ANALYSIS OF RIVER, BOREHOLE AND SWIMMING POOL

WATER BY COMPREHENSIVE GAS CHROMATOGRAPHY –

HIGH RESOLUTION TIME OF FLIGHT MASS SPECTROMETRY

Peter Gorst-Allman* and Yvette Naude#

*LECO Africa, Kempton Park, South Africa

(peter@lecoafrica.co.za)#University of Pretoria, Pretoria, South Africa

(yvette.naude@up.ac.za)

Drought Grips Southern Africa(Headlines)

• Southern Africa is in the grip of severe

drought, brought on by the recent El

Nino event (lowest rainfall in SA since

records began in 1904).

• This has placed enormous pressure on

water supplies across the country

• Many households are now forced to

collect water in buckets once a day

• Several municipalities are unable to

meet water demand

• Rivers and dams are at very low levels

Dirty water

• In 2011 the Council for Scientific and Industrial Research

reported that more than one third of 231 local

municipalities do not have the capacity to perform their

water sanitation functions

• The report warned that South Africa is heading for

disaster unless it tackles the problem of water pollution,

including its failing sewage treatment systems

• This is particularly problematic in rural areas

Present Study

• Obviously, South Africa faces serious water challenges.

With this in mind we have begun a study to examine water

quality, with particular emphasis on organic pollutants, in

rivers, dams, boreholes and swimming baths in South

Africa

Sterkfontein dam

See poster E22 on

display at 17H00 today

for new sampling

methodology.

Study Parameters

• Ten samples were collected from swimming pools, rivers and boreholes in the Pretoria and Mpumalanga areas of South Africa

• These constituted both rural and urban locations

• 1 liter samples were collected

• Extraction was performed by solid phase extraction (SPE) and by solid phase microextraction (SPME)

• The samples were analysed by Comprehensive Gas Chromatography – High Resolution Time of Flight Mass Spectrometry (GC×GC-HRT)

• The focus was on detecting endocrine disruptors (EDCs), and disinfection byproducts

What are Endocrine Disruptors?

Reference: http://www.epa.gov/endocrine/

• Endocrine disrupting chemicals (EDCs) have been defined as

exogenous agents that interfere with the production, release,

transport, metabolism, binding, action, or elimination of the natural

hormones in the body responsible for the maintenance of

homeostasis and the regulation of developmental processes.

More simply put…

• Endocrine disruptors are chemicals with the potential to interfere with

the function of endocrine systems, e.g. drugs, pesticides, polymer

additives, disinfection by-products, estrogen mimickers,

coatings materials, personal consumer products, industrial by-

products and miscellaneous pollutants

endrin 72-20-8 H01345 1985 Abalis IM, Eldefrawi ME, Eldefrawi AT. 1985. High-affinity stereospecific binding of cyclodiene insecticides and gamma-

hexachlorocyclohexane to gamma-aminobutyric acid receptors of rat brain. Pesticide Biochemistry & Physiology 24(1):95-

102.

endrin 72-20-8 H13732 2004 Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. 2004. Screening for estrogen and androgen receptor activities in

200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect 112(5):524-531.

environmental tobacco smoke n/a H17509 2006 Slotkin TA, Pinkerton KE, Seidler FJ. 2006. Perinatal environmental tobacco smoke exposure in rhesus monkeys: Critical

periods and regional selectivity for effects on brain cell development and lipid peroxidation. Environ Health Perspect

114(1):34-39.

epichlorohydrin 1-chloro-2,3-epoxypropane 106-89-8 H22563 1974 Cooper ERA, Jones AR, Jackson H. 1974. Effects of alpha-chlorohydrin and related compounds on the reproductive organs

and fertility of the male rat. J Reprod Fertil 38(2):379-386.

epichlorohydrin 1-chloro-2,3-epoxypropane 106-89870

endocrine

disruptors -8

H22614 1983 John JA, Quast JF, Murray FJ, Calhoun LG, Staples RE. 1983. Inhalation toxicity of epichlorohydrin: effects on fertility in rats

and rabbits. Toxicol Appl Pharmacol 68(3):415-423.

