Forensic Ecotoxicology: Toxicity Associated with

Halogenated Tolyltriazoles isolated from industrial cooling towers

Birkholz, D.A.1, Goudey, S.E.2, Hoeppner, S.E.3

1D.A. Birkholz Analytical Consultant, Inc., Edmonton, Alberta, Canada.

2Limnos Ltd., Calgary, Alberta, Canada.

3Life Science Forensics, Ltd., Calgary,

• Cooling towers recycle water and as such incorporate

holding ponds

• When full, ponds are discharged to the environment.

• Regulations require that the holding ponds be tested

for toxicity before discharge and demonstrated not

to be toxic. Different jurisdictions may require

differing toxicity tests (e.g. acute, chronic) in both

US and Canada.

• Several case studies presented involving:

• polyethylene plant

• straddle gas plants (2)

• fertilizer plant

• tire manufacturing

• All effluents found to be toxic

• Effluents subjected to forensic analyses

1-2L sample aliquots pH adjusted: pH 3, pH 9, pH ambient

1 L aliquot aerated Toxicity testing

1 L aliquot filtered Toxicity testing

1 -2 L aliquot passed through C18 columns

C18 column eluted with 4 mL of 25%, 50%, 75%, and two times 100% MeOH in water

3mL diluted to 200 mL with bioassay water

Toxicity testing

Toxicity testing

1 mL archived for chemical analyses

Forensic Method

Luminescent bacteria

Rainbow trout

Fathead minnow

Daphnia magna

Conc. by 3.75X (1 L)

Solid Phase Extraction

Solid Phase Extraction

Solid Phase Extraction

Polyethylene plant

Industry Sample I.D. Microtox %

cntrls or toxic

units

Daphnia Fathead

minnow

Polyethylene Cooling tower blowdown 37% NT NA

Polyethylene CTB, SPE, pH 3, 50% MeOH 52% NT NT

Polyethylene CTB, SPE, pH 3, 75% MeOH 18% T-24h T-24h

Polethylene Plant

SPE, pH 3, 75% MeOH - alkylated

Polyethylene Plant Study

• Because a corrosion inhibitor tolyltriazole and HOBr were

implicated, compounds found were the following:

• bromotolyltriazole m/z 225, 227, 146, 118,117

• dibromotolyltriazole m/z 303, 305, 307, 224, 226

• Little or no information provided by plant as to process

Typical Straddle Gas Plants

Photographs do not represent clients

Corrosion inhibitor a mixture of 5- and 4-methyl benzotriazole. Used with slime

control agents such as NaOCl and NaBr to form HOCl and HOBr

Ref: Potekhina et al (1999) Appl. Microbiol. Biotechnol., 52: 639 - 646

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Abundance

m/z 147.00 tolyltriazoles from commericial corrosion inhibitor

xx

x

After alkylation with diazomethane

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Ion 147.00 Cooling tower Effluent

After alkylation with diazomethane

Interaction between inhibitor

and metal surface enhances

with substitutents in the 5-

position of the inhibitor

molecule

Ref: Antonijevich and Petrovic (2008) Intl. J. Electrochemical Science 3: 1 - 28

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Scan 1316 (14.606 min): 2501012.D147

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51104

39 65132

911195830 14145 70 85 11496

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Scan 1635 (16.671 min): 2501012.D118

