Arch. Environ. Contam. Toxicol. 44, 77– 82 (2003)
DOI: 10.1007/s00244-002-1267-3
A R C H I V E S O F
Environmental
Contamination
a n d Toxicology
© 2003 Springer-Verlag New York Inc.
Nonylphenol and Nonylphenol Ethoxylates in Fish, Sediment, and Water from
the Kalamazoo River, Michigan
K. Kannan,1 T. L. Keith,1 C. G. Naylor,2 C. A. Staples,3 S. A. Snyder,1 J. P. Giesy1
1
Department of Zoology, 213 National Food Safety and Toxicology Center, and Institute for Environmental Toxicology, Michigan State
University, East Lansing, Michigan 48824, USA
2
Huntsman Corporation, Austin, Texas 78752, USA
3
Assessment Technologies Incorporated, Fairfax, Virginia 22030, USA
Received: 18 December 2001 /Accepted: 13 May 2002
Abstract. A survey measuring concentrations of nonylphenol
(NP) and its ethoxylates (NPEs) in fish was performed in the
Kalamazoo River, Michigan, USA, in 1999. Of 183 fish analyzed, 59% had no detectable NP or NPE. Detected concentrations were reported to range from 3.3 (limit of detection) to
29.1 ng NP/g wet weight. To further explore the means of
exposure of NP and NPE in the fish, concentrations of NP and
its mono-through tri-ethoxylates (NPE1–3) were measured in
fish, sediment, and water collected near two wastewater treatment plants on the Kalamazoo River in 2000. Samples were
analyzed using exhaustive steam distillation with concurrent
liquid extraction. Nonylphenol ethoxycarboxylates (NPE1–3C)
were also analyzed in water. Concentrations of NP and NPEs in
fish were less than the method detection limits (MDLs) in all
the samples except one fish, which contained 3.4 ng NP/g wet
weight, just above the detection limit of 3.3 ng/g. Three of 36
sediments and 1 of 24 water samples contained detectable
concentrations of NP or NPE1. NPE2, NPE3, and NPEC were
not detected in water samples.
Alkylphenol ethoxylates, particularly nonylphenol ethoxylates
(NPEs), are widely used nonionic surfactants. NPEs are used as
detergents, emulsifiers, wetting agents, and dispersing agents in
household and industrial products and in agricultural applications. These compounds enter wastewater treatment plants
(WWTPs) through domestic and industrial discharges. Efficient biological wastewater treatment processes generally remove approximately 95% of the NPEs (Naylor 1995). In the
sewage treatment process, NPEs aerobically degrade to nonylphenol (NP), mono- and di-ethoxylates (NPE1–2) and ethoxy
carboxylates (NPECs) (Figure 1). Some of the degradation
products, such as NP, are more lipophilic and tend to sorb to
organic surfaces. For instance, log10 octanol-water partitioning
coefficient (Kow) of NP is in the range of 4.2 to 4.5 (McLeese
Correspondence to: K. Kannan; email: kuruntha@msu.edu
et al. 1981; Ahel and Giger 1993) and hence are associated
mainly with final sewage sludge. Sludge is usually applied to
fields as a biosolid, incinerated, or land-filled. The remaining
residues of the biological breakdown of NPE are discharged to
receiving waters and are the presumed source of exposure to
fish and other aquatic organisms. The occurrence of NP and
NPEs in sewage treatment plant effluents (Ahel et al. 1987;
Bennie et al. 1997) and in water and sediments has been
documented (Naylor et al. 1992; Nimrod and Benson 1996;
Maguire 1999; Snyder et al. 1999; Yamashita et al. 2000;
Kannan et al. 2001). Nonylphenol ethoxycarboxylates
(NPECs1–3) are aerobic degradation products of NPEs formed
during wastewater treatment (Ahel et al. 1987; Lee et al. 1997).
Some studies have reported the occurrence of NPECs in water
(Di Corcia et al. 1994; Field and Reed 1996; Lee et al. 1998).
Only a few studies have examined the occurrence of NP and
NPEs in fish (Lye et al. 1999; Tsuda et al. 2000; Keith et al.
