Congener-specific determination of polybrominated diphenyl ethers in biosolids by GC/HRSIRMS

William C.Brumley1,2*, Mohammad A.Mottaleb2 1P.O.Box-93478, Las Vegas, NV 89193-3478, (U.S.A.) 2Baylor University, Department of Chemistry and Biochemistry-97348, Waco, TX 76798-7348, (U.S.A.) E-mail: wbrumley2000@yahoo.com


Abstract
Biosolids from eight municipal sewage treatment plants were studied from the standpoint of determining PBDE congener target analytes in each sample. Targeted analytes were subjected to high specificity through the use of high resolution selected ion recording mass spectrometry (HRSIRMS). Three ions from the molecular ion cluster of each congener group were monitored at 10000 resolution and PCB#204 was used as the internal standard. The seven congeners determined ranged from low tens of microg/kg to several mg/kg in each sample. The general presence of PBDEs indicates that biosolids serve as an environmental sink for these compounds, and the application of biosolids to land and other uses must take into consideration the presence and fate of these compounds.


INTRODUCTION

Biosolids (sewage sludge) represent the end product of bacterial digestion and applied treatments of raw sewage in a municipal sewage treatment facility[1]. The precipitated material from the aqueous solution consists of a relatively intractable mass made up of both inorganic and organic substances that have reached an environmental sink. The complete characterization of this material remains unfinished but studies by various workers have increased our knowledge about this material[2].

In recent times the interest has shifted from disposal of the biosolid material in landfills to the preferred use of processed material as a soil amendment[3]. The question of course arises as to the potential exposure to biota that would result from such an application in the environment as well as effects on human health that might arise from eating crops exposed to the amended soil by way of animals eating such crops before going to market or inhaling dust from such applications. Additionally, concern arises from any appearance of substances from biosolids entering into groundwater or ultimately into drinking water due to leachate[4].

The biosolids have usually undergone additional treatment to destroy pathogens either by heat or irradiation after the considerable bacterial degradation and additional processtreatment that has taken place. Thus, the biosolids are designated Class A or Class B depending on the extent of elimination of pathogens[5]. Some proponents advocate the additional step of composting to remove/degrade the remainder of the objectionable compounds that are currently known to reside in biosolids[6].

A number of papers have now filled in some of our questions about the types of contaminants in biosolids. The review by Rogers[1] included mention of a variety of compounds including organochlorine pesticides(e.g., aldrin), PCBs, and chlorophenols. A large presence for nonyl phenols and surfactants was also mentioned. The large and diverse class of pharmaceuticals and personal care products (PPCPs) was also mentioned and most of these compounds (e.g., antibiotics) would be found in the polar fraction of sludge components. The synthetic musks are one group of PPCPs that partition with the liphophilic fraction. Methods were given with extraction and cleanup procedures also included.

Oberg et al. described the occurrence of PBDEs in over 100 sludge samples from Sweden where the predominant tetra, penta, and hexa congeners were found as well as the decabromodiphenyl ether[7]. Ying and Kookana pointed out that high levels of triclosan in biosolids could be a concern in soil applications[8].Synthetic musks were determined in biosolids[9]. Nonylphenols, pthalates, and PCBs were determined in biosolids and soil in an effort to follow the fate of such contaminants after soil amendment using biosolids[10].

A number of papers have focused on the polar analytes (contaminants) found in biosolids. Giger et al. reported methodology for extraction and determination of antibiotics includingthe fluoroquinolone antibiotics. Extraction difficulties and relatively low recoveries were noted[11] and this contrasts with quantitative recovery of nonpolar compounds. Mottaleb and Brumley reviewed the separations used in determining PPCPs in a variety of environmental matrices[12].

There are two EPA methods relevant to our approach. Method 1668a is for PCBs and uses three ions from the molecular ion clusters for monitoring. Method 1614 for PBDEs uses two ions per molecular ion cluster. In both cases, quantitation is by isotope dilution so that extensive use is made of stable isotope-labeled compounds[13,14].

In this work we report using a new method involving GC/HRSIRMS in a survey of the occurrence of PBDE congeners in biosolid samples from eight municipal treatment facilities in the U.S.


EXPERIMENTAL

Chemicals
PBDE congeners were obtained from Accu standard (8 congener mix BDE-CM: 28, 47, 99, 100, 153, 154, 183, 209). PCB#204 was obtained from Chem Service.

