Emerging Organic Pollutants in Waste Waters and Sludge

The Handbook of Environmental Chemistry Vol.
5, Part O (2005): 1– 24
DOI 10.1007/b98605
© Springer-Verlag Berlin Heidelberg 2005
Estrogens and Progestogens in Wastewater, Sludge,

Sediments, and Soil
Marina Kuster · Maria J. López de Alda (✉) · Damià Barceló
Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18–26,
08034 Barcelona, Spain
mlaqam@cid.csic.es
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Human Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Animal Farming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Sources and Fate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3.1 Environmental Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
5 Toxicity Identification Evaluation (TIE) Approaches . . . . . . . . . . . . . . . 14
6 Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6.1 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.2 Sample Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.3 Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Abstract Estrogens and progestogens are two classes of female steroidal hormones whose
presence in the environment has been associated with the appearance of certain alarming
reproductive and development effects, such as feminization, decreased fertility, and her-
maphroditism, in living organisms exposed to these compounds. Synthetic chemicals re-
sembling these natural hormones are now well established in human medicine (mainly as
contraceptives and for treatment of hormonal disorders) and in animal farming practices
(usually as growth promoters). They are therefore produced on a large scale every year. Mainly
due to unsuccessful removal in wastewater treatment plants, they are continuously released
into the aquatic environment.Adverse effects on aquatic wildlife at concentrations as low as
~1 ng L–1 have been reported. Studies have also shown that estrogens and progestogens are
easily distributed in the environment and may accumulate in river sediments. However,
little is known about their long-term environmental impact. In this chapter, the main sources
of estrogens and progestogens, their principal pathways into the aquatic environment, and
the primary routes of exposure to these compounds are discussed. This chapter also reviews
the methods described so far for the analysis of estrogens and progestogens in wastewater,
sludge, sediments, and soils as well as the environmental levels found in these compartments.
Keywords Estrogens · Progestogens · Environmental analysis · Occurrence · Fate

2 M. Kuster et al.
Abbreviations
APCI Atmospheric-pressure chemical ionization
EC European Community
EDC Endocrine disrupting compound
EEF Molar-based 17b-estradiol equivalency factor
ELISA Enzyme-linked immunosorbent assay
ER-CALUX Estrogen receptor-mediated chemically activated luciferase gene expression
assay
ESI Electrospray ionization
EXAMS Exposure assessment modeling system
FDA United States Food and Drug Administration
GC Gas chromatography
GPC Gel-permeation chromatography
HPLC High-performance liquid chromatography
LC Liquid chromatography
LLE Liquid–liquid extraction
LOD Limit of detection
LOQ Limit of quantitation
MCF-7 Cell proliferation (E-screen)
MS Mass spectrometry
MSTFA N-methyl-N-(trimethylsilyl)trifluoroacetamide
NI Negative ion
PFPA Pentafluoropropionic anhydride
PI Positive ion
RAM Restricted access material
SIM Selected ion monitoring
SPE Solid-phase extraction
SRM Selected reaction monitoring
STP Sewage treatment plant
TIE Toxicity identification and evaluation
USEPA United States Environmental Protection Agency
WW Wastewater
WWTP Wastewater treatment plant
YES Yeast-based recombinant estrogen receptor–reporter assay
1
Introduction
Chemicals used in a wide range of applications in our modern society are

produced on a large scale worldwide. Because of their physical and chemical
properties, many of these substances or their metabolites end up in the envi-
ronment, where they can induce adverse effects on wildlife organisms. The
environmental presence of endocrine disrupting compounds has become a hot
topic to the point that it competes with other priority health concerns such as
the environmental pollution by carcinogenic compounds [1]. Among the var-
ious categories of substances with reported endocrine disrupting properties –
polychlorinated organic compounds, pesticides, organotins, alkyl phenols and
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil 3
alkyl phenol ethoxylates, phthalates, bi-phenolic compounds, fitoestrogens and

microestrogens, etc. – the group of female sexual hormones and related syn-
thetic steroids stands out because of their estrogenic potency. Many studies
have confirmed the presence of estrogens and progestogens at concentrations
of toxicological concern in the aquatic environment. Already at very low con-
centrations of ~1 ng L–1 endocrine disrupting effects, such as decreased fertil-
ity, feminization, and hermaphroditism of aquatic organisms, are assigned to
this class of steroidal hormones [2–5]. Due to their strong endocrine disrupting
potency, special attention has been given to the natural estrogens estradiol and
estrone, as well as to the synthetic estrogen ethynylestradiol [6].
Synthetic chemicals, resembling the action of natural hormones, find wide
application in both human and veterinary medicine and in animal farming
practices. Both natural and synthetic estrogens and progestogens are elimi-
nated, either as free compounds or in their conjugated form, primarily through
the urine but also in the feces. These substances enter the aquatic environment
mainly via wastewater treatment plant (WWTP) effluents (after incomplete
removal in the plant) and untreated discharges, and through runoff of sewage
sludge used in agriculture [7, 8]. Once in the waterways they may undergo a
series of processes, such as photolysis, biodegradation, and sorption to bed-
sediments, where estrogens and progestogens may persist for long periods [9].
At present, the environmental occurrence of these substances is not subjected
to regulation. However, there are concrete indications that the presence of the
most active estrogens in the aquatic environment will be regulated in the near
future. This calls for efficient and reliable analytical methods for routine mon-
itoring and control. Since the consequences linked to the presence of these
compounds in the environment were first made public, numerous analytical
methods for their quantification in different environmental matrices have been
developed. Most of these methods have focused on surface waters, while waste-
water (WW), sludge, and principally sediments and soils, have received com-
paratively less attention, probably due to the complexity of these matrices. This
chapter reviews the most advanced methods applied to the analysis of estro-
gens and progestogens in these complex matrices, together with the environ-
mental levels found in these natural systems. The main sources of the most
environmentally relevant estrogens and progestogens, their physicochemical
properties, their principal pathways into the aquatic environment, the primary
routes of exposure to these compounds, and data regarding their activity as en-
docrine disruptors are discussed in this chapter.
2
Usage
Large quantities of pharmacologically active substances are used annually in

human medicine for diagnosis, treatment, and prevention of illness or to avoid
unwanted pregnancy. In animal and fish farming, drugs are mostly adminis-
4 M. Kuster et al.
tered as food additives for preventing illness, as growth promoters or parasiti-

cides [10]. During the last five decades, the consumption of estrogens and
progestogens for some of these purposes has experienced a steady growth, and
this fact, together with the discovery of their negative ecological effects, has
contributed to the current concern about their occurrence in the environment.
2.1
Human Medicine
In terms of binding to the human estrogen receptor, estradiol is the principal

endogenous phenolic steroid estrogen, which is oxidized in the metabolic
processes to estrone and further transformed to estriol. The natural hormones
are rapidly metabolized and are therefore orally inactive or active only at very
high concentrations. Blocking the oxidation to estrone by, for instance, intro-
ducing an ethynyl group in position 17a or 17b of estradiol leads to much more
stable products, which remain longer in the body. The consequence of this
increased stability is that the so-formed synthetic steroid ethynylestradiol is
excreted up to 80% unchanged in its conjugated form [11]. Estradiol also forms
the backbone structure used in the engineering of other synthetic estrogens,
such as mestranol and estradiol valerate, also utilized in human hormone treat-
ments [11].
One of the main applications of estrogens and progestogens is in contra-
ceptives. The estrogen content in birth control pills is usually in the range of
20 to 50 mg daily [12]. As for the progestogenic content, it varies depending on
the type of contraceptive. Thus, in combined oral formulations the progesto-
genic content is in the range of 0.25 to 2 mg daily, whereas in progestogen-only
contraceptives, it is lower (30–500 mg daily).
Besides contraception, the uses of estrogens can largely be put into three
main groups: the management of the menopausal and postmenopausal syn-
drome (its widest use); physiological replacement therapy in deficiency states;
and the treatment of prostatic cancer and of breast cancer in postmenopausal
women.
In the same way as estrogens, progestogens are used in the treatment of
several other conditions such as infertility, endometriosis, in the management
of certain breast and endometrial cancers, and either alone or in combination
with estrogens in the treatment of menstrual disorders, among others. The
therapeutic doses required in the treatment of many of these diseases are
often significantly larger than those employed in contraception.
2.2
Animal Farming
Estrogens and progestogens are mainly used as growth promoters in animal

farming, and for the development of single-sex populations of fish in aquacul-
ture. Some naturally occurring sexual steroids such as estradiol, progesterone,
and testosterone, and synthetic chemicals such as zeranol (estrogenic), melen-

gestrol acetate (gestagenic), and trenbolone acetate (androgenic) have growth-
promoting effects. Due to the improvement of weight gain and feed efficiency
in meat-production animals, administration of sex steroids to cattle has been
a common practice for many years in several meat-exporting countries, in-
cluding the USA. The most widely used substances are estrogens, either in the
form of 17b-estradiol, estradiol benzoate, or the synthetic zeranol. Proges-
terone, testosterone, and the two synthetic hormones trenbolone acetate and
melengestrol acetate are generally used in combination with estrogens [13]. In
contrast, no hormone applications for use in commercial-level poultry have
been United States Food and Drug Administration (FDA)-approved since the
agency’s withdrawal of the cancer-causing hormone diethylstilbestrol in the
1950s.
In the European Community (EC), the use of hormonal substances for the
promotion of animal growth is prohibited (Directive 96/22/EC). The ban was
applied without discrimination internally and to imports from third countries
as from January 1, 1989. As a result, countries wishing to export bovine meat
and meat products to the EC were required either to have an equivalent legis-
lation or to follow a hormone-free cattle program [14].
In aquaculture, steroidal compounds are used to develop single-sex popu-
lations of fish to optimize growth. Sex determination in fish is primarily under
genetic control but may be influenced by various environmental conditions,
such as temperature, social environment, pH, stocking density, and exposure to
exogenous hormones or hormone-like chemicals [15]. Thus, all-male [16] and
all-female [17] fish stocks may be obtained through exposure to androgens and
estrogens, respectively. The potencies of sex steroids to induce sex reversal are
different for each steroid. Functional sex reversal from female to male is carried
out by using 17a-methyltestosterone, 19-norethynyltestosterone, or methylan-
drosterone (concentration range: 0.1–100 mg/kg diet). 11-Ketotestosterone and
androsterone have also been used but the dosage required is higher than those
of synthetic androgens. Phenotypical feminization is induced successfully by
using estradiol, although estrone and ethynylestradiol are used as well [18].
3
Sources and Fate
Figure 1 shows the principal routes of environmental exposure to estrogens and

progestogens. The most relevant ways by which these compounds enter the
environment and reach aquatic systems or the food chain are through WWTP
effluents, untreated discharges, and runoff of manure and sewage sludge used
in agriculture [7, 8, 10, 19–21, 45].
Human excretion is thought to be the principal source of estrogens and
progestogens. These compounds are readily adsorbed from the gastrointestinal
tract and through the skin or mucous membranes, and are metabolized in the
6 M. Kuster et al.
Fig. 1 Routes of environmental exposure to estrogens and progestogens
liver with some undergoing enterohepatic recycling. Excreted hormones and

their metabolites are found in urine, usually as water-soluble conjugates, and
a small amount of “free” estrogens occur in feces [12, 22]. The normal daily
estrogen secretion of women is 24–100 mg, depending on the menstrual cycle,
and can rise to 30 mg toward the end of pregnancy.
The excreted hormones and metabolites collected in the sewer systems end
up in WWTPs, where different processes of varying efficiency are applied. Field
data suggest that the activated sludge treatment process can consistently remove
over 85% of estradiol, estriol, and ethynylestradiol, and a lower, variable per-
centage of estrone [23]. On the contrary, the concentration of unconjugated
steroids in the effluent of WWTPs has occasionally been found to be higher
than that in the corresponding influent. Thus, many studies suggest that the
conjugated forms (mainly glucuronides and sulfates) are readily converted to
the more active free compounds in both the sewer system and WWTPs, as a
result of the activity of the b-glucuronidase and arylsulfatase enzymes present
in these systems [7, 24–27].
In activated sludge treatment works the principal mechanisms for removal
of these compounds are likely to be sorption and biodegradation. Based on the
log Kow, sorption to sludge is predicted to play an important role in the removal
of hydrophobic compounds (e.g., mestranol) from the aqueous phase. However,
this does not seem to be the case for more hydrophilic compounds, such as
estriol, estrone, and their glucuronide and sulfate conjugates. With regards to
biodegradation, the extent of which depends on factors such as nitrifying bac-
teria, sludge retention times, aeration, and temperature, some laboratory test
studies indicate that estradiol is more readily mineralized than ethynylestradiol
or estrone and that synthetic estrogens in general exhibit greater recalcitrance

