The behaviour of - Birmingham

The behaviour of The behaviour of the the toxaphene toxaphene

component component B6B6--923923

in sediment in sediment and biotaand biota

Walter Vetter Walter Vetter (w(w--vettervetter@@uniuni--hohenheimhohenheim.de).de)

University of University of HohenheimHohenheimInstitute of Food Chemistry Institute of Food Chemistry

(170)(170)

• chloropesticide used at million-ton scale [1]

• one of the dirty dozen (key-POP) [2]

• produced by the photochlorination of camphene [1]

• Wagner-Meerwein rearrangement leads to polychlorinated bornanes [1]

• mixture cannot be resolved even by MDGC [3]

Ref.:

[1] M. A. Saleh, RECT 114 (1991) 1-85

[2] UNECE

[3] W. Vetter & B. Luckas, STOTEN 170 (1995) 165

20 30 40 50 [m in]

GC/ECD chromatogram of Toxaphene

Facts on Toxaphene

Toxaphene residuesToxaphene residues in in thethe environmentenvironment

• residue pattern in environmental samples much simpler

• extensive transformation

marine mammals sediment

ClH2C CH2Cl

CH2ClCl

Cl

Cl

ClH2C CH2Cl

CH2ClCl

Cl

ClCl

B6-923

32.0 36.0 [min]

Irel

B7-1001

transformation of Cl6- and Cl7-CTTs transformation of Cl8- and Cl9-CTTs

B9-1679H3C CHCl2

CHCl2Cl

Cl

ClClCHCl2

CHCl2Cl

Cl

ClCl

40.0 42.0 44.0 46.0 48.0 50.0 52.0 [min]

Irel

H3C CHCl2

CHCl2Cl

Cl

ClCl

H3C CHCl2

CHCl2Cl

Cl

ClCl

H3C CHCl2

CHCl2Cl

Cl

ClClCHCl2

CHCl2Cl

Cl

ClCl

ClH2C CHCl2

CHCl2Cl

Cl

ClCl

40.0 42.0 44.0 46.0 48.0 50.0 52.0 [min]

IrelB8-1413

20 30 40 50 [min]

technical toxaphene

The environmental role of B6-923

• metabolite of key-congeners• very persistent due to the lack of geminal Cl

0 10.25

0.0

0.5

1.0 B8-806B7-1473B6-923

5 91.5 12time [days]

rela

tive

ECD

-res

pons

eRef: S. Ruppe, A. Neumann, W. Vetter, Environ. Toxicol. Chem. 22 (2003) 2614-2621

Transformation with dehalogenating bacteria

1.21% *2.36% *

0.38 % *

2

3

4

5

6

789

10

Cl

ClCl

Cl

ClCl

Cl

ClH

H H

HH

1

2

3

4

5

6

789

10

H

ClCl

Cl

H

Cl

Cl

ClH

H H

HH

1

2

3

4

5

6

789

10

Cl

ClCl

Cl

HCl

Cl

ClH

H H

HH

1

B8-806a B7-515b

B6-923b

-8-Cl-2-endo-Cl

-2-endo-Cl

* contribution to technical toxaphene(W. Vetter, G. Gleixner, W. Armbruster, S. Ruppe, G. A. Stern, E. Braekevelt, Chemosphere 58 (2005) 235-241)

Chirality of toxaphene

• toxaphene consists of ~1000 compounds(Korytár et al. 2003)

• ~98% chiral

• 100 compounds identified, all chiral

• four CSPs described for the GC enantioseparation(Buser & Müller, 1994; Vetter et al. 1998/2000, Jaus & Oehme 2000)

• ChiraDex (Merck) described for LC enantioseparation(Vetter & Kirchberg, 2000)

Synthesis of toxaphene enantiomers

(A)B7-515: 2x column chromatography, crystallization

K. J. Palmer, R. Y. Wong, R. E. Lundin, S. Khalifa, J. E. Casida,J. Amer. Chem. Soc. 97 (1975) 408-413

(B) B6-923: anaerobic transformation in soil, crystallization

G. Fingerling, N. Hertkorn, H. Parlar, Environ. Sci. Technol. 30 (1996) 2984-2992

(C) B6-923: synthesis, isolation, crystallization

L. K. Hansen, R. Kallenborn, A. Kiprianova, V. Nikiforov,A. Trukhin, Acta Cryst. E60 (2004) 1089-1091

Synthesis of toxaphene enantiomers

(A)B7-515: 2x column chromatography, crystallization⇒ enantiopure !