Samples
Samples were obtained from eight municipal treatment facilities and stored in glass bottles in a freezer. Several gram portions were taken and air dried in a hood. The dried material was pulverized in a ball mill grinder (Reutsch) at 20 Hz for 2 min using a glass ball and glass-lined sample vessel.

Mass spectrometry
GC/HRSIR: A Waters-MicroMass AutoSpec Premier (P) was operated at 10000 resolution in EI mode (500 microA trap current, 250 degC source, high boiling PFK calibrant, 8kV accelerating voltage, 350 V photomultipler detector). Software was MassLynx 4.1.

Method for PBDEs Extraction/Cleanup
The method uses a SW-846 approved extraction (Method 3545A)[15] of BS in methylene chloride/acetone (50/50, v/v) by pressurized liquid extraction (Dionex ASE 200 80 degC, 15 min static, 100% purge, two extractions per sample). The extract is concentrated and then fractionated on silica using SPE to isolate the fraction containing the PBDEs. A 3 ml Si SPE cartridge(Phenomenex)is washed with hexane;sample is applied in 1ml of hexane and then the sample is eluted with 2 ml hexane, 2 ml hexane/methylene chloride (50/ 50 v/v), 2 ml methylene chloride, and 2 ml acetone. The PBDEs are found in the hexane fraction and hexane/methylene chloride (90/10 v/v) fractions which are combined. Typical sample size was 0.5 g and the final volume was 1ml with 100 microL ofinternal standard added (100 pg/microL). The average recovery for this method was published previously[16].

GC/HRSIR
A 30 m 0.25 micron film 0.25 mm ID column (DB5 Agilent-JandW) was used with temperature programming: 60 degC for 1 min followed by 60-300@20 degC/min. SIR (30 msec dwell) divided into three retention time groups: Gp 1: 403.80470, 405.80265, 407.80061, 416.97063 (PFK lock mass), 429.76057 (IS), 483.71385, 485.71112, 487.70907; 5 to 13.6 min; Gp 2: 561.62367, 563.62163, 565.61958, 566.96642 (PFK lock mass), 641.53214, 643.53009, 645.52805; 13.70 to 18.0 min; Gp3: 719.44265, 719.44265 (PFK lock mass), 721.44060, 723.43856; 18.1 to 25.0 min.

RESULTS AND DISCUSSION

Confirmation of identity of PBDEs
The seven congeners included in this studyare the most common congeners determined in environmental analysis.The retention times are given in TABLE 1 for the congeners. The compounds range from tribromodiphenyl ethers to heptabromodiphenyl ether congeners, and they were addressed using three retention time groups (tri-BDE and tetra-BDE, pentaBDE and hexa-BDE, and hepta-BDE).

Confirmatory requirements are addressed using three ions from the molecular ion cluster and their relative abundances. The supporting information from the presence of additional congeners strengthens the conclusion that we are dealing with the polybrominated diphenylethers. The additional selectivity of 10000 resolution adds to our certainty as to the elemental composition, and the congener is established with the agreement of the retention time observed in sample extracts with those of standards. HRSIRMS is more selective than low resolution MS[16].


TABLE 1: Retention times of PBDE congeners in min
Congener No RT
28 12.03
47 13.11
99 14.36
100 14.02
153 16.07
154 15.36
183 18.66
IS 12.93


TABLE 2: Congener levels in biosolid samples from each of eight treatment facilities determined in triplicate (microg/kg)
Congener No123 4567 8
2843.1 55.2 8.96.3 9.8 17.56.9 21.8
471677 1627 570 284376 9201299 503
99 2471 2123652285 466 121.8 324 809
100 547 465491 60.0102 30870.8 189
153179 174 54.118.4 33.684.518.3 48.8
154142 146 46.415.7 25.873.1 17.442.2
18320.9 33.2 16.417.8 10.0 26.98.0 16.1


Biosolids analyses The analyses of the eight municipal treatment facilities were carried out in triplicate and the results are given in TABLE 2. Levels of PBDEs ranged from low microg/kg to hundreds of microg/kg or to low mg/kg levels. Congeners 47 and 99 were always found as the highest concentration contaminants among the congeners. The individual analyses of each of the biosolids samples showed some significant variations. It is suspected that biosolids material itself is highly heterogeneous so that these results must be viewed as representative but could also vary greatly as samples differ on various time scales of sampling.