in the activated sludge process [23, 27, 28].
More advanced water purification techniques, utilizing UV-irradiation,
ozonization, or activated charcoal, may significantly improve the removal of
these compounds, but these techniques are not broadly applied due to their
high cost. Thus, current European activated sludge treatment plants, with a
hydraulic residence time not greater than 14 h, can in most cases not completely
eliminate all the estrogens and progestogens from the effluent [23].
As previously mentioned, contamination of water resources by estrogens
and progestogens may also occur through runoff from manure and sewage
sludge used in agriculture. Most of the drugs used for animals end up in their
urine and feces.When this manure, or the sludge from sewage treatment plants,
is dispersed onto the field, the unmetabolized drugs present or their metabolites,
depending on their mobility in the soil system, may reach the groundwater (as
a result of leaching from fields) or the surface water in the vicinity (through
runoff) and affect terrestrial and aquatic organisms [29].
Other disposal options for the sewage sludge are landfill, dumping at sea
(forbidden in the EU since 1998) [30], and incineration. The most popular for
solid waste disposal is landfill. However, many of the disposal sites are open
dumps without protective barriers or leachate-collection systems, which rep-
resent a potential risk to the quality of the nearby groundwater.
Another increasingly important source of estrogens and progestogens in the
environment is, as mentioned before, fish farming. Treatment with steroids is
usually carried out by feeding, although in species where male sex differentia-
tion is initiated before feeding commences (e.g., salmon), other procedures are
used, such as immersion of alevins [18]. Drugs used in aquaculture as feed ad-
ditives are discharged directly into the water. It has been estimated that around
70% of the drugs administered end up in the environment surrounding the
farm, due to overfeeding, loss of appetite by diseased fish, and poor adsorption
of the drugs [31].
3.1
Environmental Distribution
The introduction of estrogens and progestogens into the environment is a func-

tion of the way several factors are combined. The manufactured quantity and
the dosage applied (amount, frequency, and duration) combined with the
excretion efficiency of the compound and its metabolites, the capability of
adsorption and desorption on soil, and the metabolic decomposition in sewage
treatment are examples of necessary factors to assess environmental exposure.
In general the fate and effect of a substance in the environment is dependent on
the distribution into the different natural systems, such as air, water, and solids
(soil, particles, sediment, and biota). Information on the physical and chemical
properties (KH, Kd, and Kow vapor pressure) of a compound may help determine
whether it is likely to concentrate in the aquatic, terrestrial, or atmospheric
8 M. Kuster et al.
environment. Table 1 lists the main physicochemical characteristics of the most

relevant estrogens and progestogens from the environmental point of view.With
regards to the water solubility, it might be worth pointing out that the steroid
solubility in WW may be markedly lower than that in distilled water [32].
Once estrogens and progestogens have reached the waterways, a series of pro-
cesses, such as, photolysis, biodegradation, and sorption to bed-sediments, can
contribute to their elimination from the environmental water. Given the relatively
low polarity of these compounds, with octanol–water partition coefficients
mostly between 103 and 105, sorption to bed-sediments appears to be a likely
process. Kd values calculated for estriol, norethindrone, and progesterone in a
Spanish river (479, 128, and 204, respectively) as the ratio between the sediment
concentration (ng kg–1) and the water concentration (ng L–1) indicate that, in fact,
these compounds exhibit a general tendency to accumulate in sediments.
Jurgens et al. [33] carried out a series of laboratory experiments to study the
behavior of estrogens in the aquatic environment and set up a model to estimate
their likely environmental concentrations in the water column and bed-sedi-
ments. According to this study, between 13 and 92% of the estrogens entering a
river system would end up in the bed-sediment compartment with the major-
ity of sorption occurring within the first 24 h of contact.
A similar approach conducted by Lai et al. [9] to investigate the partition-
ing of estrogens from water to sediments, kinetics of sorption, and the influence
of various environmental variables (salinity, total organic carbon, etc.) indi-
cated that sorption takes place rapidly within the first half hour, slows down
within the next half hour, and steadily decreases afterward. Furthermore, the
synthetic estrogens (mestranol and ethynylestradiol), with their higher Kow val-
ues, were shown to partition to the sediment to a greater extent than the natural
estrogens. At higher estrogen concentrations, there was a decrease in estrogen
removal from the aqueous phase, while higher levels of sediment induced
greater removal. The sorption of estrogens to sediments correlated to the total
organic carbon content. However, the presence of organic carbon was not a
prerequisite for sorption. Tests performed with laboratory saline water resulted
in an increase of estrogen removal from the water phase compared to unsalted
waters, which is consistent with partitioning experiments using actual field
water samples. The addition of estradiol valerate, with a particularly high Kow,
suppressed sorption of other estrogens, suggesting that it competed with other
compounds for the binding sites.
A series of experiments was also conducted by Bowman et al. [34] to ascer-
tain the effects of differing environmental factors on the sediment–water inter-
actions of natural estrogens (estradiol and estrone) under estuarine conditions.
Sorption onto sediment particles was in this case relatively slow, with sorption
equilibrium being reached in about 10 and 170 h for estrone and estradiol,
respectively. On the other hand, true partition coefficients calculated on colloids
were found to be around two orders of magnitude greater that those on sedi-
ment particles. Hence, it was concluded that under estuarine conditions, and in
comparison to other more hydrophobic compounds, both estrone and estradiol
Table 1 Physico-chemical Properties of selected Estrogens and Progestogens [(http://esc.syrres.com/interkow/physdemo.htm)]
Compound CAS number Molecular Water Log Powc Vapor Henry’s law
weight solubility a, c pressure a, b constant a, b
(mg L–1) (mm Hg) (atm-m3 mole–1)
Estradiol 000050-28-2 272.39 3.6 (27 °C) 4.01 1.26E-008 3.64E-011

Estriol 000050-27-1 288.39 441b 2.45 1.97E-010 1.33E-012
Estrone 000053-16-7 270.37 30 3.13 1.42E-007 3.8E-010
Ethinyl estradiol 000057-63-6 296.41 11.3 (27 °C) 3.67 2.67E-009 7.94E-012
Diethylstilbestrol 000056-53-1 268.36 12 5.07 1.41E-008 5.8E-012
Norethindrone 000068-22-4 298.43 7.04 2.97 7.31E-009 5.8E-010
Progesterone 000057-83-0 314.47 8.81 3.87 1.3E-006 6.49E-008
D-Norgestrel 000797-63-7 312.46 2.05b 3.48 3.93E-010 7.7E-010
a
Values are at 25 °C if not specified.
b Estimated data.
c
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
Experimental data.
9
10 M. Kuster et al.
would be expected to remain mainly in the dissolved phase and to have a strong
tendency for bioaccumulation.
Nevertheless, it is not clear yet whether sorption or biodegradation processes
play a major role. Studies conducted with activated sludge [25, 35] pointed at
biodegradation as the mechanism contributing the most to the elimination
of estrogens from the aqueous phase, while losses by sorption effects were
considered rather unlikely. However, Jurgens et al. [33], based on a designed
exposure assessment modeling system (EXAMS) model, postulate that degra-
dation processes in rivers are unimportant under average flow conditions, as
they account for only 2–8% of the input loading. This is in agreement with the
theory presented by Huang et al. [36], according to which the main removal
mechanism for hormones in WWTPs would be sorption onto particles and not
biotransformation.
Degradation studies carried out in waters from five English rivers indicate
that estradiol has a half-life of 3–27 days [33]. Estrone was found to be the first
degradation product of estradiol but no investigations of the subsequent by-
products were conducted. The poorest degradation rates were observed in the
estuary river water samples, where the high salt content might have inhibited mi-
crobial degradation. Furthermore, ethynylestradiol (half-life 46 days) was found
to be more stable than 17b-estradiol (half-life 4 days, e.g., in the River Thames).
These half-life values might correspond to ideal summer temperatures. However,
under winter conditions these compounds could be twice as persistent.
In activated sludge, the synthetic estrogens ethynylestradiol and mestranol
have been shown to remain stable and intact over 5 days, while progestogens
are already up to more than 50% disintegrated after 48 h [32].
Under the anaerobic, dark conditions normally present in the subsurface
layers of river sediments, these compounds are expected to undergo a slow
photodecomposition and biodegradation. On the other hand, desorption from
sediments has been shown to be significantly less important than sorption, with
desorption distribution coefficients two to three times lower than those obtained
for the sorption process [33]. In an environment like this, river sediments can
therefore act as sinks where estrogens and progestogens may persist for long
periods, be transported to other areas, and be eventually released by diffusion
across the sediment water-column interface or by scouring in storm events [9].
The concentration of estrogens and progestogens in bed-sediments is predicted
to increase over time; thus, bed-sediments can be anticipated as environmental
reservoirs from where these substances may eventually become bioavailable [37].
4
Occurrence
Most research of estrogens and progestogens has been conducted on water sam-
ples and less frequently on solid samples. Soils and sediments, in particular, have
received very little attention and thus literature data on these matrices are very
scarce. The same applies to the analytes investigated. Whereas free estrogens,
both natural (e.g., estradiol, estrone, estriol) and synthetic (e.g., ethynylestradiol,
mestranol, diethylstilbestrol), have often been investigated, estrogen conjugates
[38, 39] and progestogens [40] have seldom been studied, probably due to their
lower estrogenic potency. Figure 2 shows the chemical structure of the estrogens
and progestogens most frequently investigated in environmental samples.
Table 2 summarizes the literature data available on the occurrence of these two
classes of steroidal compounds in WW, sludge, and sediments.
Natural hormones (and their metabolites) have always been present in the
environment. The growing use of both natural and synthetic estrogens and
progestogens in human medicine and in livestock farming (see Sect. 2, Usage)
has led to an increase of their occurrence in natural systems. Due to steady
population growth and regional population density, an irregular distribution
of these pollutants is found. Particular concern is given to certain areas where
high levels were detected, e.g., areas adjacent to agricultural and animal farms.
Table 2 Environmental occurrence of estrogens and progestogens
Matrix (location) Compounds Concentration Ref.

(ng L–1 or ng g–1)
WATER
Wastewater
STP influent
Æ effluent
Italy Estrogens and progestogens 0.4–188 Æ 0.3–82.1 [35]
Spain Natural and synthetic estrogens <0.2–115 Æ <0.2–21.5 [40]
The Netherlands Natural and synthetic estrogens <0.5–140 Æ <0.4–47 [59]
France Natural and synthetic estrogens 2–8.6 Æ 3.8–18 [66]
SOLID SAMPLES
River sediment
Germany Natural and synthetic estrogens <0.2–2 [45]
Spain Estrogens and progestogens 0.05–22.8 [46]
UK Estrone <0.04–0.388 [42]
Activated and
digested sludge
Germany Natural and synthetic estrogens <2–49 [45]
Activated sludge
Israel Estrogen 19–64 [19]
Soil – – –
BIOTA
Rainbow trout bile
Sweden Natural and synthetic estrogens <0.1–2.5 mg/g [48]
12 M. Kuster et al.
Fig. 2 Molecular structure of the environmentally most relevant natural and synthetic
estrogens and progestogens
As previously mentioned, discharged domestic effluents represent the most