2001). The first part of this study (Keith et al. 2001) reported
the occurrence of NP in fish tissues collected from the Kalamazoo River, Michigan, USA, in 1999. Of 183 fish analyzed, 41%
had concentrations ranging from 3.3 to 29 ng NP/g wet weight
(Keith et al. 2001). Some of the fish with detectable concentrations of NP were collected near WWTPs in the cities of
Kalamazoo and Battle Creek (Figure 2). This study was conducted to further examine the occurrence of NP, nonylphenol
mono- through triethoxylates (NPE1–3) in sediment, water, and
fish tissue collected at those same sites during July–August
2000. In addition, concentrations of NPE1–3C were also analyzed in water samples.
Materials and Methods
Samples
Sampling on the Kalamazoo River was conducted up and downstream
of the Kalamazoo and Battle Creek WWTPs in Michigan (Figure 2).
The stream flow rate at the time of sampling was approximately 500
cubic feet per s. The Battle Creek WWTP employs activated sludge
treatment process (secondary), and the Kalamazoo WWTP employs
78
K. Kannan et al.
Fig. 1. Chemical structures of NP,
NPE1, and NPE1C
Fig. 2. Map of Michigan showing sampling locations and sampling scheme
tertiary treatment (trickling filter). Water, sediment, and fish were
collected during July–August 2000. Bluegill sunfish (Lepomis macrochirus) and smallmouth bass (Micropterus dolomieui) were captured
by hook and line within approximately 100 m of the WWTP effluents.
On being caught, fish were immediately placed in coolers with ice and
taken to the laboratory for extraction and analysis. Weight of the fishes
ranged from 90 to 280 g. Water samples were collected along transects
across the river. Six upstream and six downstream water samples were
collected from each location (Battle Creek samples in July, and
Kalamazoo samples in August) using clean glass jars. Each sample
was collected 15 m apart. Surface sediments were collected using a
Ponar grab sampler on transects across the river and along the river
banks (Figure 2). A total of 6 upstream and 12 downstream sediment
samples were collected from each site. The uppermost 1–2 cm were
collected from each grab sample, placed in clean glass jars, transported
to the laboratory in coolers with ice and stored at ⫺20°C until analysis.
The depth of the water column at each sediment sampling sites ranged
1–2 m. Sediment at the sampling sites were mostly gravel with little
organic content (⬍ 1%).
Standards and Reagents
p-Nonylphenol, p-cumylphenol, and 4-tert-butyl ortho-cresol of
greater than 96% purity were obtained from Schenectady International (Freeport, TX). Standards of NPE1–3 and NPE1C (nonylphenoxyacetic acid sodium salt) were obtained from Huntsman Corporation (Austin, TX). The molar responses of NPE2C and NPE3C
were assumed based on the response of NPE1C. High-purity pesticide residue– grade n-hexane, dichloromethane (DCM), and
isooctane were obtained from Burdick and Jackson (Muskegon,
MI). Organic-free water was obtained by purification of reverse
osmosis–treated water followed by Nanopure™ (Barnstead, Dubuque, IA) treatment. All glassware and stainless steel homogenization equipment were rinsed with organic-free water followed by
high-purity pesticide residue– grade acetone and n-hexane. ACS
reagent grade sodium chloride was obtained from JT Baker (Phillipsburg, NJ). Reagent-grade concentrated sulfuric acid was obtained from EM Science (Gibbstown, NJ).
NP and NPEs in Fish, Sediment, and Water
79
Fig. 3. Methylation of NPE1C
Extraction
Steam distillation with concurrent liquid-liquid extraction was used for
the determination of NP and NPE1–3 in water, sediment, and fish.
Extraction and quantitation methods of NP and NPE1–3 have been
described in detail elsewhere (Snyder et al. 2001; Keith et al. 2001).
