Alkylphenols in produced water

Analysis of alkylphenol metabolites 

in fish bile as a tool 

for monitoring produced water discharges; 

a RF-Akvamiljø 

b University of Stavanger 

Method development and utilisation 

Grete Jonsson a , Admira Cavcic b , Tone U. Stokke b , 

Jonny Beyer a and Kåre B. Jørgensen b 

Project overview 

RF - Akvamiljø 

Pollutant exposure and effects in fish related 

to the discharge of produced water in the 

North Sea oil industry 

• WP-1: Fish samples for PW effect assessment 

• WP-2: GC/MS detection of alkylphenol metabolites in bile 

• WP-3: ICP-MS detection of PW metals in bile 

• WP-4: DNA-biosensor detection of bile genotoxicity 

• WP-5: Hepatic biomarkers of PW genotoxicity in fish 

• WP-6: Endocrine, embryonic and developmental effects 

of PW in fish 

• WP-7: PW effects on thyroid hormones, steroids, 

vitamin homeostasis and behaviour in Atlantic cod 

• WP-8: Proteomics analysis of PW exposed fish 

Introduction 


Alkylphenols in produced water (PW) 

North Sea – Norwegian sector 

• >100 mill tons PW released yearly 

• 500 tons AP 

• C 0 –C 3 AP > 95% 

• C 4 –C 7 AP < 5% 


Introduction 

• Toxic 

Content 

• Project overview 

• Introduction 

• Exposure of cod to alkylphenols 

• HPLC-Fluorescence analysis 

• GC-MS analysis 

• Summary 

Why alkylphenols? 

Potentially adverse effects 

R. K. Bechmann 

Short term exposure 

Relatively high exposure concentration 

• Hormone disrupting 

Long term exposure 

Effect dependent on size and position of alkyl-group 

Introduction 

Thousands H 3 /g 

R. K. Bechmann 



Distribution of 4-t-butylphenol in cod 

5000 

4000 

3000 

2000 

1000 

0 

From: Sundt and Baussant, 2003 

Bile 

Liver 

Intestine 

Intest. cont. 

Stomach 

Stomach cont. 

8 days of water exposure 

8 days of depuration 

Gill 

Skin 

Muscle 

Pooled 

Gonad 

RF - Akvamiljø

Introduction 

Proposed metabolism of alkylphenols 

Exposure Metabolism Excretion 

R 

OH 

Rox 

OH 

Phase I 

Phase II 

Phase II 

Rox 

R 

O-conjugate 

O-conjugate 

R 

OH 

Excretion 

Excretion 

Excretion 


Target compounds 

Representative for alkylphenols in produced water 

OH OH 

CH 3 

CH 3 

4-methylphenol 2-methylphenol 3,5-dimethylphenol 2,4,6-trimethylphenol 

OH 

H3C C CH3 CH 3 

4-tbutylphenol 

Fluorescence ex/em 222/306 nm 

OH 


2-methyl-4-tbutylphenol 

CH 3 

H 3C 

OH 

OH 

C5H11 4-npentylphenol 

CH 3 

OH 

HPLC-F separation 

4-MP 

2-MP 

3,5-DMP 

2,4,6-TMP 

H 3C 

OH 

CH 3 

OH 

CH 3 

C6H13 C7H15 4-n4-nhexylphenolheptylphenol 

5 45 

Minutes 

Sample: AP mix in methanol (≈ 500 µg/l) 

Internal standard (IS): 1-Fluoronaphthalene 

4-t-BP 

4-n-C6P 4-n-C5P 4-n-C7P 4-t-B-2-MP 

IS 



Exposure of cod to alkylphenols 

Twelve exposure groups: single alkylphenols 

a mix of nine alkylphenols 

carrier control 

control 

Exposure concentration: 10 mg/kg 

Sampling: Four days after exposure (all groups) 

Sixteen days after exposure (five groups) 

HPLC-Fluorescence analysis 

of alkylphenol metabolites 

Sample preparation 

Enzymatic treatment 

Solid phase extraction 

© JB 


HPLC-separation of a mix of alkylphenols 

HPLC-F detection limits 

Utilisation 

Field exposed fish bile 

HPLC-F; instrumental LODs 

Alkylphenol µg/l 

2-methylphenol 1.5 

4-methylphenol 2.6 

3,5-dimethylphenol 0.5 

2,4,6-dimethylphenol 0.6 

4-t-butylphenol 1.1 

4-t-butyl-2-methylphenol 0.8 

4-n-pentylphenol 5.0 

4-n-hexylphenol 20.0 

4-n-heptylphenol 20.0 

LOD (limit of detection) = signal : noise = 3:1 


RF - Akvamiljø


Bile sample preparation 

Sampling 

Non-treated bile 

Conjugated metabolites 

Chemical analysis 

Illustr. Jonny Beyer 

Bile treatment Bile treatment 

HPLC-F 

Alkylphenols 

Impurities 

Enzymatic de-conjugation 


Oxidized alkylphenols 

HPLC-F 

GCMS 

Sample treatment; 

Solid Phase Extraction (SPE) 

2: Add sample 

1: Condition 3: Wash 4: Elution 

HPLC-F analysis of 

bile from fish exposed to 4-t-butylphenol 

4-t-BP 

5 45 


IS 

IS 

IS 



Standard mix 

Conjugated bile 

De-conjugated bile 


De – conjugation of bile 

Bile 

Enzyme 

HO 

OH 

HO O 

H 3C 

O 

CH 3 

O 

C OH 

CH 

Glucuronide conjugated phenol 

3 

Incubate 

2 h at 40°C 

H 3C 

OH 

CH 3 

Free phenol 

SPE recovery (%) 