significant input of estrogens and progestogens to the aquatic environment.
According to the studies carried out until now, mostly in densely populated
regions, estrogens and progestogens are normally present in domestic sewage
in the nanogram per liter range and occasionally in the microgram per liter
range (see Table 2). The nature of the compounds present in the sewage system
does, however, change as a function of their transport route.
D’Ascenzo et al. [39] have recently conducted a very comprehensive study
where the presence of the three most common natural estrogens and their
conjugated forms was investigated in female urine, in a septic tank collecting
domestic WW, and in influents and effluents of six activated sludge WWTPs.
A group of 73 women was selected to represent a typical cross section of the
female inhabitants of a Roman condominium. On average, the concentrations
of conjugated estriol, estradiol, and estrone measured in the urine were 106, 14,
and 32 mg/day, respectively.Apart from some estriol found in pregnancy urine,
free estrogens were not detected and estrogen sulfates represented 21% of the
total conjugated estrogens content. This situation, however, changed markedly
in the condominium collecting tank. Here, significant amounts of free estro-
gens were observed and the estrogen sulfate to estrogen glucuronate ratio rose
to 55/45, which was attributed to the ready deconjugation of the glucuronated
estrogens by the b-glucuronidase enzyme produced presumably in large
amounts by fecal bacteria (Escherichia coli). At the WWTP entrance, free es-
trogens and sulfated estrogens were the dominant species. Finally, the sewage
treatment was found to completely remove residues of estrogen glucuronates,
and with good efficiency (84–97%) the other analytes, but not estrone (61%)
and estrone-3-sulfate (64%).
In STP effluents, total extractable estrogens and conjugates have been de-
tected at levels up to 1 mg/L [9, 11, 26]. Despite the wide variability in terms
of removal efficiency reported for different WWTPs, a general trend has been
observed with respect to the identity of the compounds most frequently de-
tected in WWTP effluents. Thus, of the various compounds most commonly
monitored – namely, estradiol, its metabolites estriol and estrone, and the
synthetic estrogen ethynylestradiol – estrone is the most ubiquitous both in
WWTP effluents and in environmental waters in general, while the most potent
estrogens estradiol and ethynylestradiol have only occasionally been detected
[26, 40–42].As for the conjugates, the very few studies that have attempted their
determination pointed out estrone sulfates as the most abundant, while glu-
curonides are most often found below the limit of detection [26, 36, 38, 39].
Downstream of WWTPs, in the receiving river waters, the concentration
of estrogens and progestogens is normally considerably lower than that in the
corresponding effluent and decreases with distance from the WWTP [35, 36, 39,
42]. In this kind of compartment, the presence of estrogens and progestogens
has been reported to occur in the low nanogram per liter range [19, 43, 44].
In activated and digested sewage sludge, the concentration of ethynylestra-
diol (17 ng g–1), estrone (37 ng g–1), and estradiol (49 ng g–1), found in one of the
14 M. Kuster et al.
very few studies conducted in this kind of matrix (mestranol was not detected),
indicates that estrogens may remain unchanged during sludge digestion [45].
River sediments, likewise sewage sludge, have rarely been investigated
[42, 45–47] and to the authors’ knowledge there are no published reports on the
occurrence of estrogens and progestogens in either marine sediments or soils.
In one of the studies conducted with river sediments (in the UK), only the
latter of the three estrogens estradiol, ethynylestradiol, and estrone was de-
tected (>0.04–0.388 ng g–1 wet weight) [42]. Estriol and norethindrone were the
compounds most frequently detected in sediments collected in two rivers from
the northeast of Spain, where maximum concentrations were obtained for
ethynylestradiol (22.8 ng g–1 dry weight) and estrone (11.9 ng g–1) [46]. In both
studies, large concentration variations between sites and between the same site
sampled on different occasions were observed, which might be explained by the
difficulty in obtaining representative samples and the variability of factors such
as the gaseous/redox conditions influencing degradation rates. In another
study, conducted in Germany, estradiol, estrone, and ethynylestradiol were
found at concentrations up to 2 ng g–1 (estrone), whereas mestranol, a prodrug
for ethynylestradiol, was not detected [45]. Finally, neither estrogens nor
progestogens were detected in river sediments from Portugal [47].
Table 2 includes an example of bioaccumulation, as described by Larsson
et al. [48], where estrogen concentrations 4 to 6 orders of magnitude higher
than those in water were found in the bile of a rainbow trout caged downstream
of WWTPs.
5
Toxicity Identification Evaluation (TIE) Approaches
Most of the studies conducted to assess the environmental occurrence and fate
of estrogens and progestogens have focused on the determination of specific
target compounds. However, by simply following the disappearance of a sub-
stance, one cannot conclude that the environmental risk has vanished. The
derived degradation products or metabolites may also cause environmental
adverse effects. The identification of these products is a difficult task, due to the
great number of compounds that can possibly be generated, the high costs, and
the lack of analytical standards.
At the beginning of the 1990s the United States Environmental Protection
Agency (USEPA) developed the so-called TIE procedures. These approaches
were originally designed to identify the presence of health and environmentally
relevant compounds in WWs [49, 50]. However, since their introduction, they
have become established, powerful tools for determining the causative agents
of effects (such as acute toxicity, (geno)toxicity, and endocrine disrupting
potential) in aqueous and solid environmental samples.
The general scheme of TIE for the effect-based analysis is presented in Fig. 3.
As can be seen, the main purpose of TIE is to convert complex environmental
Fig. 3 General scheme of TIE procedure
extracts into fractions where the identification of the compounds responsible

for the effects observed (by means of suitable bioassays) is feasible using, e.g.,
mass spectrometric methods. A survey of the TIE procedures used for the
identification of endocrine disrupting compounds (EDCs) has been recently
published by Petrovic et al. [51].
An example of a TIE approach is that described by Desbrow et al. [7]. In this
work, the endocrine disrupting activity detected in effluents of seven UK
WWTPs by means of a yeast-based screening assay [52] was mainly attributed
to the presence of estradiol, estrone, and ethynylestradiol. However, to assess
the estrogenic activity different bioassays may be used, e.g., the yeast-based
recombinant estrogen receptor–reporter assay (YES), the MCF-7 cell prolifer-
ation (E-screen), and the estrogen receptor-mediated chemically activated
16 M. Kuster et al.
Table 3 Relative estrogenic potencies as determined by different bioassays (expressed as

EEF – the molar-based 17b-estradiol equivalency factor) (adapted from [51])
Compound/bioassay YES MCF-7 ER-CALUX
17b-estradiol 1 1 1
Estriol 3.7E-1
Ethinyl estradiol 1.9E-1–1.2 1.25–1.9 1.2
Diethylstilbestrol 4.5E-2–1.1 2.5
Estrone 1.9E-2–1E-1 1E-2 5.6E-2
luciferase gene expression assay (ER-CALUX). Table 3 lists the relative estro-
genic potencies determined for the most important estrogens by different
bioassays. As the estrogenic activity of two compounds may vary in different
approaches (bioassay classes), it needs to be stressed that the final outcome of
the TIE, with regard to the identity of the causative agent, may vary.
6
Analytical Methods
The analysis of steroid sexual hormones and related synthetic compounds

in WW, soil, sludge, and sediment samples is a challenging task. This is due to
both the complex environmental matrices and the requirement of low detection
limits. Therefore, the use of complicated, time- and labor-consuming analytical
procedures is necessary.
Many studies have reported the analysis of estrogens and progestogens
in WW and other water samples, but for solid samples, little is found in the
literature. This chapter briefly reviews the methods described for WW sam-
ples and discuss the very few methods used up to now to analyze solid sam-
ples (sediments, sludge, and soil). For more detailed data we refer to the
reviews:
– Environmental behavior and analysis of veterinary and human drugs in
soils, sediments, and sludge [53]
– Determination of endocrine disrupters in sewage treatment and receiving
waters [54]
– Review of analytical methods for the determination of estrogens and pro-
gestogens in WWs [55]
– Liquid chromatography–(tandem) mass spectrometry of selected emerging
pollutants (steroid sex hormones, drugs and alkyl phenolic surfactants) in
the aquatic environment [56]
Some of these methods are summarized in Table 4 and details concerning each
step of the analytical process are described in Sects. 6.1–6.3.
Table 4 Analytical methods described for the determination of estrogens and progestogens in environmental samples
Sample Compound Sample preparation Analytical method LOD Ref.

WWTP E3, E2, EE, E1 Speedisk-C18 and GC–MS LOQ 0.04–0.32 [66]
influent, derivatization PFPA
effluent, E2, EE, E1 SPE (C18), HPLC fraction, LLE GC–MS 0.2 [7]
and others
E2, EE, E1, aE2 SPE (SDB-XC disk, C18 or GC–MS/MS 0.1–2.4 [26]
NH2 column), HPLC fraction)
E2, E1, MES, aE2, E2-17 V, SPE (C18), silica gel, GC–MS/MS 1 [43]
16 a-OH-E1, E2-17A derivatization
E3, E2, EE, E1 SPE (Carbograph-4) HPLC–MS/MS (NI-ESI) LOQ 0.08–0.6 [35]
E3, E2, EE, E1, DES, NOR, SPE (C18 column) HPLC-DAD–MS (NI-ESI) 2–500 [57]
LEV, PROG
E2, EE, E2-17G, E2-3S SPE (C18 disk), hydrolysis, ELISA 0.1 [36]
HPLC fraction GC–MS/MS (E2) 0.2–0.4
E3, E2, EE, E1, DES, NOR, On-line SPE (PLRP-s column) HPLC-DAD–MS (NI-ESI) 10–200 [60]
LEV, PROG
E2, E3, E1, EE, DES SPE (C18 column) HPLC–MS (NI-ESI) (SIM) 200 [68]
HPLC–MS/MS (NI-ESI) 5
E2, E1 SPE (LiChrolut EN +C18 col.) + HPLC–MS (NI-ESI) 0.07–0.18 [69]
immunoaffinity extraction
Estrogens and Progestogens in Wastewater, Sludge, Sediments, and Soil
E2, E3, E1, EE, E3-3G, E2-3G, SPE (Carbograph-4) HPLC–MS/MS (NI-ESI) 0.003–15 [61]
E1-3G, E3-16G, E2-17G,
E3-3S, E2-3S, E1-3S
E1, estrone; E1-3G, estrone 3-(b-D-glucuronide); E1-3S, estrone 3-sulfate; 16a-OH-E1, 16a-hydroxyestrone; E2, 17b-estradiol; aE2, 17a-estradiol;
E2-3G,17b-estradiol 3-(b-D-glucuronide); E2-3S, 17b-estradiol 3-sulfate; E2-17A, 17b-estradiol 17-acetate;E2-17G, 17b-estradiol 17-(b-D-glucuronide);
E2-17 V, 17b-estradiol 17-valerate; E3, estriol; E3-3G, estriol 3-(b-D-glucuronide); E3-3S, estriol 3-sulfate; E3-16G, estriol 16a-(b-D-glucuronide);
17
EE, ethynylestradiol; DES, diethystilbestrol; MES, mestranol; LEV, levonorgestrel; NOR, norethindrone; PROG, progesterone; MSTFA, N-methyl-N-
(trimethylsilyl)trifluoroaceatamide; PFPA, pentafluoropropionic anhydride.
Table 4 (continued)
18
Sample Compound Sample preparation Analytical method LOD Ref.

WWTP E2, E3, E1, EE SPE (Envi-Carb col.) HPLC–MS/MS (PI-APCI) LOQ 0.5–1 [58]
influent, E2, EE, E1 SPE (SDB-XC disk, C18 or GC–MS/MS 0.1–1.8 [59]
effluent, NH2 column), HPLC fraction)
and others
E3, E2, EE, E1 SPE (Carbograph-4) HPLC–MS/MS (NI-ESI) 0.2–0.5 [59]
E2, EE, E1 Solvent extraction with GC–MS/MS 0.4–1 [42]
sonification, HPLC fractioning,
derivatization
Natural E2, EE, E1 Ultrasonic extraction with GC–MS/MS 0.04–5 [42]
river DCM, HPLC fractioning,
sediment derivatization
E3, E2, EE, E1, DES Ultrasonic extraction with HPLC–MS (NI-ESI) (SIM) 0.05–1 [46]
MeOH-acetone, SPE (C18)
E3, E2, EE, E1, DES SPE (RAM cartridges (ADS C4)) HPLC–MS (NI-ESI) (SIM) 1–5 [63]
E2, EE, E1, MES Solvent extraction, silica gel GC–MS/MS (EI) LOQ 0.2–0.4 [45]
column, SPE (RP-C18),
HPLC (RP-C18) cleanup,
derivatization MSTFA
NOR, LEV, PROG Ultrasonic extraction with HPLC–MS (PI-ESI) (SIM) 0.04 [46]
MeOH-acetone, SPE (C18)
NOR, LEV, PROG SPE (RAM cartridges (ADS C4)) HPLC–MS (PI-ESI) (SIM) 0.5 [63]
Sludge E2, EE, E1, MES Solvent extraction, GPC, GC–ion-trap MS/MS LOQ 2–4 [45]
silica gel column, derivatization
with MSTFA
M. Kuster et al.
6.1
Sampling
The choice of the sampling procedure is essential to obtain significant, repre-

sentative results of the environmental occurrence of these compounds. The
place and time of sample collection has to be planned carefully. The sampling
procedure and transport should not influence the matrix to be analyzed. In
general, exposure to light, oxygen, and high temperatures should be avoided
due to the risk of transformation of the analytes or other organic components
present in the environmental samples.
Wastewater samples have usually been collected in precleaned amber glass
containers. Both discrete and composite samples have been used for the analy-
sis of effluents and influents of WWTPs. Unpreserved samples are normally
stored at 4 °C for 48 h, or frozen [48]. Other authors add chemical agents such
as methanol, sulfuric acid, or mercuric chloride to prevent bacterial activity dur-
ing storage, and/or store the samples in supports used for extraction [26, 35, 57].
The devices used for sampling of solid samples (sludge, sediment, and soil)
are usually grab samplers or corers. Box corers or multicorers can be employed
if more detailed information on the spatial distribution of the analytes is
needed. The samples are stored in the dark at 4 °C or more commonly at –20 °C,
preferably in glass containers [53]. Very often, solid samples are also dried or
lyophilized prior to storage.
6.2
Sample Pretreatment
An essential step in the analysis of trace pollutants in environmental matrices

