the relationships appear to be site- and size-dependent. No general relationship can be attained yet. The comparison of the LAL bioassay and the chemical analysis on the 3-OHFA biomarker in this study suggests that further investigation on the potencies of endotoxins with 3-OHFAs containing different carbon chain lengths is required if the health implications are to be truly revealed. Acknowledgments This study is financially supported by the Hong Kong Research Grant Council (Project Number 614808) and the FYT Graduate School (Project ARC06/07.SC01), HKUST. The authors are also grateful to Professor Jianzhen Yu of Division of Environment and Department of Chemistry, the Hong Kong University of Science and Technology for sharing the PM2.5 mass concentrations. The authors are grateful to the reviewers’ comments and suggestions. References Carty, C.L., Gehring, U., Cyrys, J., Bischof, W., Heinrich, J., 2003. Seasonal variability of endotoxin in ambient fine particulate matter. Journal of Environmental Monitoring 5, 953e958. Castellan, R.M., Olenchock, S.A., Kinsley, K.B., Hankinson, J.L., 1987. Inhaled endotoxin and decreased spirometric values. An exposure-response relation for cotton dust. New England Journal of Medicine 317, 605e610. Douwes, J., Thorne, P.S., Pearce, N., Heederik, D., 2003. Bioaerosol health effects and exposure assessment: progress and prospects. Annals of Occupational Hygiene 47, 187e200. Fox, A., Harley, W., Feigley, C., Salzberg, D., Toole, C., Sebastian, A., Larsson, L., 2005. Large particles are responsible for elevated bacterial marker levels in school air upon occupation. Journal of Environmental Monitoring 7, 450e456. Guangdong Provincial Environmental Monitoring Centre, 2009. Pearl River Delta Regional Air Quality Monitoring Network e A Report of Monitoring Results in 2008.

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