ARTICLE IN PRESS

0043-1354/$ - se

doi:10.1016/j.w

�Correspond

E-mail addr

Water Research 38 (2004) 3693–3698

www.elsevier.com/locate/watres

Plasticizer metabolites in the environment

Owen Horna, Sandro Nallia, David Coopera,�, Jim Nicellb

aDepartment of Chemical Engineering, McGill University, 3610 University Street, Wong Building, Montreal,

Que. H3A 2B2, CanadabDepartment of Civil Engineering and Applied Mechanics, McGill University, 817 Sherbrooke Street West Montreal,

Que. H3A 2K6, Canada

Received 22 July 2003; received in revised form 17 May 2004; accepted 30 June 2004

Abstract

Earlier work with pure cultures had shown that the interaction of microbes with plasticizers leads to the formation of

metabolites including 2-ethylhexanoic acid and 2-ethylhexanol that resist further degradation. The presence of these

metabolites is now reported in a variety of environmental samples. Thus, even in a complex ecosystem, when plasticizers

are degraded, the breakdown is not complete and significant amounts of 2-ethylhexanoic acid and 2-ethylhexanol are

observed. These compounds have been shown to exhibit acute toxicity using Microtox, Daphnia, rainbow trout and

fathead minnow toxicity assays. Since it is already well established that plasticizers are ubiquitous in the environment, it

is expected that their recalcitrant metabolites will also be ubiquitous. This is of concern because, while the plasticizers

do not exhibit acute toxicity, their metabolites do.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Adipate; Phthalate; Plasticizers; Plastics; Metabolites; Toxicity

1. Introduction

Plasticizers are clear, colourless, oily liquids that are

used to impart properties such as flexibility and work-

ability to plastics. They are widely used in many

products including medical equipment, food film,

upholstery, flooring, moldings, gaskets, piping, rain-

wear, electrical wire insulation, auto trim and under-

coating, pool and pond liners and roofing systems.

Plasticizers are also used in other applications such as

paints in which special coating properties are required.

Polyvinyl chloride (PVC) is an excellent example of the

magnitude of the industrial importance of plasticizers.

The annual production of PVC is in the tens of millions

of tons of which plasticizers can account for up to 40%

e front matter r 2004 Elsevier Ltd. All rights reserve

atres.2004.06.012

ing author.

ess: david.cooper@mcgill.ca (D. Cooper).

by weight (Mersiowsky et al., 2001). The most common

plasticizer is di (2-ethylhexyl) phthalate (DEHP) whose

production is estimated at 1.4 million tonnes per year

(Bauer and Herrmann, 1997).

It has been demonstrated that plasticizers tend to

leach from solid polymer matrices into the environment

(Fromme et al., 2002). Numerous studies have con-

firmed the presence of plasticizers in air, soil and water

samples to such an extent that they are now described as

being ubiquitous in the environment (Fromme et al.,

2002; Staples et al., 1997). Because DEHP is used so

extensively, many studies have been conducted to

evaluate its degradation within the environment using

soil organisms and in a laboratory setting using pure and

mixed cultures (Cartwright et al., 2000; Cassidy and

Irvine, 1999; Ejlertsson et al., 1997; Gejlsbjerg et al.,

2001; Marttinen et al., 2003; Roslev et al., 1998; Scholz

et al., 1997). These earlier researchers had reported that

d.

ARTICLE IN PRESSO. Horn et al. / Water Research 38 (2004) 3693–36983694

DEHP is either recalcitrant or is mineralized (Cart-

wright et al., 2000; Marttinen et al., 2003). However, it

has recently been shown that the exposure of common

plasticizers including DEHP and di-2-ethylhexyl adipate

(DEHA) to pure cultures of soil microorganisms under

laboratory conditions resulted in the production and

accumulation of toxic metabolites (Nalli et al., 2003,

2004). Because millions of tonnes of plasticizers are

incorporated into plastics every year and are ultimately

released to the environment, the objective of this work

was to establish whether the toxic metabolites are also

observed in the environment.