is the pretreatment procedure. Methods that are more efficient have been de-
veloped in the last few years, facilitating subsequent chromatographic analysis.
Because of the complexity of the matrices, the sample pretreatment procedure
includes both extraction and purification of the target analytes.
Filtration is the first step of WW sample preparation because of the high
loading of organic material and suspended particles. This step is essential to
prevent clogging of the adsorbent bed by suspended solids, if solid-phase
extraction (SPE) is performed. In the case of immunochemical assays, previous
filtration avoids undesired adsorption onto antibodies. Most of the studies
reviewed employed glass-fiber filters with a pore size between 0.22 and 1.2 mm.
Investigations showed no significant loss of the analytes after this filtration
procedure [7, 36]. However, the filtration system is usually washed with an
organic solvent to ensure that no analyte is left adsorbed to the particles
[35, 58, 59].
Extraction of estrogens and progestogens is mostly performed by off-line
SPE (on-line SPE has been reported by Lopez de Alda et al. [60]), using either
disks or, more frequently, cartridges. Octadecyl (C18)-bonded silica in both car-
tridge [7, 57] and disk [36] format, graphitized carbon black [35, 58, 59], Isolut
20 M. Kuster et al.
ENV+ cartridges [48], and SDB-XC disks [26, 59] are the sorbents more widely
used. The combination of C18 and SDB has also been reported, showing good
recoveries for the investigated analytes [43]. Previous adjustment of the sample
pH is performed sometimes [43].
The occurrence of conjugated estrogens has only been investigated in a few
works [26, 36, 48, 61]. The analysis of these compounds by means of immuno-
assays requires their previous conversion to the corresponding free estrogens
by enzymatic hydrolysis [26, 36, 48]. In this case, the concentrations of the con-
jugated hormones are determined from the differences between the results
obtained for hydrolyzed and unhydrolyzed samples. It should, however, be
remarked that these enzymes convert estradiol glucuronide quantitatively into
estradiol, but convert only ca. 30% of estradiol sulfate into estradiol [36]. Levels
of the sulfate-conjugated hormones might therefore be underestimated. By
contrast, LC–MS enables the simultaneous determination of the free and con-
jugated forms without the need for hydrolysis.
Previous derivatization of the extract is necessary to improve the stability
of the compounds and the sensitivity and precision of subsequent GC–MS
analysis. Silyl derivatives formed for example with MSTFA [43], halogenated
alkene derivatives produced with heptafluorobutyric anhydride (HFBA) [36] or
pentafluoropropionic acid [58] or anhydride (PFPA), as well as acetate deriva-
tives formed using acetic anhydride [48] have been widely employed.
Solid samples are in general more difficult to handle than liquid ones.
The target analytes are extracted from their solid matrix by sonification, by
pressurized-liquid extraction (PLE), or by simple blending or stirring of the
sample with polar organic solvent solutions, e.g., methanol/acetone solutions
[42, 45–47, 62, 63]. Cleanup of the extracts was performed using SPE (C18 car-
tridges [45, 46], RAM cartridges (ADS C4) [63], silica gel [45]), gel-permeation
chromatography (GPC) [45], and semipreparative HPLC [45]. RAMs are
bifunctional sorbents that combine size exclusion and reversed-phase reten-
tion mechanisms.
6.3
Analyses
The analysis of WW samples has been dominated by the use of immunoassays

and GC–MS techniques. However, in recent years, LC–MS and LC–MS/MS have
gained in popularity, because the above-mentioned preceding hydrolysis step
(needed for immunoassay analysis) and derivatization step (needed for GC–MS
analysis) are not necessary.
Biological techniques, e.g., immunoassays, are among the most sensitive
analytical methods, but are limited by the availability of the specific antisera
and are subject to cross-reactivity. Huang et al. [36] employed an enzyme-
linked immunosorbent assay (ELISA) for determination of estradiol, its con-
jugates, and ethynylestradiol in wastewaster treatment plant effluents (see
Table 4). The reported limit of detection (LOD) of 0.1 ng L–1 reflects the sen-
sitivity of this method. Low LODs in the range of pg L–1 to 2 ng L–1 have also
been achieved by using other immunoassay [64, 65] and radioimmunoassay
[19, 22] protocols.
The analysis of estrogens and progestogens by GC–MS has been carried out
with a variety of capillary columns using helium as carrier gas [7, 26, 36, 43,
59, 66]. LODs in the range of 0.1–1.8 ng L–1 have been achieved. In terms of
sensitivity, GC– and HPLC–tandem mass spectrometry are comparable tech-
niques. However, the derivatization carried out prior to GC separation is time
consuming and can be a source of inaccuracy [7].
For the environmental analysis of estrogens and progestogens by HPLC–MS,
both electrospray ionization (ESI) in the negative-ion (NI) mode for estrogens
Fig. 4 Reconstructed SRM chromatograms obtained from the LC–ESI-MS/MS analysis of a

100 ng mL–1 standard mixture of estrogens (in the NI mode) and progestogens (in the PI
mode). Column: Purospher STAR RP-18e (125¥2 mm, 5 mm, Merck). Mobile phase: gradient
acetonitrile/water. Flow rate: 0.2 mL min–1
22 M. Kuster et al.
and in the positive-ion (PI) mode for progestogens and, to a lesser extent,
atmospheric-pressure chemical ionization (APCI) in the PI mode have been
used. Chromatographic separation has been performed on octadecyl silica
stationary phases. According to a recent study carried out to compare the per-
formance of various MS techniques (GC–MS, LC–MS, and LC–MS/MS) [67],
LC–ESI-MS/MS is the technique of choice for analysis of these compounds,
based on sensitivity and selectivity. The same study also indicates that, although
the limits of detection achieved by LC–MS in the selected ion monitoring (SIM)
mode and by LC–MS/MS in the selected reaction monitoring (SRM) mode are
in general comparable, the higher selectivity of the latter is essential to avoid
false positive determinations in the analysis of real environmental samples.
Figure 4 shows representative chromatograms obtained from the analysis of
estrogens and progestogens by LC–ESI-MS/MS.
For analysis of solid samples, GC–MS/MS [45] and more frequently HPLC–MS
have been used [46, 63]. Limits of detection vary from 0.04 to 4 ng g–1.
7
Conclusions
What is the real danger imposed by the presence of estrogens and progestogens
in aquatic systems? The studies carried out up to now indicate that the occur-
rence of these compounds in surface waters is an issue of concern, and that
wastewater treatment plant effluents play a major role in their introduction into
the environment. However, more information on their environmental presence,
transport, and fate is needed to assess their ultimate ecosystem impacts. Main
data gaps are localized in the area of environmental solid samples. Possible
future dangers from accumulation in sediments and soil are at present unpre-
dictable and should be investigated thoroughly. Besides environmental levels,
toxicological data and bioavailability and degradation studies should be avail-
able.
In the field of analysis, important progress has been made in terms of sen-
sitivity and selectivity. LC–ESI-MS/MS appears to be the technique of choice for
their determination as it provides reliable results at subnanogram per liter or
per gram levels. However, sample preparation is identified as the main bottle-
neck in the analysis of these compounds. Quite tedious and time-consuming
procedures are still required, especially in the case of complex matrices such as
sewage sludge.
To reduce the use of these sexual hormones seems to be an impossible task,
as no substitute compounds can be applied instead of estrogens and progesto-
gens to the described medicinal and farming applications. However, since
WWTP effluents constitute the most important source of these compounds, re-
mediation actions can be performed at this level by introducing more advanced
and efficient treatment processes, especially in plants receiving high inputs of
urban discharges from highly populated regions.
Acknowledgements This work was supported by the Energy, Environmental and Sustainable
Development Program (Project ARTDEMO EVK1-CT2002-00114), and by the Spanish Min-
istry of Science and Technology (Projects BQU2002-10903-E and PPQ2001-1805-CO3-01).
Maria José López de Alda acknowledges her Ramon y Cajal contract from the Spanish Min-
istry of Science and Technology.
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The Handbook of Environmental Chemistry Vol. 5, Part O (2005): 25– 51
DOI 10.1007/b98606
Organic Compounds in Paper Mill Wastewaters

A. Latorre1 · A. Rigol2 · S. Lacorte1 (✉) · D. Barceló1
1
08034 Barcelona, Catalonia, Spain
slbqam@cid.csic.es
2
Department of Analytical Chemistry, University of Barcelona, Av. Diagonal 647,
08028 Barcelona, Catalonia, Spain
1 Pulp and Paper Mill Wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2 Legislation Related to Pulp and Paper Mill Industries . . . . . . . . . . . . . . 30
3 Chemical Characterization of Pulp and Paper Mill Waters . . . . . . . . . . . . 32

3.1 Biocides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2 Resin and Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3 Surfactants and Plasticizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.4 Lignin and Hemicelluloses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.5 Chlorinated Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4 Toxicity of the Effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
5 Ain Emissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6 Removal Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
7 Conclusions and Future Recommendations . . . . . . . . . . . . . . . . . . . . 48
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Abstract This chapter is focused on the problem caused by the effluent discharges from
paper and pulp mills. At present, three aspects should be considered in paper and pulp
wastewater management: (1) the toxicity and high BOD5 of whitewaters and effluents; (2) the
lack of knowledge on specific compounds responsible for the toxicity of the liquid and solid
residue (sludge) and (3) the difficulty of treating whitewaters, which are characterized by
the presence of suspended solids, colour odour, a high organic content, and an overall high
toxicity. This chapter attempts to give an overview of organic compounds that contribute
to the toxicity of paper mill waters and effluents, their levels, toxicological characterization
and the methodologies used for their analysis. Families of compounds that are included are
natural compounds such as resin and fatty acids, lignins, lignans and carbohydrates, and
additives used during paper making such as surfactants, biocides and slimicides. In addition,
part of the chapter is devoted to describing the wastewater treatment strategies used to
decrease the toxicity and BOD5 of the effluents, which are used to indirectly phase out toxic
organic pollutants from paper and pulp whitewaters (Table 1).
Keywords Paper mill · Whitewaters · Effluents · Organic compounds · Analytical methods ·

Toxicity · Treatment
26 A. Latorre et al.
Table 1 List of acronyms ordered by compounds and techniques
Compounds
AOX Absorbable organic halides

APEO Alkylphenol ethoxylate
BOD Biochemical oxygen demand
BPA Bisphenol A
BSTFA Bis(trimethylsilyl)trifluoroacetamide
COD Chemical oxygen demand
DBNPA 2,2-Dibromo-3-nitrilopropionamide
DCM Dichloromethane
DHA Dehydroabietic acid
DS Dry solid
ECF Elementary chlorine- free
FA Fatty acids
Kj-N Kjeldahl nitrogen
LAS Linear alkylbenzene sulfonates
MBT Methylene-bis-(thyiocyanate)
MFO Mixed-function oxidase
MIB Methylisoborneol
MTBE Methyl tert-butyl ether
NOx The sum of nitrogen oxide (NO) and nitrogen dioxide (NO2) expressed
as NO2
NP Nonylphenol
NPEC Nonylphenol ethoxycarboxylate
OC Organochlorine
OP Octylphenol
OPEC Octylphenol ethoxycarboxylate
PCB Polychloroinated biphenyls
PCDBT Polychlorinated dibenzothiophene
PCDD Polychlorinated dibenzo-p-dioxins
PCDF Polychlorinated dibenzo-p-furans
PCP Pentachlorophenol
PFB Pentafluorobenzyl
RA Resin acids
TCA 2,4,6-Trichloroanisole
TCDD Tetrachloro dibenzo dioxin
TCF Totally chlorine- free
TCMTB 2-(Thiocyanomethylthio)-benzothiazole
TOC Total organic carbon
VSC Volatile sulphur compounds
VOC Volatile organic compounds
Organic Compounds in Paper Mill Wastewaters 27
Table 1 (continued)
Techniques
APCI Atmospheric pressure chemical ionization