K. J. Palmer, R. Y. Wong, R. E. Lundin, S. Khalifa, J. E. Casida,J. Amer. Chem. Soc. 97 (1975) 408-413

(B) B6-923: anaerobic transfromation in soil, crystallization⇒ enantiopure !

G. Fingerling, N. Hertkorn, H. Parlar, Environ. Sci. Technol. 30 (1996) 2984-2992

(C) B6-923: synthesis, isolation, crystallization⇒ enantiopure !

L. K. Hansen, R. Kallenborn, A. Kiprianova, V. Nikiforov,A. Trukhin, Acta Cryst. E60 (2004) 1089-1091

How and why ?

⇒ environment?

Gaining racemic B6-923

• transformation of the technical product(should lead to B6-923-enriched solution)

• LC isolation/purification of standards(should lead to rather pure B6-923)

• HPLC enantioseparation of the B6-923 enantiomers

ToxapheneToxaphene transformation transformation with anoxic sewage sludgewith anoxic sewage sludge

48.00 50.00 52.00 54.00 56.00 58.00 [min]Time-->

Irel

48.00 50.00 52.00 54.00 56.00 58.00 [min]Time-->

Irel

O c t a c h l o r o b o r n a n e sstart four weeks in sewage sludge

• simplification of the toxaphene residue pattern

• persistent (= interesting) CTTs not metabolized

Ref.: W. Vetter, D. Kirchberg, Environ. Sci. Technol. 35 (2001) 960-965

HPLC-isolation of B6-923

35.00 40.00 45.00 50.00 55.00 60.00Time--> [min]

B6 -9 2 3enables isolation of relevant CTTs in sufficient purity

⇒

(enantiomersresolved: racemic)

• result in contrast with studies by Palmer et al., Parlar et al. and Nikiforov et al. who all received enantiopure CTTs

⇒ re-crystallization or error by XRD interpretation ?

GC/ECNI-MS analysis (β-BSCD) of fractionsobtained after enantioselective HPLC

LC-enantiomer separation of CTTs (on ChiraDex)

Ref.: W. Vetter, D. Kirchberg, Environ. Sci. Technol. 35 (2001) 960-965

• 25% tert.-butyldimethylsilylated β-cyclodextrin in PS086 (modification of “BGB 172“ (BGB Analytik, Switzerland)

• only CSP reported to date which resolves the B6-923 enantiomers

• unique case even for toxaphene⇒ any other known CTT was at least enantioresolved on two CSPs

• QC/QA requires higher selectivity than GC/ECNI-MS⇒ we chose liquid chromatographic prefractioning

Enantioseparation of B6-923

Hanson Lake (Yukon, Canada)

Ref.: W. Vetter, R. Bartha, G. Stern, G. Tomy, Environ. Toxicol. Chem. 18 (1999) 2775-2781


• in 1963 treated with 6 ppb toxaphene to kill existing fish

• attempts to restock with sport fish failed



• in 1963 treated with 6 ppb toxaphene to kill existing fish

• attempts to restock with sport fish failed

• today: toxapheneconcentration in sediment ~ 1 mg/kg


Enantioselective determination of B6-923

Cl

Cl

H H

Cl

HH

ClH

H

H H

Cl

Cl

HH

H 1

4

3

2

5

6

79

8

10

Sediment Hanson Lake (Yukon, Canada)