2. Experimental

2.1. Reagents

DEHP (99%), di (2-ethylhexyl) adipate (DEHA,

99%), 2-ethylhexanol (99%+) and 2-ethylhexanoic acid

(99%) were purchased from Sigma Aldrich (St. Louis,

MO). Lab grade acetone, HPLC grade water, HPLC

grade chloroform and hydrochloric acid were purchased

from Fischer Scientific (Montreal, QC)

2.2. Toxicity studies

The Daphnia (Daphnia magna) and the rainbow trout

(Oncorhynchus mykiss) toxicity assays of reagent grade

2-ethylhexanol and 2-ethylhexanoic acid were conducted

by Bodycote Material Testing Canada (Pointe-Claire,

QC, Canada). The lethal concentrations for 50%

mortality (LC50) were obtained by exposure to serial

dilutions (100%, 50%, 25%, 12.5% and 6.25% by

volume) of solutions of 300 mg/L 2-ethylhexanol or

1700 mg/L 2-ethylhexanoic acid. These solutions were

prepared with the pure reagents in de-chlorinated

municipal water. The LC50 was calculated using the

mortality after a 48 h period for the Daphnia and a 96 h

period for the rainbow trout.

The pure reagents were sent to Bodycote Material

Testing Canada (Ste- Foy, QC, Canada) for the fathead

minnow (Pimephales promelas) toxicity assay. Solutions

of 15 mg/L 2-ethylhexanol and 86 mg/L 2-ethylhexanoic

acid in carbon filtered municipal water with 88 mg/L

CaCO3 were prepared. The same serial dilutions noted

above were tested to obtain the 25% inhibition

concentration (IC25) representing the concentration that

would elicit a decrease in 25% of the organism’s body

weight over the course of a 7-day study. The IC25 was

estimated using linear interpolation.

Acute toxicity of these compounds based on the 5 min

Microtox toxicity assay has been previously reported

(Nalli et al., 2003). The assay involved the short-term

incubation of the putative toxic compound with the

luminescent marine bacterium Photobacterium phosphor-

eum. The concentration of toxicant that caused a 50%

decrease in light output is reported as the effective

concentration, EC50, and is presented as percent by

volume of the whole product mixture. The toxicity of a

sample increases as the EC50 decreases.

2.3. Sample collection and preparation

Samples were selected to reflect a wide range of water

quality from pure to highly contaminated. All the

samples were collected in 10 L Pyrexs glass reagent

bottles (Fisher Scientific, Montreal, QC). Before sam-

pling, the bottles were rinsed three times with lab grade

acetone, distilled water, and HPLC grade chloroform

for a total of nine washes. Aluminum foil was used as a

barrier between plastic screw tops and the samples to

avoid potential contamination of samples with plastici-

zers. The bottles were stored at 4 1C. Sediment and snow

samples were collected using 200 mL glass bottles, rinsed

as described above, and then transferred to 10 L reagent

bottles. All samples were collected manually as grab

samples.

Undisturbed snow was sampled from a green space in

downtown Montreal within 5 h of falling. The St.

Lawrence River site was at the downstream end of the

island of Montreal. River samples, including sediment

samples, were taken within 2 m of the shoreline. Water

samples were drawn from a creek that drains an

industrial area on the Island of Montreal and passes

through parkland (Bois-de-Liesse Park, Montreal, QC)

Landfill leachate was also collected from a landfill

located inside the boundaries of the city of Montreal.

The Miron landfill site was chosen to provide an

example of the type of contamination that could be

generated from typical urban waste. The sample was

collected through a sampling port on a pipe that

delivered the leachate that had been collected

from the landfill to an aeration basin. A sample of

HPLC-grade water from an unopened bottle was used as

a control.

The pH of the samples was reduced to 1.5 using 10 M

HCl and then the samples were extracted twice using

100 mL of HPLC-grade chloroform. The chloroform

from the combined extracts was evaporated and the

residue was re-dissolved in 1 mL of chloroform. Snow

was melted and then extracted using the same proce-

dure. The river sediment samples were extracted using

100 mL of HPLC grade chloroform in a Soxhlet

extraction apparatus. The chloroform extract was

prepared as above.