CZE Capillary zone electrophoresis
ECD Electronic-capture detector
EROD Ethoxyresorufin-O-deethylase
ESIP Electrospray ionization
FID Flame ionization detector
HRGC High-resolution gas chromatography
HRMS High-resolution mass spectrometry
IS Internal standard
LLE Liquid–liquid extraction
MEKC Micellar electrokinetic chromatography
SPME Solid-phase micro extraction
TU Toxicity units
1
Pulp and Paper Mill Wastewaters
The pulp and paper industry is the sixth largest polluter (after the oil, cement,
leather, textile and steel industries), discharging a variety of gaseous, liquid
and solid wastes into the environment [1]. The main environmental issues are
emissions to water and air, sludge build-up and energy consumption. It is the
pollution of water bodies, however, which is of major concern because large
volumes of wastewater are generated for each metric ton of paper produced, de-
pending on the raw material, finished product and extent of water reuse.
Untreated paper mill effluent discharges cause considerable damage to the
receiving waters, since they have high biochemical oxygen demand (BOD),
chemical oxygen demand (COD), chlorinated compounds (measured as ad-
sorbable organic halides, AOX), suspended solids (mainly fibres), fatty acids,
tannins, resin acids, lignin and its derivatives, sulphur and sulphur compounds,
etc. [1]. While some of these pollutants are naturally occurring wood extrac-
tives (tannins, resin acids, lignin), others are xenobiotic compounds that are
formed during the process of pulping and paper making (chlorinated lignins,
resin acids and phenols, dioxins and furans) [2, 3]. Some of the pollutants listed
above, notably polychlorinated dibenzodioxins (PCDD) and dibenzofurans
(PCDF), are recalcitrant to degradation and tend to persist in nature.
The accumulation of organic matter in whitewaters is due to the paper-mak-

ing process itself. Figure 1 depicts a mass stream overview of a pulp and paper
mill. In paper mills, paper is made from wood, pulp or recycled paper by mix-
ing the raw material with water to a fibre suspension which is ground, diluted
and evenly distributed on the wire of a paper machine. On the wire, the pulp
is dewatered and the paper sheets start to form, with a final dryness of 90–95%.
Only 60–70% of the fibres are retained on the wire, and the rest end up in white-
waters that must be recovered and recycled to the paper machine to achieve
maximum productivity. Due to this process, whitewater allows build-up of
organic matter in the system, which causes a number of troubles in paper pro-
duction such as neutralization of cationic retention chemicals due to the anionic
nature of organic matter, growth of microorganisms due to high concentrations
of organic matter (causing depletion of oxygen and production of hydrogen
sulphide), formation of biofilms and generation of “stickies” due to lipophilic
extractives which are accumulated in the paper [4].
In addition, whitewaters produce corrosion in the paper machine. The type
and amount of organic compounds in whitewater and effluents depend on the
raw material, paper-making process, additives used and type of energy supply.
On average, paper production generates from 10 to 50 m3 of wastewater per ton
Fig. 1 Mass stream overview of a pulp and paper mill. The presence of some substances
depends on the raw material, paper-making process, additives used and type of energy
supply
of paper [5]. Wastewater is either released untreated, treated or recycled. Pulp

and paper mill effluents discharged into freshwater, estuarine and marine
ecosystems alter aquatic habitats, affect aquatic life and adversely impact
human health. Chronic sublethal toxic effects measured as increased liver
mixed-function oxidase activity (MFO) and symptoms of altered reproductive
capacity in fish and aquatic invertebrates have been detected in the discharge
of treated pulp mill effluents [6]. In addition to that, large amounts of solid
wastes are generated as by-products of the wastewater treatment plants. Mean
emission levels are 0.3–1 kg/ton paper [5]. Primary sludge (solids removed dur-
ing physical wastewater treatment prior to biological treatment) is rich in wood
fibre and volatile solids. Secondary sludge (product of biological treatment)
may contain organochlorine compounds (chlorophenols, chlorocatechols,
chloroguaiacols, chlorovanillins and chlorosyringaldheydes) as well as trace
levels of some dioxin and furan congeners, generated by chlorine bleaching [7].
There is ample evidence of the adverse health and environmental effects linked
to organochlorinated compounds, often related to endocrine disruption
(growth retardation, thyroid dysfunction, decreased fertility, feminization or
masculinization of biota, etc.) [8].
Table 2 reports some common parameters used for the characterization of
paper mill effluents. Biodegradable organic carbon, associated with families of
non-chlorinated organic materials, is measured by biochemical and chemical
oxygen demand (BOD, COD) methods. Some of the compounds found in
paper mill effluents and sludge, including chlorinated compounds and several
wood extractive constituents found in pulp liquors, are refractory (resistant to
rapid biological degradation) and thus not measurable by the BOD5 analytical
method.At present, there is a lack of information on the characterization of the
Table 2 Average levels of several parameters used for the characterization of whitewaters
and sludge
Type of pollutant Typical example Levels
Air emissions Malodorous gases of reduced 0.3–3 kg/t

sulphur compounds, measured of air-dried pulp
as total reduced sulphur
Particulate matter 75–150 kg/t
Sulphur oxides/nitrogen oxides 0.5–30/1–3 kg/t
Volatile organic compounds (VOCs) 15 kg/t
Liquid effluents Biochemical oxygen demand (BOD) 10–40 kg/t
Chemical oxygen demand (COD) 10–60 kg/t
Absorbable organic halides (AOX) 0–4 kg/t
Kj-N 3–13 mg/L
P total 0.5–1.8 mg/L
Solid wastes Sludge from primary and 50–550 kg/t
secondary treatment
toxic organic fraction of pulp and paper mill effluents to evaluate their poten-
tial effects towards the environment, and the specific compounds, groups of
compounds or organic fractions that cause harmful effects should be deter-
mined. The objectives of the proposed chapter are to describe the methods
used to characterize pulp and paper mill effluents chemically and toxicologi-
cally, with the ultimate goal of obtaining a deep knowledge on the compounds
responsible for toxicity, their concentration in the different types of industries
and treatment methods used for their removal.
2
Legislation Related to Pulp and Paper Mill Industries
As highlighted by the EU, in order to improve the management and control

of industrial effluents, the Council Directive 96/61/EC on integrated pollu-
tion prevention and control (IPPC Directive) [5] is implementing the best
available technologies to ensure a high level of protection of the environ-
ment. The IPPC Directive has started an exchange of information between EU
member states and the pulp and paper industries concerning the best available
techniques aimed at: (1) improving the quality of water by installation of
treatment plants; (2) minimizing water consumption, since the pulp and paper
mill industry is the second highest consumer of fresh water in Europe, gener-
ating six billion m3 of wastewater annually; (3) resolving the chronic toxicity
and ecotoxicity associated with paper and pulp effluents; and (4) reducing
the amounts of additives used (or substituting less toxic compounds). As a
result, this sector still requires data and investment to improve water quality,
which will be performed by revising and establishing emission limits. To assess
these necessities, and to support the European IPPC Directives [5], new
analytical techniques are emerging to determine those pollutants which may
induce toxicity, may negatively influence water treatment or may affect paper
production.
Historically, the pulp and paper industry throughout the world has been re-
garded as particularly polluting to aquatic environments. Until the 1950s, it was
common for pulp mills and many other industries to discharge untreated, toxic
effluents directly into rivers and seas [9]. Nowadays, the development of new
technologies directed to the treatment of industrial wastewaters is a European
Community priority, which aims to reduce the organic pollution generated by
industrial activities, and in the last instance, to reuse effluent waters. Since 1999,
member states have been encouraged to comply with Directive 76/464/CEE [10]
(and daughter Directives 86/280/CEE, 88/347/CEE and 90/415/CEE), as well as
the Water Framework Directive (WFD) [11] related to the monitoring of toxic,
persistent compounds with high accumulation potential. The IPPC Directive
[5] have the objective of reviewing and implementing strategies and measures
to control the sources of pollution and improve the quality of water. Some
European countries (e.g. Portugal, France, Germany) have started to analyse the
Table 3 Current national discharge limits and proposal levels for production of bleached Kraft and bleached sulphite pulp [5]
Country Type of pulp COD BOD5 TSS AOX
Austria Bleached Kraft pulp Exist: 30 kg/t Exist: 3 kg/t Exist: 5 kg/t Exist: 2.5 kg/t
New: 20 kg/t New: 2 kg/t New: 2.5 kg/t New: 0.25 kg/t
Bleached sulphite pulp Exist: 40 kg/t Exist: 3 kg/t Exist: 5 kg/t Exist: 0.2 kg/t
New: 25 kg/t New: 2 kg/t New: 2.5 kg/t New: 0.1 kg/t
France Bleached Kraft pulp Exist: 65 kg/t Exist: 3.98 kg/t Exist: 6.5 kg/t 1 kg/t (yearly average)
New: 20 kg/t New: 3 kg/t New: 5 kg/t
Bleached sulphite pulp Exist: 45 kg/t Exist: 6.5 kg/t Exist: 6.5 kg/t 1 kg/t (yearly average)
New: 35 kg/t New: 5 kg/t New: 5 kg/t
Germany Bleached Kraft pulp Exist: 40 kg/t Exist: 35 mg/L No values Exist: 0 kg/t
New: 25 kg/t New: 30 mg/L (part of COD)

Bleached sulphite pulp Exist: 40 kg/t Exist: 35 mg/L No values Exist: 2.5 kg/t
New: 25 kg/t New: 30 mg/L (part of COD) New: 0.25 kg/t
Ireland Bleached Kraft pulp No limit 90% removal or 50 mg/L No limit 0.1 mg/L
Bleached sulphite pulp No limit 90% removal or 50 mg/L No limit 0.1 mg/L
Italy Bleached Kraft pulp 160 mg/L 40 mg/L 80 mg/L No requirements
Bleached sulphite pulp 160 mg/L 40 mg/L 80 mg/L No requirements
United Kingdom Bleached Kraft pulp No achievable guidance 10–50 mg/L 10–50 mg/L <1.5 kg/t
levels proposed
Bleached sulphite pulp No achievable guidance 10–50 mg/L 10–50 mg/L <1.5 kg/t
levels proposed
31
levels of various priority pollutants in surface waters, sediments and biota

according to the European policy. Most countries still lack relevant and precise
data on the pollution generated by the paper and pulp industries, and identi-
fication of the specific compounds responsible for the toxicity of the effluent is
a subject still to be covered.
Environmental limits or guidelines for the pulp and paper industry vary
significantly between European countries, despite efforts to create a more
uniform system [5]. As an example the discharge limits for bleached Kraft and
bleached sulphite pulp are given in Table 3. In some countries, such as Austria
or France, there is different legislation depending on the type of paper mill.
Normally, the most restrictive corresponds to the bleached Kraft pulp. In other
countries, such as Ireland, there are no requirements or limits for some para-
meters.
In 1997, the US EPA finalized a new set of federal guidelines, the so-called
cluster rules [12]. The guidelines contain limits for 12 different types of mills,
each of them distinguished by four different technical levels depending on the
type of technology applied. Limit values are given as pollutant load expressed
as kilograms of pollutant per tonne of product, distinguishing maximum values
per day and average monthly values. The regulations apply to any pulp, paper
or paperboard mill that discharges process waters. Compared to recent permit
requirements in Europe (see Table 3) the limitations for existing mills set in the
US cluster rules are lenient.
There is special legislation for other countries. For example Canada, one
of the most important pulp and paper producing countries, has set several
general guidelines, and the actual limits and guidelines can be different in each
state [5]. The wastewater limit systems in Canada are mostly based on load
kilograms per tonne production. Different limits are applied for different types
of mills. Some parameters are measured as concentrations. Toxicity limits are
also used.
3
Chemical Characterization of Pulp and Paper Mill Waters
The analysis of organic pollutants in whitewaters and effluents requires

procedures which can eliminate suspended matter and fibres and still permit
the extraction and efficient recovery of target analytes. Whitewaters and efflu-
ents, especially from closed-cycle systems, are characterized by a very high
total organic content (TOC) (up to 5,000 mg/L), by the presence of particulate
matter and by the formation of microfibres which, if not eliminated, may affect
the extraction efficiency. To remove suspended matter and particles, sample
filtration through 1, 0.7 and 0.45 mm filters is necessary, or otherwise centrifu-
gation at 2,000 rpm for 20 min. Most apolar compounds might be retained
in the particulate fraction of the sample and thus, the filter should also be
extracted [13]. Due to the large amount of organic matter, the extraction pro-
Fig. 2 Chemical structure of organic compounds identified in paper mill effluents

cedure must be adapted to this matrix and the chemistry of the target com-
pounds should be considered. The chemical structure of the most representa-
tive compounds of each family are shown in Fig. 2. Table 4 summarizes the
extraction methods for different families of chemicals generally found in
paper mill effluents, pointing out the detection limit and the percentage of
recovery.
3.1
Biocides
Depending on the type of paper produced, it is common to dose biocides for

wood preservation and during paper making to decrease the problems related
to microbial, fungal and algal growth. This means an undesired handling of
toxic chemicals and the risk of negative effects in the receiving water, as bio-
cides are discharged with the wastewater. Furthermore, high concentrations of
biocides in whitewater may diminish the efficiency of secondary treatment,
available in some paper mills. There are two main classes of biocides [14]. On
the one hand, there are oxidizing agents such as chlorine dioxide and hydrogen
peroxide. These happen to be the same chemicals that are widely used for pulp
bleaching. The oxidizing action either kills the bacteria and fungi outright or
it weakens the cell walls so that they are more susceptible to the other main
classes of biocides. The other class involves highly toxic organic chemicals, such
as thiocyanates, isothiazolins, cyanobutane, dithiocarbamate and bromo com-
pounds, which are used for wood preservation in place of traditional
chlorophenols [15].A possible third category consists of materials that have an
ability to inhibit biological film formation, e.g. surfactants such as alkyl sulpho-
succinates.
The size and type of the paper mill (recycle, Kraft, pulp, etc.) and open/
closed circuits are crucial to determine the type of biocides to be used and their
doses. The fate of biocides is as follows: a fraction will degrade (chemically
or biologically), a fraction will remain in the circulating waters and finally, a
fraction will be present in the effluent or remain in the solid matter. An addi-
tional problem is that, due to their physicochemical properties, some biocides
may be fibre retentive and can accumulate in the final paper product [16].
Little information is available on biocides in pulp and paper mill whitewaters
and effluents due to the complexity of the water matrix, but generally LC tech-
niques are employed since paper mill biocides are usually highly soluble
and polar compounds. A recent study recommends the use of LC–ESI-MS in
the positive-ion mode for the determination of TCMTB and DBNPA in effluent
waters [17]. Figure 3 indicates the average level of biocides found in paper mill
waters. DBNPA and TCMTB were detected in process waters of a recycling
paper mill at concentrations of 8–116 and 2–4 mg/L, respectively [18]. MBT
was detected at concentrations of 0.19 and 0.02 mg/L in whitewater and
primary effluent, respectively, after SPE and LC-UV detection [17]. The same
method was found to be suitable for determining this product in paper, after
Table 4 Current methods for the isolation and analysis of toxic compounds found in papermill waters
Compounds Matrix Method LOD Recoveries Ref.