epichlorohydrin 1-chloro-2,3-epoxypropane 106-89-8 H08226 1983 Kluwe WM, Gupta BN, Lamb JC 4th. 1983. The comparative effects of 1,2-dibromo-3-chloropropane (DBCP) and its

metabolites, 3-chloro-1,2-propaneoxide (epichlorohydrin), 3-chloro-1,2- propanediol (alphachlorohydrin), and oxalic acid, on

the urogenital system of male rats. Toxicol Appl Pharmacol 70(1):67-86.

epichlorohydrin 1-chloro-2,3-epoxypropane 106-89-8 H08259 1989 Toth GP, Zenick H, Smith MK. 1989. Effects of epichlorohydrin on male and female reproduction in Long- Evans rats.

Fundam Appl Toxicol 13(1):16-25.

epichlorohydrin 1-chloro-2,3-epoxypropane 106-89-8 H08256 1990 Slott VL, Suarez JD, Simmons JE, Perreault SD. 1990. Acute inhalation exposure to epichlorohydrin transiently decreases

rat sperm velocity. Fundam Appl Toxicol 15(3):597-606.

EPN ethyl p-nitrophenyl

benzenethionophosphonate

2104-64-5 H13732 2004 Kojima H, Katsura E, Takeuchi S, Niiyama K, Kobayashi K. 2004. Screening for estrogen and androgen receptor activities in

200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ Health Perspect 112(5):524-531.

epofenonane 57342-02-6 W14671 2005 Oda S, Tatarazako N, Watanabe H, Morita M, Iguchi T. 2005. Production of male neonates in Daphnia magna (Cladocera,

Crustacea) exposed to juvenile hormones and their analogs. Chemosphere 61(8):1168-1174.

epoxiconazole 133855-98-8

(formerly 106325-08-

0)

H19711 2006 Trosken ER, Adamska M, Arand M, Zarn JA, Patten C, Volkel W, Lutz WK. 2006. Comparison of lanosterol-14 alpha-

demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles. Toxicology 228(1):24-32.

TEDX (The Endocrine Disruption Exchange, Inc.) LIST

1038 EDCs TO DATE

February 2016

Is an organization that focuses primarily on the human health and environmental problems caused by

low-dose and/or ambient exposure to chemicals that interfere with development and function, called

endocrine disruptors. TEDX was founded by Dr. Theo Colborn, writer and lecturer on the human

health and environmental threat posed by endocrine disruptors and other industrially-produced

chemicals at low concentrations in the environment. http://www.endocrinedisruption.com

• Disinfection byproducts form when disinfectants, such as chlorine, react with

compounds present in water.

• These disinfectants react with naturally present fulvic and humic acids, amino

acids, and other organic matter, to produce a range of DBPs such as the

trihalomethanes (THMs), haloacetic acids (HAAs), halonitromethanes,

haloacetonitriles, haloamides, and others.

• Swimming pools using chlorine have been found to contain trihalomethanes,

generally below the current EU standard for drinking water (100 µg/l; 0.1 ppm),

though concentrations of trihalomethanes (mainly chloroform) of up to 0.43 ppm

have been measured.

• In addition, trichloramine has been detected in the air above swimming pools,

and is suspected to cause increased asthma in swimmers. It is formed by the

reaction of urea with chlorine and gives the indoor swimming pool its distinctive

odour.

• The species and concentrations of DBPs vary according to the type of

disinfectant used, the dose, the concentration of organic matter, the time since

dosing, temperature, and pH of the water.

What are Disinfection Byproducts?

• There is concern over possible human health risks, epidemiology

studies indicate a possible risk of bladder cancer, and some DBPs

cause cancer in laboratory animals.

• There are also concerns about possible reproductive &

developmental effects (from epidemiology studies).