Tap water was sampled continuously from the

Montreal water distribution system. A total of 44.3 L,

at a flowrate of 1 L/h, was passed through 100 mL of

HPLC grade chloroform. The chloroform extract was

then prepared as above.

ARTICLE IN PRESSO. Horn et al. / Water Research 38 (2004) 3693–3698 3695

2.4. Gas chromatography/mass spectrometry

A Varian CP-3800 gas chromatograph with an FID

detector was used to quantify the concentrations of

DEHP and DEHA. The column used was the CP-Sil 5

CB 15 m� 0.53 mm (Varian, St-Laurent, QC). The

settings on the GC were as follows: an injector

temperature of 250 1C, an initial column temperature

of 40 1C, a hold time 2 min, a first temperature ramp rate

of 10 1C per minute to 150 1C and a second temperature

ramp rate 20 1C per minute to a final temperature of

300 1C. The detector temperature was 300 1C.

The quantification of the metabolites 2-ethylhexanol

and 2-ethylhexanoic acid was performed using a gas

chromatograph/mass spectrometer (Thermo Quest mod-

el TRACE GC 2000/Finnigan POLARIS), because of its

superior sensitivity to these compounds. The GC/MS

contained an RTX-5 MS internal diameter 0.25 mm

column (Resteck) and the settings on the GC were as

follows: an injector temperature of 275 1C, an initial

column temperature of 60 1C, a hold time of 1 min, a

first ramp rate of 5 1C per minute to 100 1C and a second

ramp rate of 20 1C per minute to a final temperature of

340 1C. Calibration curves were obtained for each

compound. The 95% confidence limits on the analysis

of DEHA, DEHP, 2-ethylhexanol and 2-ethylhexanoic

acid were plus or minus 5, 4, 0.1 and 0.4 mg/L,

respectively, based on six samples each.

Identification of the compounds was based on a

comparison to the GC retention times and the fragmen-

tation patterns from GC- mass spectrometry of plasti-

cizer and metabolite standards.

3. Results and discussion

Plasticizers such as DEHP or DEHA can be degraded

by a common soil bacterium in the presence of an easily

used carbon source (Nalli et al., 2003). The degradation

was not complete and resulted in the production of

metabolites including 2-ethylhexanol, 2-ethylhexanoic

acid and monoesters such as 2-ethylhexyl phthalate. A

proposed pathway for the degradation of DEHP or

DEHA is shown in Fig. 1. The final metabolite for

both DEHP and DEHA was 2-ethylhexanoic acid,

which was observed to be particularly resistant to

further degradation.

Initial work using the Microtox assay showed that

these metabolites were more toxic than the original

plasticizers. Toxicity tests have now been extended to

include assays based on various aquatic organisms

including fathead minnows, rainbow trout and Daphnia.

The results shown in Table 1 confirm that 2-ethylhex-

anoic acid and 2-ethylhexanol exhibit acute aquatic

toxicity. Notably, previous reports have also linked the

presence of 2-ethylhexanoic acid to peroxisome prolif-

eration of liver cells in rats and mice, which resulted in

tumour growth and death (Astill et al., 1996; Keith et

al., 1992; Lhuguenot et al., 1985; Sundberg et al., 1994).

The above results could have important implications

with respect to potential environmental impact. How-

ever, the production of metabolites by pure cultures

would not necessarily lead to their accumulation in the

environment if the whole ecology of a site results in

further degradation of these compounds. To assess this

possibility, a number of sites around the island of

Montreal, Canada were sampled. All of the samples

were found to contain the most common plasticizers

(Table 2), which is consistent with earlier work in which

plasticizers were found to be ubiquitous (Fromme et al.,

2002; Staples et al., 1997). Neither plasticizers nor any of

the possible metabolites were detected in the control

sample.