(%)
Extraction Separation Detection
DBNPA Paper food packaging Hot water extraction MEKC UV 1700 mg/g 53.0 16
TCMTB Paper-recycling SPE LC MS 1.5 mg/L n.r. 17
DBNPA process waters 80 mg/L n.r.
MDC Fortified tissue paper Hot water extraction LC UV n.r. 51 18
Secondary-treated effluent followed by SPE 0.01 mg/L 88
TCMTB Surface-treated lumber Extraction with ACN LC UV n.r. 96.2 19
RA and FA Paper mill effluents LLE with DCM, followed GC MS n.r. n.r. 21
by derivatization step
FA esters Primary effluent SPE GC MS n.r. n.r. 25

LLE with DCM n.r. n.r.
RA Paper mill effluents and SPE, followed by GC MS n.r. 75 26
river waters derivatization step LC Fluoresc. 0.001–0.02 91–95 mg/L
RA and FA Effluents from bleaching LLE with MTBE, followed GC FID n.r. n.r. 27, 28
processes by derivatization step
RA and FA Whitewater, effluents LLE with MTBE LC (APCI)-MS 0.3–32 mg/L 70–101 23
Direct injection 0.5–81.3 mg/L 75–95
RA and FA Wood resin Extraction with DCM, LC MS 6–12 mg n.r. 29
followed by injected
derivatization step
RA and FA Paper-recycling process LLE with MTBE, GC MS 0.007–0.2 81–106 17, 32
waters followed by mg/L
35
derivatization step
36
Table 4 (continued)

(%)
NP1EC, OP1EC Paper-recycling SPE LC ESI-MS 10–80 mg/L <90 17

NP, OP, LAS process waters
BPA Wastewater from paper SPE GC MS n.r. n.r. 35
production
NPEC Paper mill effluents SPE GC MS 0.2–2 mg/L 90–110 36
Hemicelluloses Pulp fibres 1% Acid methanolysis GC FID n.r. n.r. 38

with HCl, followed
by a silylation step
Hydrolysis and MS n.r. n.r. 39
methylation reaction
Lignin Paper mill effluents Permanganate CZE UV 0.55–2.9 mg/L n.r. 46
degradation, followed MS 0.05–0.30 n.r.
by LLE with
acetone/DCM
Lignin Paper mill effluents CuO degradation, GC MS n.r. n.r. 48
followed by a
derivatization step
OC, PCB, CDPE, Sediments from pulp GPC HRGC HRMS n.r. n.r. 53
PAH, PCDD, and paper mill
PCDF
PCDBT Bleached pulp mill Soxhlet extraction of HRGC HRMS <10 ng/L n.r. 54
effluents filters with toluene
A. Latorre et al.
Table 4 (continued)

(%)
OC, PCB, Nesting along GPC GC EDC n.r. n.r. 55

PCDD, PCDF contaminated rivers HRGC HRMS n.r. n.r.
Chlorophenols, Effluent from bleaching LLE with diethyl GC FID n.r. n.r. 27
chloroguaiacols, processes ether/acetone, followed
chlorovanillin, by derivatization step
chlorocatechol
Chlorophenols, Pulp mill effluents SPE LC Ampero- 0.4–6 mg/L 84–100 37
chloroguaiacols, metric
chlorosyringol electrode
Chlorophenols, Contaminated sediment Dean–Stark Soxhlet GC MS n.r. n.r. 52

chloroguaiacols, extraction
chlorovanillin,
chlorocatechol
Chlorophenols, Sediment Extraction with GC MS n.r. n.r. 57
chloroguaiacols, N-hexane, followed by
chlorocatechol, derivatization step
VSC Air, water and sediments Cryogenic trap GC FID 10 pg/L n.r. 68
VSC Pulp mill effluents HS-SPME GC MS 0.7–5 ng/L n.r. 69
LLE with DCM GC MS n.r. n.r. 70
MIB, geosmin River LLE with Hexane GC MS 0.5 ng/L 97–103 71
Drinking water HS-SPME GC MS 1.2–3.3 ng/L n.r. 73

37
Fig. 3 Levels of different families of organic compounds in paper mill effluent waters
extraction with boiling water. The LC-UV detector was also applied to deter-
mine TCMTB and chlorophenols [19] from lumber surfaces after extraction of
sticks (1¥2 mm) with acetonitrile. Micellar electrokinetic chromatography
(MEKC) was optimized for the determination of ten biocides in paper food
packaging, with the inherent advantages of rapidity, simplicity and no use of
toxic reagents [16].
3.2
Resin and Fatty Acids
Wood extractives include lipophilic (fatty and resin acids, sterols, steryl esters
and triglycerides) and hydrophilic (lignans, low-molecular-mass lignins, lignin-
like substances and hemicelluloses) compounds that dissolve in whitewaters
during paper production. Among them, resin and fatty acids have a high ten-
dency to form pitch deposits and stickies that alter the machine functioning
and decrease the paper physical properties (tensile strength, opacity, brightness,
etc.) [20]. On the other hand, wood extractives accumulated in whitewaters
can end up in the effluent, and are potential toxicants to biota. Figure 3 shows
the concentrations of resin and fatty acids in whitewaters. The levels detected de-
pend on the paper-making procedure. Concentrations of 20 to 400 mg/L

have been detected in water [16, 20, 21] and 1,474 mg/g d.w. in sediment [22].
These compounds are not removed by primary flocculation whereas a 50%
decrease or more is observed after biological treatment, showing the effective-
ness of this treatment in reducing the concentration of these type of com-
pound [23].
The analysis of resin and fatty acids is fully reviewed elsewhere [24]. The
structures of the most representative resin and fatty acids, dehydroabietic and
palmitic acids, are shown in Fig. 2. Liquid–liquid extraction (LLE) has been
used to extract resin and fatty acids [25]; methyl tert-butyl ether provides an
excellent solvent for the extraction of these compounds [26–29]. Whitewater
pH is generally between 6 and 8, and extraction can be performed under neu-
tral or alkaline conditions [30], although acidic conditions avoid microbial
growth during sample storage [28]. The determination of resin acids in wood
extractives has been traditionally performed by GC with a flame ionization de-
tector (GC-FID) on either a DB1 or HP5 analytical column [31]. This procedure
permits determination of the different compounds (resin acids and hemicel-
luloses) at microgram per liter levels. With non-selective detectors, compound
identification is performed by retention time comparison against a standard.
However, the analysis of complex water matrices leads to interferences and
coelutions, which make identification and quantification difficult. Therefore,
two columns of different polarity should be used for compound confirmation.
This situation changes if an MS detector is used. Spectra with molecular frag-
ment or cluster ions are generated which provide structural information on the
ionized compound. With GC, derivatization of the extract is needed either
using methylation agents such as diazomethane, which presents severe health
hazards, or bis(trimethylsilyl)trifluoroacetamide (BSTFA) combined with tri-
methylchlorosilane. Another derivative of resin acids (RAs) is the pentafluo-
robenzyl (PFB) esters. Figure 4a shows a GC–MS chromatogram which permits
the complete separation of 15 resin and fatty acids, after derivatization with
BSTFA. The main problem is that the derivatized extract has a very short half-
life, generally of 12 h, and the sample has to be derivatized once more if the
injection sequence fails. In addition, the derivative may affect the long-term
performance of the GC–MS system which will necessitate extra cleaning.
GC–MS permits identification of resin and fatty acids from whitewaters of dif-
ferent paper mills [32].
Given the poor volatility of resin and fatty acids, liquid chromatography cou-
pled to mass spectrometry in the negative-ion mode can be used without the
need for derivatization [31, 34]. APCI and ESI are suitable interfaces, although
no fragmentation is observed even at high fragmentor voltages. Figure 4b
shows a LC–MS chromatogram. There is coelution of non-aromatic RAs when
using either C18 or C8 columns, but this is not a problem since the concentra-
tion of total RAs indicates the quality of paper mill water.
Depending on the type of the paper mill process the concentration of resin
and fatty acids range wideley. For example, downstream of a bleached Kraft mill
b
Fig. 4a, b (a) GC–MS total ion chromatogram in EI of a standard containing resin and fatty
acids at 7 mg/mL, after derivatization with BSTFA. (b) LC–APCI-MS total ion chromatogram
of the same standard. Identification numbers: 1=palmitic acid; 2=margaric acid; 3=linoleic
acid; 4=oleic acid; 5=stearic acid; 6=pimaric acid; 7=sandarocopimaric acid; 8=isopimaric
acid; 9=palustric acid; 10=levopimaric acid; 11=dehydroabietic acid; 12=abietic acid;
13=neoabietic acid; 14=chlorodehydroabietic acid; and 15=dichlorodehydroabietic acid
effluent discharge, the concentration of resin acids found in river sediment is

139 mg/g. [22]. This level is ten times lower compared with the level found in the
sludge of this Kraft mill. A similar behaviour was observed for the analysis of
resin and fatty acids in effluent and river water closed to the paper mill. The
concentration decreases in most cases, due to degradation and dilution. Only
for some resin acids, such as dehydroabietic acid, was the concentration
observed 11 km downstream similar to the level found in the source, due to
their high stability [26].
3.3
Surfactants and Plasticizers
Surfactants, such as linear alkylbenzene sulfonates (LAS) and alkylphenol

ethoxylates, are present in whitewaters because of their use as cleaning agents
or as additives in antifoamers, deinkers, dispersants, etc. The non-ionic sur-
factants alkylphenol ethoxylates (APEO) degrade to nonylphenol (NP) or to a
lesser extent, to octylphenol (OP), which are considered as persistent environ-

mental pollutants. LAS and APEO have been detected in whitewaters of paper
mills at concentrations up to 5,000 mg/L for the former [16] and from 0.3 to
10 mg/L for NP and OP [35]. The total concentration of nonylphenol ethoxy-
carboxylates (NPEC) in paper mill effluents ranged from below detection to
1,300 mg/L [36]. These compounds can also be analysed by LC–MS, with the
additional advantage that long-chain alkylphenol ethoxylates and carboxylates
can be simultaneously determined [34]. Due to the complexity of the matrix,
the identification and quantification of compounds should be controlled by the
addition of an adequate surrogate or internal standard. Heptylphenol can be
used for the analysis of alkylphenols.
Paper mill whitewaters and effluents are rich in bisphenol A (BPA), which is
used in great quantities for the production of epoxy resins and polycarbonate
plastics. Its presence in effluents has been reported as a result of its use in the
manufacture of thermal paper or due to migration from plastic containers at
the high water temperatures of whitewaters [35]. This compound is preferably
analysed by GC–MS. The levels encountered in paper mill effluents are between
28 and 72 mg/L [36, 37]. Another study revealed levels up to 226 mg/L [33]. Spe-
cial in vitro test systems and animal experiments have demonstrated a weak
oestrogenicity for BPA. Since aquatic wildlife could be endangered by paper
mill waste discharges at the concentration that BPA is found, its survey in paper
mill effluents should be taken into consideration.
3.4
Lignin and Hemicelluloses
Wood consists mainly of cellulose, hemicelluloses and lignin in various pro-