147

77

5191

39

10463

325745 8570 133110

Straddle Gas Plant A

Sample I.D. Compound I.D. Est. Conc

ug/L

Trout toxic

units

Microtox

toxic units

SPE, pH 3.0, 50% MeOH Bromotolyltriazole 18.8 1.0 1.0

Dibromotolytriazole 13.6 1.0 1.0

Tribromotolyltriazole 0.64 1.0 1.0

Total 33.0

SPE, pH 3.0, 75% MeOH Bromotolyltriazole 6.9 2.8 1.7

Dibromotolyltriazole 61.5 2.8 1.7

Tribromotolyltriazole 19.6 2.8 1.7

Total 88.0

88/33 = 2.67

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Abundance

Ion 225.00 bromotolyltriazole

Ion 303.00 dibromotolyltriazole

Ion 381.00 tribromotolyltriazole

Straddle Plant A 50% MeOH - alkylated

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Abundance

Ion 225.00 bromotolyltriazole

Ion 303.00 dibromotolyltriazole

Ion 381.00 tribromotolytriazole

75% MeOH

Straddle Gas Plant A -75% MeOH-alkylated

Straddle Gas Plant B

Industry Sample I.D. Microtox %

Cntrl

Trout

Straddle Gas Plant P-1 SPE, amb pH, 75% MeOH 31 toxic

Straddle Gas Plant P-2 SPE, amb pH, 75% MeOH 32 toxic

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Abundance

Ion 181.00 chlorotolyltriazole

Ion 215.00 dichlorotolyltriazole

Ion 259.00 chlorobromotolyltriazole

Ion 293.00 dichlorobromotolyltriazole

Ion 337.00 chlorodibromotolyltriazole

P1 75% MeOHMicrotox 32% c

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Abundance

Ion 181.00 chlorotolyltriazole

Ion 215.00 dichlorotolyltriazole

Ion 259.00 bromochlorotolyltriazole

Ion 293.00 bromodichlorotolyltriazole

Ion 337.00 dibromochlorotolyltriazole

P2 75% MeOH

Microtox 31% c

Straddle Gas Plant B

Industry Sample Compounds Found

Straddle Gas Plant B SPE, pH 3.0, 75% MeOH Chlorotolyltriazole

SPE, pH 3.0, 75% MeOH Dichlorotolyltriazole

SPE, pH 3.0, 75% MeOH Bromochlorotolyltriazole

SPE, pH 3.0, 75% MeOH Bromodichlorotolytriazole

SPE, pH 3.0, 75% MeOH Dibromochlorotolytriazole

Major compounds found

Fertilizer Plant

Industry Sample I.D. Microtox

% cntrls

FHM

TU

Trout

TU

Fertilizer Plant Cooling water 100 1.3 9.1

CW SPE, pH 8.0, 50% MeOH 9 5.3 21

CW SPE, pH 8.0, 75% MeOH 66 1.3 NA

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Abundance

Ion 215.00 dichlorotolyltriazole

Ion 259.00 chlorobromotolyltriazole

Ion 303.00 dibromotolytriazole

Ion 337.00 chlorodibromotolytriazole

Ion 381.00 tribromotolytriazole

Ion 293.00 dichlorobromotolyltriazole

Plant 36 - 50% MeOH - alkylated

FHM 5.3 TU

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Abundance

Ion 215.00 dichlorotolytriazole

Ion 259.00 chlorobromotolytriazole

Ion 303.00 dibromotolytriazole

Ion 337.00 chlorodibromotolyltriazole

Ion 381.00 tribromotolytriazole

Ion 293.00 dichlorobromotolytriazole

Plant 36 - 75% MeOH - alkylated

FHM 1.3 TU

Fertilizer PlantSample I.D. Compounds Est. Conc

ug/L

Trout

TU

FHM

TU

SPE, pH 8.0, 50% MeOH Dichlorotolyltriazole 4.6 21 5.3

Bromochlorotolyltriazole 74.6 21 5.3

Dibromochlorotolyltriazole 120 21 5.3

Bromodichlorotolyltriazole 12 21 5.3

Dibromotolyltriazole 5.3 21 5.3

Tribromotolyltriazole 6.4 21 5.3

Total 223

SPE, pH 8.0, 75% MeOH Dichlorotolyltriazole 3.7 NA 1.3

Bromochlorotolyltriazole 42 NA 1.3

Dibromotolyltriazole 2.6 NA 1.3

Dibromochlorotolyltriazole 30 NA 1.3

Tribromotolyltriazole 1.3 NA 1.3

Bromodichlorotolytriazole 3.7 NA 1.3

Total 83.3

223/83.3 = 2.67, 120/30 = 4.0, TU=5.3/1.3 = 4.0

Tire Manufacturer

Sample I.D. Compounds Est. Conc.

ug/L

Trout TU

SPE, pH 3.0, 75% MeOH Tolyltriazole 149 21-27

Chlorotolyltriazole 1562 21-27

Dichlorotolytriazole 104 21-27

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Abundance

Ion 133.00 tolyltriazole underivatized Ion 167.00 chlorotolyltriazole underivatized