Fish were cut at the midsection from the pectoral to dorsal fin, which
included digestive and excretory system, and 20 g of this cross-section
was homogenized (Blender 700, Waring, New Hartford, CN). This
area of the fish was chosen for analysis because NP was likely to
accumulate in the digestive/excretory system. The homogenized sample was poured into a 2-L boiling flask. Twenty grams of sodium
chloride, 3 ml concentrated sulfuric acid, and 500 ng of the surrogate
standard, p-cumylphenol, were added. The sample was extracted using
a Nielsen-Kryger improved version steam-distillation column (Ace
Glass, Vineland, NJ) for a combined total of 3 h. The resulting extract
was concentrated to 1 ml using a nitrogen evaporator (Organomation
Associates, Berlin, MA). To further remove lipids from the sample
extract, a Perkin-Elmer (Norwalk, CT) series 200 autosampler and
binary pump and a Hewlett Packard (Palo Alto, CA) 1046A fluorescence detector were employed to separate lipids from the compounds
of interest. Eight hundred microliters of the isooctane extract was
fractionated using a Phenomenex Luna 5 ␮m silica column (250
mm ⫻ 4.6 mm; Torrance, CA) by isocratic elution with 12% 1:4
methanol: DCM and 88% hexane at a rate of 0.65 ml/min. Fluorescence detection was used to determine surrogate recovery during this
fractionation. A fraction of high-performance liquid chromatography
(HPLC) effluent was collected between 7 and 16 min, 3.0 ␮g 4-tertbutyl ortho-cresol added as an internal standard, and concentrated
under nitrogen to 100 ␮l isooctane. Compounds of interest were
identified and quantified using a Hewlett Packard 5890 series II plus
gas chromatograph (GC) and a HP 5972 mass selective detector (MSD)
operated under EI mode. Separation was accomplished using a 30-m
DB-17MS capillary column (0.25 mm ID, 0.25 ␮m film; J&W Scientific,
Folsom, CA). The GC was held at 100°C for 2 min and ramped to 300°C
at 4°C/min. The MSD was operated in selected ion monitoring (SIM)
mode with three ions monitored for each compound of interest. Internal
standard recoveries were greater than 80%. Matrix spike recoveries were
greater than 70% for NP and NPE1–2 and greater than 17% for NPE3.
Method detection limits (MDLs) for NP, NPE1, NPE2, and NPE3 were
3.3, 16.8, 18.2, and 20.6 ng/g wet weight, respectively.
For sediment and water samples, 100 g wet weight and 500 ml,
respectively, were used for extraction as described. Twigs and pebbles
were removed from sediments prior to extraction. The detection limits of
NP, NPE1, NPE2, and NPE3 in sediment were 5.5, 20, 25, and 30 ng/g dry
weight, respectively. An aliquot of sediment sample was dried at 60°C for
48 h to determine the moisture content. Sediment from both locations
contained polycyclic aromatic hydrocarbons (PAHs) at ppm levels. PAHs
are known to interfere with the analysis of NP when HPLC with fluorescence detection is used. Therefore, NP concentrations were confirmed
with GC/MSD with SIM. The detection limits of NP, NPE1, NPE2, and
NPE3 in water were 2.6, 2.7, 16, and 18 ng/ml, respectively.
The method described by Field and Reed (1996) with slight modifications was used for the determination of NPE1C in water. The
method involves solid phase extraction of water with anion-exchange
Empore disks® (3M). Water samples (500 ml) were passed through
25-mm anion-exchange Empore disks using a vacuum manifold. Disks
were then placed in a 2-ml gas chromatography autosampler vial. One
hundred microliters of iodomethane (CH3I; Aldrich) and 900 ␮l acetonitrile were added to the autosampler vial, which was then heated at
80°C for 1 h in a sand bath. Derivatization with iodomethane converts
nonylphenoxyacetate to its methyl ester (Figure 3). The methyl ester
was analyzed using a GC/MSD with selected ion monitoring (Figure
4). To determine the accuracy and precision, matrix spike and recovery
experiments were performed using deionized water. The MDL for the
derivatized NPE1C was 1.7 ng/ml. MDLs for NPE2C and NPE3C were
10.5 and 8.7 ng/ml, respectively.
Results
Fish
Ten bluegill sunfish at the Battle Creek WWTP location and
eight sunfish and two smallmouth bass at the Kalamazoo
WWTP location were analyzed for NP and NPE residues
(Table 1). For the bluegill sunfish collected in Battle Creek,
concentrations of NP, NPE1, NPE2 and NPE3, were all less
than their MDLs of 3.3, 16.8, 18.2, and 20.6 ng/g wet weight,
respectively. This contrasts with results of the July–November
1999 in Battle Creek sampling, which found a mean concentration of NP in 26 bluegill sunfish and 2 smallmouth bass of
17.2 ng/g wet weight (range: ⬍3.3–29.1 ng/g) (Keith et al.
2001). For the fish collected near the Kalamazoo WWTP
effluents in early July or early August, 9 of 10 contained no
detectable concentrations of NP and NPEs. One bluegill sunfish collected downstream of the Kalamazoo WWTP effluent
contained 3.4 ng NP/g wet weight, which is just above the
MDL of 3.3 ng/g. This is different from the results for fish
collected in 1999 in which rock bass (Ambloplites rupestris),
the only species analyzed at that time, contained detectable
concentrations of NP in 70% of the samples with a mean
concentration of 12.3 ng NP/g wet weight (Keith et al. 2001).