portions. The amounts and compositions of these component groups depend
primarily on the wood species [38].
In chemical pulping, a significant part of the hemicelluloses is dissolved from
the fibres into the pulping liquor. The rest remains in the fibre or is adsorbed into
it, significantly affecting the properties of the cellulose fibres or paper produced
[39]. It has been demonstrated that the presence of soft or hardwood hemicel-
luloses in the cellulosic pulp can improve some features of paper making. The
plasticity and the high superficial area conferred by hemicelluloses result in an
increased binding among the fibres and a higher tensile strength in the paper
sheet. However, high amounts of hemicelluloses seem to be deleterious to the
mechanical properties of the paper due to a decrease in the individual fibre re-
sistance, and to the optical properties due to the low opacity in the paper sheet
[40]. Some studies have demonstrated a relationship between the degradation of
hemicellulose components, such arabinose and mannose, and wood strength
losses. The significant reduction in strength observed during incipient decay
of wood by brown rot fungi is therefore likely to be due to hemicellulose de-
composition [41]. A basic method for the analysis of hemicelluloses is the de-
termination of their constituent sugar residues obtained by acid hydrolysis
or methanolysis (hydrochloric acid in anhydrous methanol). The liberated mo-

nosaccharides are converted into the corresponding methyl glycosides, and
carboxyl groups of uronic acids are esterified with methyl groups. Thereby,
methanolysis gives the advantage of a reasonable stability of the released methyl
glycosides, and allows simultaneous analysis of acid and neutral sugars by cap-
illary GC–MS or LC after suitable derivatization [42].
On the other hand, lignins are natural polymers in plant cell walls and rep-
resent, after cellulose, the most abundant polymer in nature, with a very com-
plex structure. The lignin composition will be different not only among plants
of different origin, but also among different tissues of an individual plant [43].
It is formed by removal of water from sugars to create aromatic structures.
Lignin resists attack by most microorganisms, and it is a main component
of pulp and paper mill effluent waters. Lignin produces coloured waters [44],
unlike hemicellulose, and is an undesirable polymer whose removal during
pulping requires high amounts of energy and chemicals [45]. Extracted lignins
from non-wood fibres are potential raw materials for new industrial applica-
tions [43]. As a result, lignin should be monitored.
In order to obtain information about the structure of lignin there were some
studies based on the oxidative treatment of the molecule by potassium perman-
ganate [46]. This method involves the selective degradation of all aliphatic side
chains attached to aromatic groups in lignin, resulting in the formation of a mix-
ture. The identification of these as well as the amount of each individual acid pro-
vides information about the substitution pattern in a particular lignin. From the
degradation product obtained it is possible to deduce the structure of lignin. Up
to now, the mixture of aromatic acids obtained from permanganate oxidation of
lignin has been analysed by GC after esterification [47], but Javor et al. developed
a new methodology using capillary zone electrophoresis (CZE), which provides
rapid results with the avoidance of time-consuming preparation of esters of the
resulting aromatic acids. Another methodology, based on the CuO degradation
of lignin, is also considered a suitable technique for their analysis [48].After CuO
degradation of lignin, nine products corresponding to three lignin units (p-hy-
droxyphenyl, guaiacyl and syringyl) could be identified. The degradation prod-
ucts can be easily derivatized, separated by GC and identified by MS.
3.5
Chlorinated Compounds
Since the late 1970s, much emphasis was put on the role of chlorinated sub-
stances formed in the bleach plants. Bleaching effluents from bleached chem-
ical pulp plants are one of the remaining pollution problems of pulp mills due
to the large amounts of chlorinated organic matter discharged into the envi-
ronment [49]. The bleaching of chemical pulp is accomplished in several stages,
to some of which chlorine is added in different forms. The chlorine reacts with
lignin and other organic matter present in the pulp giving chlorinated com-
pounds. During the last decade, there has been a drastic decrease in the use of
molecular chlorine as bleaching agent, which has been replaced by chlorine

dioxide, molecular oxygen, peroxide and ozone. This has led to a decrease in ad-
sorbable organic halides (AOX), which is the main parameter used by regulatory
agencies to determine the discharge of chlorinated organics [50]. Reduction of
AOX has also been achieved by the installation of treatment plants. Current
trends are directed towards closed-cycle systems using either elementary chlo-
rine-free (ECF) or totally chlorine-free (TCF) bleaching pulp. However, this is
only possible if no chlorinated agents have been used within the process. For
some time chlorophenols, and especially pentachlorophenol (PCP), were used
as wood preservatives, and as a result they have been encountered in the water
[51] and sediments [52] of several paper mills. However their use is now re-
stricted and the wood chain is organized so that wood is rapidly consumed and
the use of fungicides is minimized.
The presence of chlorine and chlorinated compounds is also the source of
dioxins and furans during paper making, and these compounds have been
detected in sediments in the vicinity of a pulp and paper mill [53] and in
effluents, along with polychlorinated dibenzothiophenes [54]. A recent study
found high concentrations of PCDD and PCDF along with PCP in nestling tis-
sue (Tachycineta bicolor) collected downstream of paper pulp mills, suggesting
that the primary source of contaminants was the use of PCP for timber preser-
vation [55]. In addition, it has been shown that dioxins bioaccumulate in fish
downstream of pulp and paper mills [56]. The levels of chlorinated compounds
of different families are shown in Fig. 3.
The survey of PCP and other chlorinated compounds has been traditionally
performed by the measurement of AOX, which gives a measure of the total chlo-
rinated organic compounds [57]. Typical AOX levels are between 0.01–0.1 kg/t.
However, to specifically determine the different families of organochlorinated
compounds in paper mill whitewaters and effluents, several analytical methods
have been developed. Current official methods for the analysis of chlorophenols,
e.g. US-EPA 604, 625 and 8041, are based on LLE followed by GC using electron
capture detection (ECD) or MS. However, there is a general trend to use SPE and
LC to avoid the use of toxic organic solvents and derivatization procedures. A
complete review of LC methods for the analysis of chlorophenols is given else-
where [58]. Levels of chlorinated organic compounds in paper mill waters are
between 1 and 100 mg/L, as shown Fig. 3. The analytical protocol for the analy-
sis of dioxins and furans is well established and follows the EPA method 8280A.
4
Toxicity of the Effluents
Some effects have been observed in fauna living close to paper mill discharges,
such as skin and physiological diseases in fish and a decrease in the number of
juveniles, changes in communities and population structure, changes in growth
rates, and delayed sexual maturation and reproduction, among others [2, 58, 59].
In addition, oxygen depletion is common in such effluents, causing anoxia to fish

and other aquatic specimens. The toxicity of paper mill whitewaters and efflu-
ents can be measured using acute toxicity tests such as Microtox or ToxAlert.
These tests measure the bioluminescence inhibition of Vibrio fischeri caused by
the presence of different toxicants in the water sample. Toxic substances will
cause changes in cell structures and/or metabolic pathways of marine Vibrio
fischeri, which are rapidly reflected in a bioluminescence decrease. The LC50
value of several surfactants, resin acids, fatty acids and biocides has been
determined by ToxAlert using individual compounds and mixtures, and the
combination of chemical analysis and effect studies permitted the toxicity of
whitewaters and effluents of several paper mills to be assessed [33]. Figure 5
represents the percentage of bioluminescence inhibition using ToxAlert and the
total organic load of sample (sum of resin and fatty acids, surfactants and bio-
cides) of an untreated effluent and whitewaters corresponding to different pa-
per mills which had undergone several treatments. In cases where the concen-
tration of organic compounds was high, a high percentage of bioluminescence
inhibition was observed. On the other hand, in four samples the organic load
was low, as well as the percentage of inhibition using ToxAlert. However, un-
treated Kraft and print paper showed a low organic compound load and a high
toxicity. This is attributed to the presence of other compounds not considered
Fig. 5 Total organic composition (resin and fatty acids, biocides and surfactants) and per-
centage of bioluminescence inhibition of several types of waters (recycle, Kraft, print board
in open and closed circuit) submitted to primary or biological treatment. Identification
letters: A=effluent; B=recycle untreated; C=recycle primary treatment; D=Kraft untreated;
E=Kraft biologically treated; F=print paper untreated; G=print paper biologically treated;
H=board untreated; I=board biologically treated; J=board closed loop; and K=board
biologically treated
or detected with chemical analysis. Nevertheless it can be concluded that the

combination of chemical analysis and bioluminescence inhibition assay permits
evaluation of the quality and efficiency of treatments.
The measurement of the ethoxyresorufin-O-deethylase (EROD) activity
is another sensitive parameter to detect the effects of paper mill industrial
effluents on living organisms in the receiving waters. The EROD activity is a
measure of the activity of the cytochrome P-450 enzyme system, which plays
a central role in the transformation and elimination of xenobiotics. Increased
EROD activity has been shown as far as 40 km from pulp mills, and EROD in-
duction in fish caused by pulp mill effluents remains after biological treatment
[60]. It is specified that EROD activity and erythrocytic nuclear abnormalities
are induced by abietic and dehydroabietic acid [60].
However, it is difficult to identify the chemical compounds that are respon-
sible for these effects. LC50 values have been tested in several fish species and
levels below 2 mg/L have been reported for resin acids [61] and below 0.1 mg/L
for some biocides used in the paper industry, such as MBT and TCMTB [34].
Wood extractives (resin and fatty acids, sterols, etc.), diterpene alcohols and
juvabiones account for 70–100% of the toxicity in various paper mill effluent
streams [62]. However, toxicity depends on the treatment [63] and recent
papers relate toxic effects towards aquatic biota due to the presence of resin
acids in a secondary-treated bleached Kraft pulp mill effluent [64], and due to
nonylphenol polyethoxy carboxylate metabolites of non-ionic surfactants in a
US paper mill effluent [65]. Moreover, resin acids and, to a smaller extent, un-
saturated fatty acids have been reported as major contributors to the toxicity
of paper industry effluents to aquatic organisms, causing chronic sublethal tox-
icity, genotoxicity and potential bioaccumulation in fish tissues [65]. Endocrine
disruption is being highlighted in modern toxicology. Relatively little is known
about the potential endocrine effects of paper mill effluents on aquatic organ-
isms. Field surveys on fish in proximity to sewage plants show hermaphrodism
and laboratory studies also confirm this phenomenon [66].
5
Air Emissions
The environmental impact of Kraft (sulphate) pulp mills associated with at-
mospheric pollution is due to the emissions of volatile reduced sulphur com-
pounds (VSC) [67]. VSC are formed as a result of the anaerobic decomposition
of organic matter, such as hydrogen sulphide (H2S), methyl mercaptan (CH3SH),
dimethyl mercaptan (CH3SCH3) and dimethyl disulphide (CH3SSCH3). They are
formed in water, and due to their volatility can be emitted to air. In general,
these compounds have very low olfactive detection levels. This explains their
detection by humans even in small quantities and at great distances from
the emission sources. At encountered levels the toxicity of these compounds is
negligible. However, being a nuisance, they are subjected to particular attention
as the pulp and paper industry is continuously faced with more stringent
air emission limits concerning gaseous pollutants. Most of the techniques for
the analysis of VSC in aqueous matrices use the purge-and-trap method with
cryogenic trapping of these analytes in glass tubes [68]. The use of solid-phase
microextraction (SPME), working in the headspace (HS) mode, seems to be
a good alternative to the traditional techniques [69, 70]. Abalos et al. [69]
analysed effluents from a recycled paper mill, obtaining levels between 7 and
24 mg/L, with dimethyl sulphide being the main compound detected, which may
originate from the sodium hydrosulphite and sodium metabisulphite used as
bleaching reagents during the process. Lower levels were found in a bleached
Kraft pulp mill effluent, with values around 0.5–2 mg/L [71].
The release of mill wastewater effluents may be a significant contributor to
mill odours. One example of this pollution is the presence of two terpenoids,
geosmin (trans-1,10-dimethyl-trans-9-decalol) and 2-methylisoborneol (MIB),
caused by the presence of actinomycetes (bacteria) and blue-green algae
(cyanobacteria). Both of these compounds are associated with water from spring
runoff and/or eutrophic systems [72], and are responsible for the majority of the
reported taste and odour events in surface waters close to paper mills. Current
methods for detection and quantification at low levels require large sample
volumes (100–1,000 mL) and intensive sample concentration procedures [71].
Recently, a HS-SPME–GC method was developed that minimized sample ma-
nipulation and time consumption [73]. Watson et al. analysed different paper
mill wastewater treatment plants, obtaining a wide range of concentrations
depending on the sampling point, with levels between 13 ng/L and 127 mg/L.
Other compounds, such as 2,4,6-trichloroanisole (TCA), 2-isopropyl-3-
methoxypyrazine and 2-isobutyl-3-methoxypyrazine, were found downstream
of a pulp mill effluent, and were considered as off-flavours. These compounds
are by-products of chlorination, or can be produced by actinomycetes or other
biota [74].
6
Removal Strategies
For both environmental and economic reasons, many paper mills aim at lower
water consumption and a decrease of water discharge. These can be achieved by
recycling water but unfortunately, closing the water system is far from easy
because an increased recycle of whitewaters leads to an accumulation of solu-
ble organic matter and salts in the paper mill. The advantages and disadvantages
of closed-cycle systems in paper mills are shown in Table 4 [61]. However, the
problems derived from a build-up of organic matter in the whitewater systems
has forced many mills that have been trying a closed-cycle approach to open up
their systems again and continue to discharge great amounts of wastewater.
The first effect of paper mill wastewater discharge is the depletion of oxygen
in the receiving waters, caused by oxygen-consuming microbial degradation of
readily biodegradable organic matter being discharged. This has led to a rapid
interest in developing new methods for in-mill treatment of whitewater to re-
move organic matter.A review of the treatment of pulp and paper mill effluents
indicates processes that minimize the discharge of wastewater into the environ-
ment [75]. Evaporation techniques and chemical treatment are very costly
operations, and membrane filtration often suffers from fouling problems, which
decrease the efficiency and increase the operating costs [76]. Biological treat-
ment is undisputedly the most effective and economical way of removing great
amounts of organic matter from wastewaters.
The possibility of using in-mill biotreatment was proposed in the 1980s, and
during the 1990s biological treatment and reuse of recycle fibre mill process
water was applied in some mills [77, 78]. The objective of these treatments is
to reduce BOD, which is the direct cause of oxygen consumption. However,
being rather conventional, biological treatment plants operating under normal
biological conditions (<40 °C, pH around neutral) require extensive modifica-
tions of the environmental conditions, such as cooling. This is costly and highly
undesirable as it causes heat losses and less efficient production in the paper
mill, which is optimally operated at higher temperatures. The insight into this
has led to a number of studies on the possibility of operating in-mill treatment
at higher temperatures [79–81]. It has even been possible to treat acidic white-
water with high efficiency at a pH as low as 3.5 [81]. Effluents from the mill are
treated in bioprocesses such as aerated lagoons or activated sludge, whereas
whitewaters undergo an anaerobic treatment followed by activated sludge. The
efficiency of the treatments is controlled through measurements of generic
parameters such as COD and BOD. It is assumed that removing as much of the
organic matter as possible will solve the problem. BOD is removed to a great
extent, generally more than 95%. Still, several problems related to the reuse of
biologically treated whitewaters have been encountered:
– Biological treatment removes the bulk of the organic matter, but the fraction
remaining, often dominated by lignin, makes biotreatment difficult. This
gives a significant increase in the colour of the treated water, and unaccept-
able colouring of the product for such paper qualities for which the colour
is important.
– Aerobic biotreatment effectively eliminates odours from organic acids and
sulphide. However, in cases where biotreated water has been reused in
paper production, the product has suffered from a weak “soily” smell that is
unacceptable and has ruled out the continued use of biotreated water.
– It is often necessary to dose nutrients into the bioprocess to achieve a
good performance. However, this leads to nutrients entering the whitewa-
ter system with the reused water. As microbial activities in the whitewater
systems are generally nutrient limited, the increased supply of nutrients
may lead to a considerably increased growth of microorganisms and in-
creased slime problems, rather than the decrease that is the aim of biotreat-
ment [81].
– In cases where water is discharged, considerable effects on life in the receiv-