Most of the environmental samples also contained

one or more of the metabolites, which is consistent with

recent studies of the degradation of these plasticizers

using pure cultures (Nalli et al., 2003). This work had

shown that 2-ethylhexanoic acid was a very intractable

metabolite and, thus, it was not surprising to find

appreciable amounts of it in the environment. However,

it was surprising to find that these samples also

contained significant amounts of the less stable meta-

bolite, 2-ethylhexanol. This is important because the

alcohol has a higher acute toxicity than the acid (Table

1). This was unexpected because biological systems have

been shown to be able to easily oxidize alcohols

including 2-ethylhexanol to the corresponding car-

boxylic acids (Cheung et al., 2003; French et al., 2002).

River sediments had the highest concentrations of the

alcohol and acid. This is consistent with the fact that

these compounds are relatively hydrophobic and thus

likely to partition into the sediments. The concentrations

of metabolites in the sediment were so large that it was

even possible to observe a monoester, which is the first

metabolite in the series shown in Fig. 1. This was

surprising because the monoesters were seldom observed

even in the laboratory experiments conducted with pure

microorganisms and with high concentrations of plasti-

cizers (Nalli et al., 2003).

The samples of snow and tap water were expected to

have the lowest concentrations of the compounds of

interest. In fact, even these were contaminated. The data

for the sample of snow revealed that precipitation can

act as a means of introducing plasticizers into surface

waters. This sample also contained appreciable

amounts of the most intractable metabolite. The tap

water contained small amounts of the plasticizers and

the acid metabolite. It is probable that this

metabolite originated from the degradation of the

plasticizers, but it is unknown whether this compound

entered the water during processing or distribution or if

it came from the water source (St. Lawrence River) and

ARTICLE IN PRESS

O

O

O

O

OH

O

O

O

OH

OH

OH

O

OH

O

H

O

OH

O

R R+

+ R

R = or

Fig. 1. Mechanism for the production 2-ethylhexanol and 2-ethylhexanoic acid by the biological degradation of DEHP and DEHA.

Table 1

Comparison of 2-ethylhexanol and 2-ethylhexanoic acid toxicities using four acute toxicity assays

Compounds investigated Microtoxa Fathead minnow Rainbow trout Daphnia

EC50 (mg/L) IC25 (mg/L) LC50 (mg/L) LC50 (mg/L)

2-ethylhexanoic acid 43 23.0 (12–25)b 150 (110–220)b 120 (0–430)b

2-ethylhexanol 7.8 415 27 (20–39)b 100 (78–160)b

aToxicity data obtained from Nalli et al. (2003).bConfidence intervals for toxic concentrations obtained by bootstrapping.

O. Horn et al. / Water Research 38 (2004) 3693–36983696

passed through the treatment system. The samples of the

landfill leachate contained plasticizers in detectable

amounts, but neither 2-ethylhexanol nor 2-ethylhexa-

noic acid were detected. Since the production of

metabolites has only been observed during aerobic

growth, the absence of these compounds in the leachate

could be attributed to the anaerobic conditions of the

landfill.

The ubiquity of these metabolites and their

presence in significant concentrations indicate that these

ARTICLE IN PRESS

Table 2

Plasticizer and metabolite concentrations measured in environmental samples from the urban area of Montreal, Canada

Source Concentration (parts per billion)a

DEHAb DEHPc 2-ethylhexanol 2-ethylhexanoic acid

HPLC grade water 0.0 0.0 0.0 0.0

Tap water 5.1 4.6 0.0 0.050

Melted snow 150 130 0.0 6.7

River water 14 180 0.85 3.2

River sedimentd 4400 110,000 100 110

Creek water 5.8 47 0.49 1.2

Landfill leachate 25 62 0.0 0.0

aIn mg/L for water samples and mg/kg for sediment samples.bDi-(2-ethylhexyl) adipate.cDi-(2-ethylhexyl) phthalate.dTrace amounts of mono 2-ethylhexyl phthalate were also observed .