29 31 33 35 [min]

m/z 309

m/z 307

A1734W.D

Irel

• B6-923 racemic

• sediment concentration ~ 1 mg/kg

fractionated on silica



Cl

Cl

H H

Cl

HH

ClH

H

H H

Cl

Cl

HH

H 1

4

3

2

5

6

79

8

10• B6-923

racemic

30 31 32 33 34 35 [min]

m/z 307, m/z 309

A1734W.D

Sediment Hanson Lake (Yukon, Canada)

SIM-Masses of hepta- and octachlorobornanes⇒ no response, no interference except isomer possible⇒ no B6-923 isomer >5% of B6-923 detected by non-

chiral analysis⇒ racemic composition of B6-923 verified


Toxaphene in age-dated sediment core slices from Hanson Lake (Yukon Territories, Canada)

19921987/84

197919731968196419591954194619351864

0 1 2 3 4 5 6 7 8 9Concentration ( µg/g)

treatment of lakewith toxaphene inJuly, 1963

Median age of each slice

• production started in 1946

• data before 1946 owing to porewaterdiffusion

• to some amount also valid for data between 1946 and 1963



Cl

Cl

HH

Cl

HH

ClH

H

H H

Cl

Cl

HH

H1

4

3

2

5

6

79

8

10

Sediment Hanson Lake, age-Dated

• LC-fractioning (8g silica, n-hexane)• GC/ECNI-MS-SIM

0.90

0.95

1.00

1.05

ER

1992 1985 1979 1973 1968 1964 1959 1954 1946 1935

year

m/z

307

309

• no significant deviation from the racemate



Terry Creek (GA, USA)

• located close to a former toxaphene plant

• today: strongly polluted with toxaphene

⇒ no fishing/swimming

Ref.: W. Vetter, K. A. Maruya, Environ. Sci. Technol. 34 (2000) 1627-1635


Cl

Cl

HH

Cl

HH

ClH

H

H H

Cl

Cl

HH

H1

4

3

2

5

6

79

8

10

Terry Creek (GA, USA) sediment*

29 31 33 35

A2295W.D

[min]

Irel

m/z 309

m/z 307• B6-923

racemic

• established at sites with different history

• confirmed in lab-experiments with dehalogenating bacteria

* Ref.: W. Vetter, K. A. Maruya, Environ. Sci. Technol. 34 (2000) 1627-1635

Enantioselective determination of B6-923in biota from Terry Creek (GA, USA)

30.0 40.0 50.0 60.0 [min]

longnose gar

30.0 40.0 50.0 60.0 [min]

grass shrimp

A2115W.DA2146W.D

25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 [min]

striped mullet

A2148W.D

25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0 [min]

seatrout

A2150W.D

Ref.: W. Vetter, K. Maruya, Environ. Sci. Technol. 34 (2000) 1627-1635

B6-923racemic in sediment, non-racemic in fish

Cl

Cl

H H

Cl

H H

Cl H

H

H H

Cl

Cl

H H

H 1

4

3

2

5

6

7 9

8

10

• alteration of ER in biota compared to sediment is a direct proof of biotransformation

• 2nd eluting B6-923 enantiomerless stable

• ER in fish must represent equilibrium between uptake and elimination

• proof by elimination study with fish

sediment

fish

m/z 309


B6-923 elimination study with fish

Cl

Cl

H H

Cl

H H

Cl H

H

H H

Cl

Cl

H H

H 1

4

3

2

5

6

7 9

8

10

sediment

fish

• equilibrium between uptake and elimination

• without utpake only elimination

• 2nd enantiomer should be eliminated faster

m/z 309


ToxapheneToxaphene eliminationelimination studystudywithwith fishfish ((FundulusFundulus sp.sp.))

weight: 2-5 g25.00 30.00 35.00 40.00 45.00 50.00 55.00 [min]

B6-923

sample at start

Ref.: W. Vetter, K. Smalling, K. Maruya, ES&T 35 (2001) 4444-4448

ToxapheneToxaphene eliminationelimination studystudywithwith fishfish ((FundulusFundulus sp.sp.))