Considerable variation is sometimes observed in the results for the three sampling set and this is attributed primarily to heterogeneity in the biosolids themselves.


TABLE 3: Precision for determination of a single extract (sample #3, third replicate)
Congener #microg/kg / %RSD
289.84 / 1.1
47591 / 6.5
99729 / 4.6
100165 / 4.1
15360.3 / 6.0
15450.3 / 4.4
18320.0 / 6.5


TABLE 4: Supplemental data showing all three determinations of congener levels in biosolids from eight treatment facillities determined in triplicate (three separate portions of dried, groundmaterial taken through the extraction, cleanup, and final separation/determination independently) in microg/kg.
Sample#6 Congener# 1 2 3 4 5 6 7 8
28 33.1 59.1 8.3 6.3 10.4 19.7 6.4 21.9
28 28.3 64.3 8.8 7.2 9.4 17.9 7.9 19.0
28 68.0 42.1 9.7 5.5 9.7 14.8 6.5 24.4
47 1311 1747 576 273 391 1001 268 674
47 1161 1775 538 328 352 1038 362 568
47 2559 1359 595 252 386 722 268 826
99 1565 2333 578 288 532 1244 296 719
99 1390 2586 665 330 486 1620 403 630
99 4458 1451 713 237 381 791 272 1079
100 362 491 130 61.4 117 328 62.9 168
100 311 568 155 69.2 105 378 88.4 147
100 968 330 162 49.4 83.4 218 61.0 251
153 115 170 49.1 17.6 42.2 91.4 18.7 46.5
153 107 240 54.5 21.7 32.5 108 22.9 36.8
153 317 112 58.7 15.8 26.0 54.2 13.3 63.1
154 99.2 143 42.6 15.6 29.8 79.9 16.3 39.1
154 91.1 196 47.2 17.6 24.8 88.0 21.4 32.9
154 236 98.1 49.3 13.9 22.8 51.3 14.5 54.5
183 10.9 27.2 14.6 12.7 11.3 28.7 7.9 14.9
183 17.4 50.3 15.6 11.4 10.0 32.4 9.3 12.9
183 34.4 22.1 19.1 29.4 8.7 19.7 6.8 20.6


The reproducibility of a single determination is given in TABLE 3 for sample #3 (#3 replicate). This establishes a good estimate of precision for the single extract and can be compared to the variation found among replicate extractions of the same sample if so desired.

The precision of determination may not be as good as for isotope dilution studies where perhaps 2% precision or better may be obtained, but the RSDs are quite acceptable for this simpler and less costly approach. Supplemental data in TABLE 4 show the three independent determinations for each congener for all eight municipal sitesand indicate sample heterogeneity.

The implications of these contaminant levels centers upon concern with the possible introduction of these contaminants, that have reached an environmental sink, back into the environment as a result of the application of biosolids to landfills or agricultural use. Leachate may also represent an additional path backinto environmental transport processes.

Data examples
The following figures 1-3 illustrate the response for retention time groups 1, 2, and 3 respectively in sample 3 (third independent analysis). In general, the specificity is high for determining the congeners in biosolids despite the complexity of the matrix[16].There are an enormous number of compounds present in this matrix, and this remains a fair characterization of the lipophilic fraction itself. The PBDEs exhibit some advantages because of their negative mass defect (high resolution monitoring thus eliminates many compounds with positive mass defect) and their relatively high molecular weights and longerretention times. Nevertheless, interferences were observed in low resolution monitoring with at least one congener group. Negative ion approaches also represent greater selectivity as does HRMS, but specificity may be lacking for lower brominated congeners since there may be little or no molecular anion produced[17].



Figure 1: Group 1 total ion current responses (including m/z 405.80265 and m/z 485.71112 congener groups) showing congeners # at RT=12.05 (#28) and 13.15 (#47) and internal standard at RT=12.96.



Figure 2: Group 2 total ion current responses (including m/z 563.62163 and m/z 643.53009) showing congeners # at RT=14.06 (#100) , 14.40 (#99) , 15.40 (#154) , and 16.11 (#153).