• Research studies have tried to:

o Comprehensively identify DBPs formed from different

disinfectants

o Test for toxicity,

o Understand their formation,

o Minimize or eliminate them in drinking water

Disinfection Byproducts – What are the issues?

Solid Phase Extraction (SPE)

• Add methanol (10 ml) to the water sample (500 ml).

• Condition a Resprep C18 (Restek Corporation) 500 mg cartridge with acetonitrile:dichloromethane (5 ml, 1:1, v/v), then methanol (5 ml), followed by organic-free water (3 ml).

• Pass water (500 ml) slowly through the SPE cartridge using the Restek Vacuum Manifold (Restek Corporation).

• Dry the SPE tube under vacuum for approximately 30 minutes.

• Slowly elute acetonitrile:dichloromethane (5 ml, 1:1, v/v) into a 12 mL clean glass vial.

• Slowly elute n-hexane (2 ml) into the same 12 mL glass vial.

• Evaporate to dryness under a gentle stream of nitrogen.

• Reconstitute the dried residue in n-hexane (1 ml), vortex, and pipet into a 2 ml autosampler vial.

• Inject either 1µl or 10 ul (large volume injection) for GCxGC-HRTOFMS analysis.

Prepare cartridge, load sample, elute with appropriate solvent

Solid Phase Microextraction (SPME)(i) Extraction

• For the determination of volatile organic compounds, swimming pool or

surface water (30 ml) was sampled in a 40 ml glass vial, sealed with a

screw cap lined with a Teflon® septum.

• Sodium chloride was added to the water sample at 25% w/v, and the

sample was placed in a water bath at 35°C.

• Volatile components were adsorbed

onto a SPME device fitted with a

2-50/30µm DVB/Carboxen/PDMS

StableFlex fibre.

• The fibre was exposed to the

headspace above the sample

for 30 min.

Solid Phase Microextraction (SPME)(ii) Desorption

• After extraction the SPME device was removed from the vial and

desorbed in the injection port of a GC x GC-HRT for 1.5 min at 250 °C

in the splitless mode.

• The fibre was conditioned between extractions by heating it in a GC

injection port (split flow mode 50:1) for 20 min at 250 °C.

Folded Flight Path of up to 40 m yields ultra-high resolution

Vernchikov et.al.US Patent 7385187

Allows ultra-fast capture of high resolution spectra

LECO PEGASUS GC-HRT

AD

R=25,00032 Reflections

20 m Flight Path

High Resolution

Lenses

Mirrors

Mirrors

AD

R=50,00064 Reflections

40 m Flight Path

Ultra-High Resolution

Folded Flight Path™

The instrument has two

modes of operation

LECO Pegasus®-HRT Performance

Mass Accuracy <1 ppm

Mass Range 10-1500 m/z

Resolving Power up to 50,000

Detection Limit 1 pg OFN on column

Linear Dynamic Range >3.5 orders

Data Acquisition Speed* Up to 200 sps

Ionization EI, PCI

*The high acquisition rate is essential for GCxGC

Instrumental Conditions (GCxGC-HRT) Detector: LECO Pegasus HRT 4D

Inlet Temperature: 250°C

Split Mode: Splitless

Carrier Gas: Helium, 1.0 ml/min corrected constant flow

Column 1 : Rxi-5MS, 30 m x 0.25 mm ID x 0.25 µm film thickness

Column 2: Rxi-17Sil MS, 2 m x 0.25 mm ID x 0.25 µm film thickness

Guard Column (TL): Rxi-Guard, 0.8 m x 0.25 mm ID

Column 1 Oven: 40ºC for 1 min, to 300ºC at 10ºC/min, hold 3 min.

Column 2 Oven Offset: 10ºC (relative to primary oven)

Modulator Offset: 25°C (relative to 2nd oven)

Modulation Period: 4.0 s (Hot pulse 1.0 s)

Transfer Line Temperature: 280ºC

Total Run Time: 30 min

0.6 m of the secondary column is in the second oven. The remainder passes through the modulator into the primary oven.