Cl6 - Cl7 Cl8 - Cl9

weight: 2-5 g25.00 30.00 35.00 40.00 45.00 50.00 55.00 [min]

B6-923

25.00 30.00 35.00 40.00 45.00 50.00 55.00 [min]

sample at start

after 60 days

no B6-923



⇒ follows first order kinetics⇒ determination of half life in fish

36.50 36.50

start 3 days

Cl

Cl

H H

Cl

HH

ClH

H

H H

Cl

Cl

HH

H 1

4

3

2

5

6

79

8

10

enantiospecific elimination rate relim

dC/dt = -kC C=Coe-kt

Enantiomer t 1/2

B6-923-1 9.3 days

B6-923-2 4.7 days

36.50

14 days


B6B6--923 throughput by fish923 throughput by fish

• toxaphene-uptake via food is racemic(food web study: W .Vetter & K. Maruya, ES&T 34 (2000) 1627-1635)

• ER-shift is compensated by uptake of food• constant ER in starting sample enables

calculation of the “throughput“• application of 1st order kinetics:

– 91% of the original B6-923 pool transformed within 7 days

– model fish transforms 680 ng B6-923/day – bio mass 75 kg: elimination of 10.2 mg/day

Repetition of Repetition of thethe eliminationeliminationstudystudy at at lowerlower waterwater temperaturetemperature

5

10

15

20

25

30

0 10 20 30 40 50 60 70Time (d)

Tem

pera

ture

(o C)

Warm Exp. (April-June)Cold Exp (Nov-Jan)

Ref.: K. Maruya, K. Smalling, W. Vetter, Environ. Sci. Technol. 39 (2005) 3999

Repetition of Repetition of thethe eliminationeliminationstudystudy at at lowerlower waterwater temperaturetemperature

3 6 3 7 3 8 3 9

I r e l

[ m i n ]

.

3 6 3 7 3 8 3 9

I r e l

3 6 3 7 3 8 3 9

I r e l

3 6 3 7 3 8 3 9

I r e l

E2

[min]

3 6 3 7 3 8 3 9

I r e l

E2

3 6 3 7 3 8 3 9

I r e l

E2

three time zero samples

• relatively constant ER• slower overall-elimination at lower temperature

day 3 day 11 day 64

Time-dependent concentrations of B6-923 in the elimination study with fish

10

100

1000

0 20 40 60 80

Time (d)

E1

E2

E1+ E2

Conc.[ng/g]

• corrected for growth by normalization with B9-1679


Half-lives (t½) and first-order elimination rate constants (ke) for B6-923 and B7-1001 in Fundulus heteroclitus during cool and warm water conditions

1327t ½ B7-1001 (d)

-0.0532-0.02561ke B7-1001 (d-1)

714t ½ B6-923 (d)

-0.0983-0.0503ke B6-923 (d-1)

25 oC15 oC


Elimination of B6Elimination of B6--923 923 fromfrom fishfish at at high/high/lowlow waterwater temperaturetemperature

>100120 (120+0)n.d. (0+0)64 120 (120+0)41

>100110 (110+0)28 150 (150+0)20

612014 7.320011

2.62607 3.25801.63603 1.6512201.3830 0

ER[ng/g] sum(E1+E2)

ER[ng/g] sum(E1+E2)

15 °C25 °Ctime (d)

Classification of toxaphene congeners based on elimination kinetics and potential for biotransformation

B8-1414B9-1679B6-923Penta-2; B7-515

congenerexamples

yes(non-racemic)

no(racemic)

yes(non-racemic)

no(racemic)

evidence of enantio-selectivity

slow, high activationbarrier

slow, lowactivationbarrier

rapid, high activationbarrier

rapid, lowactivationbarrier

eliminationprocessesa

IVIIIIIIClassi-fication

a temperature dependence of elimination is represented by the Arrhenius equation k ae –Ea/RT

where k = reaction rate constant, Ea = activation energy (barrier), R = universal gas constant and T [K]

Thanks to co-workersand partners

thank you

for your attention !

Keith Maruya

Kelly Smalling

Gary Stern

GreggTomy

Steffen RuppeElke Stoll

Doreen Kirchberg

Richard Bartha

The behaviour of - Birmingham

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