75% MeOH

underivatized

N

N

N

H

CH3

m/z 133

N

N

N

H

CH3

m/z 167

Cl

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Abundance

Ion 147.00 alkylated tolyltriazole

Ion 181.00 alkylated chloro-tolyltriazole

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Abundance

Ion 147.00 tolyltriazole

Ion 181.00 chlorotolyltriazole

Ion 215.00 dichlorotolyltriazole

75% MeOH TMfg - alkylated

RT = 21 – 27 TU

Halogenated Tolyltriazoles and Toxicity

Industry Compound Class Est Conc

ug/L

Trout

TU

FHM

TU

Microtox

TU

Gas Plant Bromotolyltriazoles 33 1.0 NA 1.0

Gas Plant Bromotolyltriazoles 88 2.8 NA 1.7

Fertilizer Bromochlorotolyltriazoles 223 21 5.3 11.1

Fertilizer Bromochlorotolyltriazoles 83 NA 1.3 NA

Tire Mfg Chlorotolyltriazoles 1815 21-27 NA NA

Polyethylene Bromotolyltriazoles NA NA NA 5.6

Cooling Tower Simulation

A

B

C

D

E

Lab Simulation

and HPLC fractionation

Lab simulation and HPLC

HPLC FRACTION Trout toxicity – 24h Major components

A 30% mortality monobromotolyltriazole

B 30% mortality monobromotolytriazole

C 60% mortality dibromotolytriazole

D 60% mortality dibromotolyltriazole and tribromotolyltriazole

E 30% mortality tribromotolyltriazole

Conclusions• Corrosion inhibitors (tolyltriazole used in combination with biocides (HOCl, HOBr, and

stablilized bromine) form halogenated tolyltriazoles.

• These compounds are toxic and persistent

• Rainbow trout (mandated Canadian bioassay) are particularly sensitive to these compounds.

• Luminescent bacteria (Microtox) are also sensitive to these compounds

• Fathead minnow are least sensitive (mandated American bioassay)

• Brominated tolyltriazoles and chlorobromotolyltriazoles are the most toxic. Toxic responses are seen below 100 ug/L

• Chlorinated tolyltriazoles are the least toxic. Toxic responses are in the order of 1000 ug/L

• Cooling towers using tolyltriazole and biocides (HOBr, HOCl) can form either chlorinated or brominated tolyltriazoles depending on maintenance program

• Cooling towers using chlorotolyltriazole and biocides (HOBr, HOCl) form chlorobromotolyltriazoles.

• Reaction with halogens represents a significant component of triazole consumption in a cooling water system.

Conclusions

• pKa of 5-chlorobenzotriazole is 6.12 ,i.e. a weak acid. As the number of halogens increases so

does the acidity

• consistent with efficient trapping on C18 at pH 3.0

• Rainbow trout LC50 for 4(-5)methylbenzotriazole is 21.4 mg/L therefore halogenated moieties

are 2-orders of magnitude more toxic

• 5-methlbenzotriazole is readily biodegradable (77% after 28 days). Also binds with metal

• 4-methylbenzotriazole is recalcitrant

• biodegradation of halogenated tolyltriazoles unknown

• partition coefficient for 4(-5)-methylbenzotriazole is 1.71

• partition coefficient for halogenated TT is unknown - more halogens more lipophilic

Additional Reading

Patrrick J. Downey and B. J. Venables (2005)

Case Study 6.15: Identification of toxic reaction

products in cooling tower chemicals. Pages 228 - 237

Contact Information• Detlef (Deib) Birkholz, MSc, PhD, P.Chem.

• D.A. Birkholz, Analytical Consultant, Inc.

Phone: 587-597-5197

Email: birkholz@ualberta.ca

• Calgary contacts: Paracel Laboratories

c/o Dr. Milan Ralitsch

Bay F 1423 – 45 Avenue N.E.

Calgary, Alberta T2E 2P3

Phone 403-776-4443 OR

Life Science Forensics, c/o Stephanie Hoeppner,

Phone: 587 437-8278