Sediment
NP and NPE were not detected in any of the six sediment
samples from upstream of the Battle Creek WWTP (Tables 1
and 2). Three of 12 downstream sediment samples nearest
80
K. Kannan et al.
Fig. 4. GC chromatogram and mass
spectrum of NPEC derivative
(from 0 to approximately 30 m) the effluent pipe contained
measurable concentrations of NP (15.3, 7.0, and 5.8 ng NP/g
dry weight), but no NPEs were detected. Similarly, none of the
six sediments from upstream of the Kalamazoo WWTP contained detectable concentrations of NP or NPEs. In 2 of the 12
downstream sediment samples nearest the effluent pipe, NP
was found at 2– 4 ng/g dry weight, and NPEs were not detected
in any downstream sediment sample.
Water
Results of the analysis of water samples are shown in Table 1.
Samples collected upstream of the Battle Creek WWTP con-
tained no detectable concentrations of NP, NPEs, or NPECs.
Two of the 12 downstream samples closest to the effluent
contained trace concentrations of NP and NPE1, which were
quantified below their MDL. Although recovery of matrix
spike or internal standard was high (⬎ 87%), no NPEC compounds were detected at concentrations greater than the MDL
for the derivatized NPE1C of 1.7 ␮g/L in either upstream or
downstream samples collected near the Battle Creek WWTP.
At the Kalamazoo WWTP, no detectable concentrations of NP,
NPE, or NPEC were found. In 3 of the 12 samples collected
downstream of the Kalamazoo WWTP, trace concentrations of
NP and NPE1 were quantified below their MDLs, but not
NPE2–3 or NPEC. The sample collected closest to the effluent
contained a concentration of 1.1 ␮g/L NP and 2.0 ␮g/L NPE1.
NP and NPEs in Fish, Sediment, and Water
81
Table 1. Concentrations of NP, NPEs, and NPECs in water, sediment, and fish from the Kalamazoo River
Sample
Fish (ng/g wet weight)
Battle Creek WWTP
Kalamazoo WWTP
Sediment (ng/g dry weight)
Battle Creek WWTP (up)
Battle Creek WWTP (down)
Kalamazoo WWTP (up)
Kalamazoo WWTP (down)
Water (␮g/L)
Battle Creek WWTP (up)
Battle Creek WWTP (down)
Kalamazoo WWTP (up)
Kalamazoo WWTP (down)
NP
NPE1
NPE2
NPE3
NPE1C
⬍ 3.3 (n ⫽ 10)
⬍ 3.3 (n ⫽ 9);
3.4 (n ⫽ 1)
⬍ 16.8 (n ⫽ 10)
⬍ 16.8 (n ⫽ 10)
⬍ 18.2 (n ⫽ 10)
⬍ 18.2 (n ⫽ 10)
⬍ 20.6 (n ⫽ 10)
⬍ 20.6 (n ⫽ 10)
NA
NA
⬍ 5.5 (n ⫽ 6)
⬍ 5.5 (n ⫽ 9);
5.8 to 15.3 (n ⫽ 3)
⬍ 5.5 (n ⫽ 6)
⬍ 5.5 (n ⫽ 12)
⬍ 20.1 (n ⫽ 6)
⬍ 20.1 (n ⫽ 12)
⬍ 24.5 (n ⫽ 6)
⬍ 24.5 (n ⫽ 12)
⬍ 30.1 (n ⫽ 6)
⬍ 30.1 (n ⫽ 12)
NA
⬍ 20.1 (n ⫽ 6)
⬍ 20.1 (n ⫽ 12)
⬍ 24.5 (n ⫽ 6)
⬍ 24.5 (n ⫽ 12)
⬍ 30.1 (n ⫽ 6)
⬍ 30.1 (n ⫽ 12)
NA
⬍ 2.7 (n ⫽ 6)
⬍ 2.7 (n ⫽ 6)
⬍ 2.7 (n ⫽ 6)
⬍ 2.7 (n ⫽ 5)
2.0 (n ⫽ 1)
⬍ 15.6 (n ⫽ 6)
⬍ 15.6 (n ⫽ 6)
⬍ 15.6 (n ⫽ 6)
⬍ 15.6 (n ⫽ 6)
⬍ 17.8 (n ⫽ 6)
⬍ 17.8 (n ⫽ 6)
⬍ 17.8 (n ⫽ 6)
⬍ 17.8 (n ⫽ 6)
⬍ 1.7 (n ⫽ 6)
⬍ 1.7 (n ⫽ 6)
⬍ 1.7 (n ⫽ 6)
⬍ 1.7 (n ⫽ 6)
⬍ 2.6 (n ⫽ 6)
⬍ 2.6 (n ⫽ 6)
⬍ 2.6 (n ⫽ 6)
⬍ 2.6 (n ⫽ 5)
1.1 (n ⫽ 1)
NA ⫽ not analyzed; fish samples were collected within 100 m of outfalls.