CH 3 


Envi- OASIS 

Alkylphenol C18 Phenyl Carb MAX 

2-methylphenol 88 87 135 ip 

4-methylphenol 193* 251* 129* ip 

3,5-dimethylphenol 93 72 95 85 

2,4,6-dimethylphenol 72 72 44 64 

4-t-butylphenol 77 76 99 69 

4-t-butyl-2-methylphenol 79 80 96 73 

4-n-pentylphenol 95 nd nd 78 

4-n-hexylphenol 81 nd nd 94 

4-n-heptylphenol 71 nd nd 87 

* 4-methylphenol excreted in control bile 

nd: not determined 

ip: large interfering peaks 


SPE packing material 



bile from fish exposed to 4-n-pentylphenol 

5 45 


IS 

IS 

IS 

4-n-C 5P 

Standard mix 



RF - Akvamiljø


bile from fish exposed to a mix of nine alkylphenols 

SIM 



8 

C 1 

C 1 C2 

C 3 

C 4 

IS 

IS 

Standard mix 


* 

** * * 

* IS * 


5 Minutes 

45 

C 5 

C 5 

C 6 C 7 

* * 


HPLC-F analysis of field samples 

0 30 


1 

2 

2 

C1 C1 C 1 

C 1 

3 4 

1 3 

C 2 

C 2 

C 3 

C 3 

C 4 

C 4 

GC-separation 

Time (min) 

5 

6 

TMS-derivatives 

10 Time (min) 

30 

4 

5 

6 

C 5 

C 5 

Standard mix 

German Bight 

PW exposed bile 

Spiked 

PW exposed bile 


Concentration: ca 1 mg/kg 

1: 2-methylphenol 

2: 4-methylphenol 

3: 3,5-dimethylphenol 

4: 2,4,6-trimethylphenol 

5: 4-tertbutylphenol 

6: 2-methyl-4-tertbutylphenol 

30 


Alkylphenol metabolite concentration 

in bile from fish exposed to single compounds 

Concentration (µg/g bile) 

200 

100 

0 

n = 4 or 5 

2-MP 3,5-DMP 2,4,6-TMP 4-t-B-2-MP 

Four days after exposure 

Sixteen days after exposure 

GC-MS analysis 

of alkylphenol metabolites 

GC-separation 

Sample preparation 

Enzymatic treatment 

Solid phase extraction 

Liquid liquid extraction 

Derivatization 


Derivatized versus underivatized alkylphenols 

GC-MS detection limits 

Instrumental limits of detection (LOD) pg injected 

Phenols TMS-ethers 

2-methylphenol 15 0.4 

4-methylphenol 40 0.4 

3,5-dimethylphenol 10 0.3 

2,4,6-trimethylphenol 18 1.3 

4-tertbutylphenol 30 1.2 

2-methyl-4-tertbutylphenol 15 0.3 


RF - Akvamiljø

SIM 

2-MP 

4-MP 

IS 1 

3,5-DMP 

IS 2 

GC-separation 

Sample: TMS-derivatives of 9 alkylphenols and 5 internal standards 

2,4,6-TMP 

4-t-BP 

4-t-B-2-MP 

IS 4 

IS 3 

IS 5 

4-n-C 7P 

4-n-C6P 4-n-C5P 16 Time (min) 

46 

BSTFA 

TMSI 

Peak area Derivatization efficiency 

Low TMSI Double peaks 

for 4-t-butylphenol 

Increasing amount of derivatization reagent 

Recovery (%) 

SPE packing material: OASIS MAX (anion exchange) 

Solvent: Methyl-tertbutylether 



Alkylphenol off-column on-column 

derivatization derivatization 

2-methylphenol 78 89 

4-methylphenol 151 134 

3,5-dimethylphenol 94 86 

2,4,6-dimethylphenol 22 66 

4-t-butylphenol 75 67 

4-t-butyl-2-methylphenol 36 69 

4-n-pentylphenol 84 86 

4-n-hexylphenol 80 87 

4-n-heptylphenol 77 80 


Derivatisation efficiency 

Two trimethylsilylation (TMS) reagents tested 

BSTFA: Bis(trimethylsilyl)trifluoroacetamide 

TMSI: Trimethylsilylimidazole 

OH 


+ 

BSTFA 

or 

TMSI 

2 hours at 60°C 

15 min at room temp. 

CH 3 

H3C Si CH3 O 



On- and off-column TMS-derivatization 

Off-column 

derivatization 

On-column derivatization 

3: Wash 4: Dry column 5a: Elution 5b: Derivatize 6b: Elution 


Derivatized alkylphenols 

Impurities 

HPLC-F 

Summary 

6a: 

Derivatization 


• Knowledge about the metabolism of C 1 -C 7 alkylphenols 

• Analysis of bile from fish exposed to simple and known 

compounds 

• Not sufficiently specific for analyses of bile from fish 

exposed to complex mixtures (field samples) 

RF - Akvamiljø

GC-MS 

Summary 

• Derivatization improve GC separation 

• Derivatization improve detection limits 

• On-column derivatization results in good recoveries 

• On-column derivatization result in improved 

separation of matrix interferences from alkylphenols 


Acknowledgement 

to 

NFR - PROOF 

for financial support 

RF - Akvamiljø

Alkylphenols in produced water

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