Detector: LECO Pegasus HRT 4D

Acquisition Rate: 60 spectra/s

Stored Mass Range: 45 to 520 Da

Extraction Frequency: 2 kHz

Relative Detector Offset: 200 V

Source Temperature: 200ºC

Electron Energy: 70 eV

Instrumental Conditions (GCxGC-HRT)

Swimming Pool Water (LV)

GC×GC-HRT Chromatogram (TIC): Primary axis 180 s – 1670 s: Secondary axis 1 – 3 s

GC×GC-HRT Chromatogram (TIC): Primary axis 180 s – 1670 s: Secondary axis 1 – 3 s

Swimming Pool Water (SPME)

High Resolution Deconvolution

Three peaks

0.15 s apart

Spectral Quality

Mass accuracy:

0.22 ppm (CHCl2)

0.77 ppm (CH[37]Cl[81]Br)

Selected compounds found in swimming pool

water by GCxGC-HRTCompound Class RT1 (s) RT2 (s) Similarity Formula Mass Acc (ppm)

Trichloromethane Trihalomethane 199.98 1.48 863 C2HCl2 0.22

Bromodichloromethane Trihalomethane 203.97 1.67 960 CHCl2 1.20

Dichloroacetonitrile Disinfection byproduct 207.97 1.73 941 C2HClN -1.35

Dichloronitromethane Disinfection byproduct 235.93 1.75 902 CHCl2 1.20

Dibromochloromethane Trihalomethane 275.88 1.85 959 CHBrCl -0.52

1,1,1-Trichloro-2-propanone Disinfection byproduct 319.83 1.76 873 C3H3Cl2O 0.43

Tribromomethane Trihalomethane 363.77 2.02 940 CHBr3 -0.54

1,4-Dichlorobenzene EDC 491.61 1.85 923 C6H4Cl2 0.26

1-Chlorooctane Halogenated compound 535.55 1.60 882 C4H8Cl -2.24

Hexachloroethane Potential carcinogen 551.54 1.80 930 C2Cl5 -1.08

1-Bromooctane Halogenated compound 611.46 1.64 872 C4H8Br -0.66

Trichloronitromethane Disinfection byproduct /pesticide 651.26 2.26 895 CCl3 0.77

Terpineol Fragrance 659.40 1.75 787 C10H16 -0.38

Diethyl phthalate Plasticizer 979.00 2.02 934 C12H14O4 0.53

Benzophenone Sunscreen additive 1006.82 2.19 868 C13H10O -0.56

Caffeine CNS stimulant / Human pollution 1150.64 2.53 773 C8H10N4O2 0.38

Dibutyl phthalate Suspected teratogen & EDC 1214.70 1.94 933 C8H5O3 -0.14

DCPA Herbicide 1238.53 1.97 778 C9H3Cl4O3 2.65

Oxybenzone Sunscreen additive 1258.50 2.24 879 C14H11O3 1.43

Pyrene PAH 1318.58 2.53 810 C16H10 -1.00

Red = EDC: Blue = DBP

Selected compounds found in river water by

GCxGC-HRTCompound Class RT1 (s) RT2 (s) Similarity Formula Mass Acc (ppm)

Tetrachloroethylene CNS depressant / Possible carcinogen 283.87 1.66 704 C2Cl4 -1.09