Table 2. Concentrations of NP in sediments (ng/g, dry weight
along the gradient from sewage outfall
Location
Battle Creek
Kalamazoo
Distance from
Outfall (m)
0
15
30
ⱖ 45
⬎0
Concentration
(ng/g, dry weight)
15.3
7.0
5.8
⬍ 5.5
⬍ 5.5
Discussion
The Kalamazoo River basin receives effluent discharges from
numerous sources, ranging from small municipalities to urban
areas. The most common biodegradation intermediates detected in wastewater effluents are lower ethoxylates, ether
carboxylates and NP (Snyder et al. 1999). It is expected that
fish and other aquatic organisms inhabiting the Kalamazoo
River would be exposed to NPE residues that enter the river via
permitted discharges. The results of this and an earlier study
suggest that concentrations of NP and NPE in fish taken from
the Kalamazoo River are relatively low. Overall, the two studies measured NP and NPE concentrations in 203 fish. Of these
fish, 65% had no detectable residues of NP or NPE. For the fish
with detectable NP and NPE residues, concentrations ranged
from 3.3 to 29.1 ng/g wet weight.
Concentrations of NP and NPE in fish collected from the
same river in different years gave varying results. This difference in tissue residues between sampling events could be
explained by a number of parameters. These include temporal
variations in wastewater concentrations of NP and NPE in
WWTP effluents, the volume of influent and effluent, river
water flows, climate and fish migratory behavior. For example,
despite the occurrence of NP in water, concentrations in fish
from several rivers and lakes in Japan were not detectable
(Tsuda et al. 2000).
NP concentrations in sediments ranged from less than the
MDL to 15.3 ng NP/g, dry weight. NP concentrations in
sediments were less than those reported for 30 rivers influenced
by municipal and industrial wastewater effluents (Naylor et al.
1992). Relatively lesser concentrations of NP in sediment
could be due to the small organic carbon content of sediments
(⬍ 1%). Concentrations in sediments from the Battle Creek
WWTP were greater than those from the Kalamazoo WWTP.
This could be due to differences in the wastewater treatment
process. The Battle Creek WWTP employs only secondary
treatment, whereas the Kalamazoo WWTP employs tertiary
treatment. Sediment NP concentrations downstream of the Battle Creek WWTP decreased with distance from the outfall
(Table 2). Concentrations of NP declined to near background
concentrations in sediments collected 1 km downstream from
WWTPs in the Detroit River (Bennett and Metcalfe 2000).
Only one water sample collected downstream of the Kalamazoo WWTP contained a measurable concentration of NP (1.1
␮g/L) and NPE1 (2.0 ␮g/L). Effluents from most WWTPs in
Michigan contained NP concentrations lower than 1 ␮g NP/L
(Snyder et al. 1999). NPEC were not detected in Kalamazoo
River water samples collected within 100 m downstream of the
WWTP discharges. Concentrations of NPEC in river water
collected from Wisconsin ranged from less than the MDL to
13.5 ␮g NPEC/L. The greatest concentration of 13.5 ␮g
NPEC/L, which was predominated by NPE2C (87%), was
detected in water collected downstream of a WWTP in Fox
River (Field and Reed 1996).
Overall, the results of this study show that NP and NPE
residues in fish tissue collected from a typical watershed in the
Great Lakes region of the United States are detected only about
35% of the time. This includes fish species that are bottomdwelling. Detectable concentrations were relatively low, ranging from about 3 to 29 ng/g wet weight.
Acknowledgments. Funding for this project was provided by the
Alkylphenol Ethoxylate Research Council (APERC).
82
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