Figure 3:Group 3 m/z 721.4406 ion current response showing congener #183 at RT=18.71.


Two figures below of mass spectra illustrate the agreement of spectra obtained from the monitored ions for a standard and for spectra obtained from sample extracts forcongener#99.Thetheoreticalrelative abundance for the most abundant ions of the molecular ion cluster is 51.2:100:97.8, and observed experimental values are within 2% of the calculated values.



Figure 4: Mass spectrum of a standard of congener #99.


Figure 5: Mass spectrum of congener #99 obtained from a biosolid sample 3 (replicate 3).


Detection of other congeners and specificity
In addition to the monitored congeners, additional responses attributable to PBDEs were observed within the group windows. Specifically, in the first group window additional responses were observed for tribromo-BDEs at RT=11.88 and 12.07 min., and tetrabromo BDEs were observed at RT=12.71 and 13.33 min. In the second group window an additional response was observed for a pentaBDE at RT=15.12 min and responses were observed for hexaBDEs at RT=15.45 and 16.15 min.

The silica gel cleanup affords an extract that has removed the more polar coextractives that include the fecal sterols and sterones[16]. This enhances the specificity of the method but still allows detection of additional congeners beyond the commonly occurring compounds. The additional congeners were estimated at levels below 100 ppb based on responses of similar congeners.

CONCLUSION

These results confirm the important contribution that PBDEs make to the contaminant levels of biosolids in a ubiquitous manner. Biosolids thus constitute an important environmental sink for PBDEs. The levels reported should enable risk assessors to evaluate the application of biosolids to land use in conjunction with the potential exposure of biota to these compounds. This method differs from Method 1614 by only using a single internal standard and thus is much cheaper to carry out, while it maintains greater confirmative power using three ions from the molecular ion cluster.

REFERENCES

[1] H.R.Rogers; Sci.Total Environ., 185,3 (1996).
[2] T.A.Ternes, N.Herrmann, M.Bonerz, T.Knacker, H.Siegrist, A.Joss; Water Res., 38, 4075 (2004).
[3] G.A.OConnor; Sci.Total Environ., 185, 71 (1996).
[4] M.J.La Guardia, R.C.Hale, E.Harvey, T.M.Mainor; Environ.Sci.Technol., 35, 4798 (2001).
[5] Guidance for Controlling Potential Risks to Workers Exposed to Class B Biosolids, Publication Number 2002-149, DHHS, NIOSH, CDC, July 2002, assessed at http://www.cdc.gov/ nio sh/ docs/ 2002149/ pdfs/ 2002-149.pdf on 05/16/2007.
[6] J.V.Fermante J.Meggan; Pollution Engineering, 29, 40 (1997).
[7] K.Oberg, K.Warman, T.Oberg; Chemosphere, 48, 805 (2002).
[8] G.G.Ying, R.S.Kookana; Environ. Internat., 33, 1099 (2007).
[9] L.I.Osemwengie; J.Environ.Monitoring, 8, 897 (2006).
[10] R.Gibson, M.J.Wang, E.Padgett, A.J.Beck; Analysis of 4-nonylphenols, phthalates, and polychlorinated biphenys in soils and biosolids, Chemosphere, 61, 1336 (2005).
[11] W.Giger, A.C.Alder, E.M.Golet, H.P.E.Kohler, C.S.McArdell, E.Molnar, H.Siegrist, M.J.F.Suter; Chimia, 57, 485 (2003).
[12] M.A.Mottaleb, W.C.Brumley; Trends Chromatogr., 2, 11 (2006).
[13] U.S.EPA Method 1668A (PCBs), accessed on 04/ 17/2008 at http://www.epa.gov/Region3/1668a.pdf.
[14] U.S.EPA Method 1614 (PBDEs), accessed on 04/ 17/2008 at http://www.epa.gov/waterscience/meth ods/method/files/1614.pdf.
[15] U.S.EPAMethod 3545A (Pressurized Fluid Extraction) of SW 846, accessed on 03/20/2008 at http:// www.epa.gov/sw-846/3 _series.htm.
[16] W.C.Brumley, K.E.Varner, L.A.Riddick; Environ. Science,An Indian J., 3(3), (2008).
[17] W.C.Brumley; Anal.Chem., Indian J., 7(7), 429 (2008).