O. Horn et al. / Water Research 38 (2004) 3693–3698 3697

compounds resist degradation in environments where

mixed microbial populations would be present. There

are several recent references, which briefly mention the

observation of either the alcohol or the acid in studies

involving analyses of samples of precipitation and

indoor air. However the sources of these compounds

were not suggested (Balestrini and Polesello, 1999;

Sartin et al., 2001; Sunesson et al., 1996). The presence

of the 2-ethylhexanoic acid in these and in our own

samples substantiates concerns about the fate of the

plasticizers in the environment.

It must be acknowledged that there are other possible

sources of the alcohol and acid besides the metabolism

of plasticizers. For example, they could originate from

plasticizer production processes, the use of lubricants

and surfactants, as well as the pharmaceutical industry

(Staples, 2001). However, it has been claimed that these

sources are well contained and that the main sources of

exposure would be due to accidental spills (Staples,

2001). These sources could not account for the

quantities observed in our studies nor their presence in

many different environmental samples. In addition, the

mono-ester observed in the sediment sample could only

come from the metabolism of the di-ester, since the half-

life of DEHP degradation by abiotic alkaline hydrolysis

has been reported to exceed 1000 years at neutral pH

(Wolfe et al., 1980). Thus, these observations support

the argument that the metabolites that were observed in

the environment originated from the biodegradation of

plasticizers.

4. Conclusions

As plasticizers themselves are ubiquitous and can be

partially degraded by common soil bacteria, we con-

clude that plasticizer metabolites will also be wide-

spread. While the levels of metabolites observed in this

study may not result in acute aquatic toxicity, it is likely

that their intractability will lead to a tendency for them

to persist in the environment.

Acknowledgements

This research was supported by grants from the EJLB

Foundation and the Natural Sciences and Engineering

Research Council of Canada. Nalli and Horn acknowl-

edge financial support received through the Eugenie

Ulmer Lamothe fund of the Department of Chemical

Engineering, McGill University.

References

Astill, B.D., Gingell, R., Guest, D., Hellwig, J., Hodgson, J.R.,

Kuettler, K., Mellert, W., Murphy, S.R., Sielken Jr., R.L.,

Tyler, T.R., 1996. Oncogenicity testing of 2-ethylhexanol in

Fischer 344 rats and B6C3F1 mice. Fund. Appl. Toxicol. 31

(1), 29–41.

Balestrini, R., Polesello, S., 1999. Identification of an unusual

contaminant in wet deposition samples: 2-ethylhexanoic

acid. Ann. Chim. (Rome) 89 (7–8), 631–636.

Bauer, M.J., Herrmann, R., 1997. Estimation of the environ-

mental contamination by phthalic acid esters leaching from

household wastes. Sci. Total Environ. 208 (1,2), 49–57.

Cartwright, C.D., Thompson, I.P., Burns, R.G., 2000. Degra-

dation and impact of phthalate plasticizers on soil microbial

communities. Environ. Toxicol. Chem. 19 (5), 1253–1261.

Cassidy, D.P., Irvine, R.L., 1999. Use of calcium peroxide to

provide oxygen for contaminant biodegradation in a

saturated soil. J. Hazard. Mater. 69 (1), 25–39.

Cheung, C., Davies, N.G., Hoog, J.-O., Hotchkiss, S.A.M.,

Smith Pease, C.K., 2003. Species variations in cutaneous

alcohol dehydrogenases and aldehyde dehydrogenases may

ARTICLE IN PRESSO. Horn et al. / Water Research 38 (2004) 3693–36983698

impact on toxicological assessments of alcohols and

aldehydes. Toxicology 184 (2–3), 97–112.

Ejlertsson, J., Alnervik, M., Jonsson, S., Svensson, B.H., 1997.

Influence of water solubility, side-chain degradability, and

side-chain structure on the degradation of phthalic acid

esters under methanogenic conditions. Environ. Sci. Tech-

nol. 31 (10), 2761–2764.

French, K.J., Rock, D.A., Rock, D.A., Manchester, J.I.,

Goldstein, B.M., Jones, J.P., 2002. Active site mutations

of cytochrome P450cam alter the binding, coupling, and

oxidation of the foreign substrates (R)- and (S)-2-ethylhex-

anol. Archi. Biochem. Biophys. 398 (2), 188–197.