o-Xylene Aromatic 339.80 1.72 910 C8H10 0.33

Camphene Essential Oil 419.70 1.63 892 C10H16 0.82

1,2,3-Trimethylbenzene Aromatic 439.68 1.70 748 C9H12 -0.88

Phenol Synthetic precursor 455.66 1.94 872 C6H6O 0.53

o-Cymene Essential Oil 495.61 1.70 743 C10H14 0.73

1-Chlorooctane Halogenated compound 527.57 1.61 810 C4H8Cl -1.15

Benzaldehyde Flavour, fragrance, pharmaceutical 431.54 2.05 869 C7H6O -0.26

1-Octanol Precursor to perfumes 535.56 1.67 904 C8H18O -0.32

Benzoic acid Food preservative (Na salt) 619.45 1.904 919 C7H6O2 0.88

Camphor Essential Oil 619.50 1.87 631 C7H11 -1.16

Naphthalene PAH 659.45 2.07 915 C10H8 -1.71

Biphenyl Heat transfer medium (eutectic mix) 823.25 2.06 871 C12H10 -0.42

Diphenylether Heat transfer medium (eutectic mix) 839.23 2.06 760 C12H10O -1.27

Diethyl phthalate Phthalate 935.06 2.30 906 C8H5O3 1.20

Benzophenone Sunscreen additive 1011.01 2.23 873 C13H10O 0.77

Phenanthrene PAH 1118.88 2.33 894 C14H10 0.92

Thioxanthene Derivatives used as antipsychotics 1162.82 2.24 692 C13H10S 0.00

Pyrene PAH 1290.66 2.41 829 C16H10 0.24

Diisooctyl phthalate Phthalate 1494.41 1.79 705 C8H5O3 1.20

Red = EDC

Selected compounds found in the Braamfontein Spruit

water by GCxGC-HRT

Compound Class RT1 (s) RT2 (s) Similarity Formula Mass Acc (ppm)

Benzothiazole Numerous medicinal activities 679.42 2.05 578 C7H5NS -1.08

Phenanthrene PAH 1098.90 2.10 852 C14H10 -1.19

Caffeine CNS stimulant (psychoactive) 1134.86 2.33 773 C8H10N4O2 1.04

Diphenylsulphone High temperature solvent 1186.79 2.31 756 C12H10O2S 1.39

Dibutylphthalate Phthalate 1198.78 1.74 930 C16H22O4 (149) 0.67

Triclosan Antibacterial and antifungal agent 1286.67 1.94 834 C12H7Cl3O2 0.93

Bis(2-ethylhexyl)phthalate Phthalate 1498.40 1.62 917 C24H38O4 (M-111) 0.36

Cholestanol Biomarker for human faecal matter 1758.08 2.20 756 C27H48O (M-33) 0.00

Cholesta-3,5-diene Semiochemical 1782.05 2.26 701 C27H44 -0.15

Selected compounds found in the LECO Borehole water

by GCxGC-HRTCompound Class RT1 (s) RT2 (s) Similarity Formula Mass Acc (ppm)

Dibutylphthalate Phthalate 1198.78 1.72 940 C16H22O4 (149) 0.00

Methyl dehydroabietate Essential oil 1222.74 1.68 700 C21H38O2 (239) -1.37

10,18-Bisnorabieta-8,11,13-triene Essential oil 1254.70 1.72 808 C18H26 0.21

Pyrene PAH 1270.68 2.14 706 C16H10 0.38

Fluoranthene PAH 1302.65 2.21 607 C16H10 -0.28

Red = EDC

Conclusions

• GC×GC-HRT is a powerful tool for the comprehensive analysis and

chemical characterization of analytes in complex matrices.

• The combination of high resolution front-end separation with high

resolution time-of-flight mass spectrometry makes possible the

identification of compounds previously unknown in these samples

(Increased peak capacity + increased selectivity = increased confidence in

identification).

• High resolution permits minimization of false positives and negatives and

facilitates confident quantitation

• Mass accuracy allows for confident identification and selective detection

• EI in combination with HRCI facilitates this further by providing molecular

ions.

• Automated high resolution deconvolution makes it easier to find peaks –

both knowns and unknowns

Related Work

Today 12 September 2016

Poster E22

A NEW PASSIVE SAMPLER USING SILICONE RUBBER FOR THE

ANALYSIS OF SURFACE WATER BY COMPREHENSIVE GAS

CHROMATOGRAPHY – TIME OF FLIGHT MASS SPECTROMETRY

Yvette Naudé1 , Peter Gorst-Allman2 , Egmont Rohwer1

1Department of Chemistry - University of Pretoria, 2LECO Africa, Kempton Park, South Africa