Determination of Alkylphenols in Coastal Waters, Sediments and Tissues by
Steam Distillation Extraction and Gas Chromatography-Mass Spectrometry
Chin-Yuan Cheng1, Li-Lian Liu2, Wang-Hsien Ding1*
1
Department of Chemistry, National Central University, Chung-Li, Taiwan 320
2
Institute of Marine Biology, National Sun Yat-sen University, Kaohsiung, Taiwan 804
The contamination of soil, water and organism by organic pollutants is a seriously
environmental problem. The sources of these organic pollutants are mostly produced by
man-made chemicals such as alkylphenol polyethoxylates. Alkylphenol polyethoxylates
(APnEOs, n = number of ethoxy units) are nonionic surfactants wildly used in the textile
industries, dyeing industries, household detergents and other commercially applications.
Approximately 80% of the APnEOs are nonylphenol polyethoxylates (NPnEOs) and another
20% are mainly octylphenol polyethoxylates (OPnEOs). The biodegradation of APnEOs
under aerobic conditions leads shortening ethoxy chain and yields mainly to the formations of
alkylphenol mono- and diethoxylates, whereas under anaerobic conditions fully
deethoxylated alkylphenols (APs) is also formed. Indeed, APs have been estimated that they
are persistence organic pollutions which are not easily degraded by microorganisms. APnEOs
and their relative metabolites have been demonstrated as endocrine disruptors which can
cause estrogenic effects in fish and underwater organisms. Consequently, it is necessary to
develop a reliable method for routinely determining the levels of alkylphenolic compounds in
the environment.
NPnEOs are a major class of nonionic surfactants widespread used in Taiwan and an
unusual survey was elucidated the concentration of NPnEOs in household detergents and
cleaners. The results have been shown that NPnEOs were detected in 41% of 90 household
detergents at concentrations from 0.2 to 21%. The highest concentration of NPnEOs was
detected in a laundry detergent especially designed for washing socks. In Taiwan, the
wastewater of APnEOs was directly discharged into the rivers due to Taiwanese deficient
municipal wastewater treatment. We can realize that APs and their short ethoxy chain
metabolites had been impacted Taiwanese aquatic environment. The most comprehensive
survey from Taiwan reported concentrations of nonylphenol isomers (NPs) in 107 samples
from 40 rivers that are affected by municipal or industrial wastewater effluents and more than
54% of the samples had detectable levels of NPs in the range 0.89 to 50.0 g/L. In the
sediments, the 19 samples of 6 rivers were found NPs in 74% and the concentrations were
detected in the range 250 to 8580 μg/g. The concentrations of NPs in Taiwanese rivers and
sediments are higher than those of other countries, however the reports of APs concentrations
in the estuary waters and tissues are unknown. The coastal cultivation undergoes a few affect
by the tidal current and the species of tissues along the coast can show that how degrees of
pollution in the local area. The goal of this study is setting the levels of APs in oysters
(Crassostrea gigas) and snails (Thais clavigera, T. rufotincta, T. keluo) as an indicator that
shows the degrees of pollution corresponding to the local rivers.
The tissues (oysters and snails), coastal waters and estuary sediments were collected in
Tai-shi and Chi-ku. Before stored at 0℃, the tissues were freeze-dried and the coastal waters
were acidified to pH 3.0 with hydrochloric acid. The coastal waters were extracted by Oasis
HLB solid-phase extraction (SPE). The SPE procedures were conditioned by 3 ml methyl
tertiary-butyl ether (MTBE), 3 ml methanol, 3 ml deionized water (pH 3.0), then eluted by 3
ml MTBE finally. The extracts dried by sodium sulfate anhydrous and evaporated to dryness
using a gentle stream of purified nitrogen. The residues were redissolved in 100 μl
dichloromethane containing chryene-d12 as internal standard. For solid samples (sediments
and tissues), the tissues and sediments were extracted by steam distillation extraction (SDE).
This modified Nielson-Kryger steam distillation technique for extraction of organics from
solid samples, based on the vapor pressure and water solubility of analyte, has been reported
as a simple and highly efficient method to extract semi-volatile compounds from solid
materials and 4 ml n-hexane was added to the apparatus as extraction solvent. The SDE
method minimized the matrices of large molecular weight interferences (i.e., lipids or humic
acids) and used far less solvent than other extraction methods (i.e., Soxhlet extraction). The
extraction parameters were optimized using APs spiked Kaolin samples and the average
recoveries of APs were above 80% with RSD below 9%. Before SDE, liquid media
(deionized water) were adjusted to pH 7.0, transferred the samples and liquid media into 250
ml round-bottom flask with proper sodium chloride added to increase recoveries of APs. The
SDE was run for 1 hour and the SDE extracts were treated as coastal water extracts which
extracted after SPE procedures. The alkylphenolic compounds were identified and quantitated
by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode.
The extracts (1 l) were injected with the injection temperature at 300C in the splitless mode.
A DB-5MS capillary column (30 m  0.25 mm I.D., 0.25 m film thickness, J&W, CA, USA)
was used. The GC temperature program was as follows: 70C for 2 min, followed by a
temperature ramp at 30C/min to 130C, then temperature ramp at 8.5C/min to 300C, and
hold for 5 min, and total 29 min. The calibration curve of APs ranged 0.1 g/mL to 10 g/mL
and the correlation coefficients exceeded 0.997. The quantitation limit of this method was 10
ng of the alkylphenolic compounds (S/N>10).
APnEOs and their metabolites were discharged into the bay following the downstream,
however the concentrations of APs were too low to detect in the coastal water samples at
Tai-shi and Chi-ku. Therefore, alkylphenolic compounds may become associated with
organic matter in sediments. In Tai-shi, the concentrations of NPs in estuary sediments ranged
from 2 to 26 ng/g (n=15, wet weight); in Chi-ku, the concentrations of NPs in estuary
sediments ranged from <1 to 137ng/g (n=15, wet weight). The highest concentrations of NPs
in estuary sediments were detected near the oyster farms. Hence, the results showed APs
maybe accumulated in aquatic species, including oysters and snails.
In Tai-shi, the total concentrations of OP and NPs in oyster and snail tissues ranged from
150 to 400 ng/g and 450 to 870 ng/g (n=5, dry weight), respectively; In Chi-ku, the total
concentrations of OP and NPs in oyster and snail tissues ranged from 100 to 780 ng/g and
280 to 1410 ng/g (n=7, dry weight), respectively. The levels of APs in tissues are higher than
those of other countries. The risk assessment for marine organisms and human health can be
estimated on the basis of these results.
The results in this study shows that APs tend to bioaccumulate in lipids of organisms or
adsorb onto sediment in the environment because of the physicochemical properties indicate
that APs are hydrophobic substances. These data represent APs in estuary sediments and
tissues have a potential harmful impact on the environment. The appropriate action is to ban
the use of APnEOs in commercially or industrial applications because of APs exert estrogenic
effects in oysters and snail along the Taiwanese coast.