Fromme, H., Kuchler, T., Otto, T., Pilz, K., Muller, J.,

Wenzel, A., 2002. Occurrence of phthalates and bisphenol A

and F in the environment. Water. Res. 36 (6),

1429–1438.

Gejlsbjerg, B., Klinge, C., Madsen, T., 2001. Mineralization of

organic contaminants in sludge-soil mixtures. Environ.

Toxicol. Chem. 20 (4), 698–705.

Keith, Y., Cornu, M.C., Canning, P.M., Foster, J., Lhuguenot,

J.C., Elcombe, C.R., 1992. Peroxisome proliferation due to

bis(2-ethylhexyl) adipate, 2-ethylhexanol and 2-ethylhexa-

noic acid. Arch. Toxicol. 66 (5), 321–326.

Lhuguenot, J.C., Mitchell, A.M., Milner, G., Lock, E.A.,

Elcombe, C.R., 1985. The metabolism of di(2-ethylhexyl)

phthalate (DEHP) and mono(2-ethylhexyl) phthalate

(MEHP) in rats: in vivo and in vitro dose and time

dependency of metabolism. Toxicol. Appl. Pharmacol. 80

(1), 11–22.

Marttinen, S.K., Kettunen, R.H., Sormunen, K.M., Rintala,

J.A., 2003. Removal of bis(2-ethylhexyl) phthalate

at a sewage treatment plant. Water. Res. 37 (6),

1385–1393.

Mersiowsky, I., Weller, M., Ejlertsson, J., 2001. Fate of

plasticized PVC products under landfill conditions: a

laboratory-scale landfill simulation reactor study. Water

Res. 35 (13), 3063–3070.

Nalli, S., Cooper, D.G., Nicell, J.A., 2003. Biodegradation of

plasticizers by Rhodococcus rhodochrous. Biodegradation

13 (5), 343–352.

Nalli, S., Horn, O. J., Grochowalski, A. G., Cooper, D. G.,

Nicell, J. A., 2004. Origin of 2-ethylhexanol as a VOC.

Indoor Air, Submitted for publication.

Roslev, P., Madsen, P.L., Thyme, J.B., Henriksen, K., 1998.

Degradation of phthalate and di-(2-ethylhexyl)phthalate by

indigenous and inoculated microorganisms in sludge-

amended soil. Appl. Environ. Microbiol. 64 (12),

4711–4719.

Sartin, J.H., Halsall, C.J., Davison, B., Owen, S., Hewitt, C.N.,

2001. Determination of biogenic volatile organic com-

pounds (C8–C16) in the coastal atmosphere at Mace Head,

Ireland. Anal. Chim. Acta 428 (1), 61–72.

Scholz, N., Diefenbach, R., Rademacher, I., Linnemann, D.,

1997. Biodegradation of DEHP, DBP, and DINP: poorly

water soluble and widely used phthalate plasticizers. Bull.

Environ. Contam. Toxicol. 58 (4), 527–534.

Staples, C.A., 2001. A review of the environmental fate and

aquatic effects of a series of C4 and C8 oxo-process

chemicals. Chemosphere 45 (3), 339–346.

Staples, C.A., Peterson, D.R., Parkerton, T.F., Adams, W.J.,

1997. The environmental fate of phthalate esters: a literature

review. Chemosphere 35 (4), 667–749.

Sundberg, C., Wachtmeister, C.A., Lundgren, B., DePierre,

J.W., 1994. Comparison of the potencies of (+)- and (-)-2-

ethylhexanoic acid in causing peroxisome proliferation and

related biological effects in mouse liver. Chirality 6 (1),

17–24.

Sunesson, A.-L., Nilsson, C.-A., Andersson, B., Blomquist, G.,

1996. Volatile metabolites produced by two fungal species

cultivated on building materials. Ann. Occup. Hyg. 40 (4),

397–410.

Wolfe, N.L., Steen, W.C., Burns, L.A., 1980. Phthalate ester

hydrolysis: linear free energy relationships. Chemosphere 9

(7–8), 403–408.