ing waters, especially from chemical pulping effluents, have been encountered.
Pathological changes in fish have been observed, as well as effects on physi-
ological and biochemical parameters. It is obvious that these serious effects
on the ecosystem are not due to readily biodegradable organic matter, but
rather to compounds more resistant to biological treatment.
These factors have stopped the installation of biological treatment plants at a

number of large Kraft mills, which now continue to discharge great volumes of
untreated effluent. However, there is a growing tendency to install advanced
post-treatment stages to deal with the remaining problems. The combination
of biological treatment and membrane filtration has found a special interest.
Pauly and Kappen [82] studied the combination of thermophilic anaerobic
treatment and ultrafiltration, and found the biotreatment improved the per-
formance of filtration. However, the problems with odour remained. The im-
proved performance of membrane filtration after biological treatment was also
observed by Nuortila-Jokinen [76], in which case biotreatment was to be con-
sidered more of a pre-treatment and filtration the main treatment. However,
combined biotreatment and advanced filtration, such as ultrafiltration and
nano-filtration, are expensive solutions and large amounts of reject streams
are formed which have to be further tested. Therefore, combined processes are
undoubtedly needed, and it is important to identify the most cost-effective
solutions that will give a satisfactory result for each type of paper production.
Effective technologies should be directed towards (1) elimination of the organic
compounds responsible for the toxicity of paper and pulp effluents and related
emissions and (2) reduction of the amount of solid waste going to landfills.
7
Conclusions and Future Recommendations
The pulp and paper industry is the greatest industrial polluter in terms of waste-
water volumes and organic discharge. Compounds encountered in whitewaters
are natural wood components such as resin and fatty acids, and additives added
in the process such as wood preservatives, biocides and surfactants and plasti-
cizers. Since the introduction of the best available technologies and according
to the IPPC Directive, there has been an improvement in the pulp and paper
sector such as minimization of the use of chlorine, additives, energy and fresh
water which has lead to a reduction of emissions of toxic compounds to water,
air and sludge. Generic parameters such as COD, BOD, AOX, total suspended
soils, SO2 and NOx are systematically controlled and maximum discharge lim-
its are well satisfied. However, a recent IPPC Reference Document on the pulp
and paper industry indicates that there is insufficient information on the
organic composition of whitewaters, effluents and sludge from pulp and paper
mills, and on the sampling and analytical methods that should be used for their
characterization. As indicated in this document, water quality among the

different pulp and paper mills can only be assessed by measuring legislated
parameters (BOD, COD, metals etc.) and the specific organic/inorganic com-
position, and by the toxicological characterization of whitewaters, effluents and
solid waste. This chapter has attempted to give an overview of the organic com-
pounds present in pulp paper mill whitewater, the levels encountered and their
toxicological implication. It has also highlighted the treatments performed and
the tools which are nowadays used to remove COD, toxicity and organic load
of pulp and paper mill whitewater for an environmentally friendly paper pro-
duction process. Recently, much effort has been devoted to correlating toxicity
studies and the chemical characterization of pulp and paper mill effluents. This
permits a much stricter control of the treatment that should be performed and
of the quality of the water, which still in many cases is discharged to the envi-
ronment.
Acknowledgements This study has been supported by the EU Energy, Environmental and
Sustainable Development Program (CLOSEDCYCLE, Contract No. EVK1-2000-00749) and
Ministerio de Ciencia y Tecnología (PPQ2000-3007-CE). T. Welander and A. Malmqvist are
acknowledged for providing information on pulp and paper mill treatments.
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DOI 10.1007/b98607
Evaluation of Pesticides in Wastewaters. A Combined

(Chemical and Biological) Analytical Approach
M. D. Hernando1 · I. Ferrer2 · A. Agüera2 · A. R. Fernandez-Alba2 (✉)
1
2
Department of Analytical Chemistry, University of Almería, 04120 Almería, Spain
amadeo@ual.es
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2 Chemical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.1 Sample Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.1.1 Liquid–Liquid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1.2 Solid-Phase Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2.1.3 Semipermeable Membrane Devices and Other Membrane Processes . . . . . . 57
2.2 Cleanup Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.3 Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.3.1 Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.3.2 Liquid Chromatography–Mass Spectrometry (LC–MS) . . . . . . . . . . . . . 64
3 Toxicity Biological Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

3.1 Bioassays Applied to Evaluate the Toxicity of Pesticides . . . . . . . . . . . . . 66
3.1.1 Acute Toxicity Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
3.1.2 Chronic Toxicity Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2 Toxicity Studies of Wastewater Containing Pesticides . . . . . . . . . . . . . . 70
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Abstract The current status of the analysis of pesticides in wastewater by chromatographic

techniques and toxicity bioassays is reviewed and evaluated. When using chromatographic
techniques, the low concentrations of pesticides present and the complexity of the waste-
water matrices require a sample concentration step prior to measurement. Also, cleanup
techniques need to be applied for better detection of the analytes and to avoid ion suppres-
sion. The most commonly used methods of analysis for the detection of pesticides in waste-
water samples involve GC–MS and LC–MS. However, an evaluation only based on chemical
analysis may be insufficient without information related to the negative effects generated.
Bioassays play an important role in the detection and screening of the toxic effects of
pesticides in complex samples such as wastewaters. They provide a response that relates to
the overall effects (synergism, antagonism) of the chemicals present in wastewaters and they
assess the short- (acute) and long-term (chronic) effects. Therefore, both chemical and
biological analytical strategies are relevant to the correct evaluation of pesticides in waste-
waters, their behavior during wastewater treatment, and the reuse of water resources.
Keywords Pesticides · Wastewater · GC–MS · LC–MS · Toxicity bioassays

54 M. D. Hernando et al.
1
Introduction
Pesticides in wastewaters come typically from point sources of contamination

such as disposal sites and landfills where industrial or agricultural wastes are
buried without any consideration, as well as discharges from industrial effluents
from pesticide production plants. Furthermore, nonpoint sources derived from
regular agricultural activities, especially in intensive agricultural areas, and
accidental spills can also be significant. Urban use of pesticides is also possible
in large cities where the use of herbicides and insecticides may result in runoff
into the sewers. These sewers in turn may expel pesticides into wastewater
treatment plants (WWTPs).
Due to the partial to complete resistance of many pesticides to biodegrada-
tion during the wastewater treatment processes, these compounds can escape
elimination in WWTPs and enter into the aquatic environment. As a conse-
quence, their evaluation represents an important objective in the efficiency of
WWTPs and water quality.
Until the beginning of the 1990s, halogenated, nonpolar pesticides were the
focus of interest and a part of intensive water monitoring programs in many de-
veloped countries, and subsequently a drastic reduction of emission was achieved
after adoption of appropriate measures [1]. Today, in industrialized countries we
can consider the presence of these compounds as having less importance. But
they are used as effective pesticides (e.g., lindane, DDT) and still represent a big
issue in developing countries in terms of the environment and human pollution
[2–4]. Awareness of the presence of nonpolar pesticides in wastewaters is
achieved mainly through the use of gas chromatography. Conversely, a “new”gen-
eration of pesticides considered as “emerging contaminants” with a wide range
of structures and typically with high polarity has only been recognized for the
last few years. As a consequence of this, high polarity and sometimes thermally
labile LC-based methods are generally more suitable for their analysis. Therefore,
the interest in evaluating these compounds in wastewater clearly remains, as
is shown by the inclusion of an important number of pesticides in the list of 33
priority substances issued in the last EU Water Framework Directive [5].
In general, we can consider the analytical methods for pesticides well doc-
umented and evaluated as a consequence of the important routine monitoring
programs for food and drinking water [6–13]. Nevertheless, the complex nature
of wastewaters is a great limitation to chemical analyses in their ability to
totally evaluate pesticide content in the low microgram per liter range (or even
below that).A second point of interest is the large number of pesticides, around
800, on the market with a very wide range of structures and physicochemical
properties, which makes it very difficult to develop adequate target multiresidue
methods that cover enough of them, even without taking into consideration the
formation of possible transformation products.
Consequently, there is a lack of knowledge concerning this kind of pollution
and a need to apply sophisticated and powerful analytical techniques to per-
Evaluation of Pesticides in Wastewaters 55
form adequate identification and quantification of pesticides in wastewaters. In

such situations, chemical analysis based on the concentrations of a limited
number of compounds has serious limitations, even if the treated effluents
meet the threshold concentration levels for discharge. The major one is the
inability to account for the contribution of the negative effects of the target
pollutants in the mixture, and to make feasible an effective evaluation treatment
process to allow the reuse of wastewater. Furthermore, wastewaters are not pol-
luted by a single chemical, but rather by a mixture of numerous chemicals, and
this fact can be the main reason for the toxic impacts of wastewater samples.
The mixtures of pesticides and other pollutants may cause toxicity even if each
individual chemical is below its threshold concentration because of interactive
effects among them. It means that the combined effect of various chemicals
can be the result of additive effects of individual chemicals, or they can even
produce a greater toxic effect showing synergism [14, 15].
In the light of these limitations, effective additional tools able to assess the
biological responses of the pesticides present, as well as their interaction with
the other chemicals, have to be introduced to complete the evaluation of waste-
waters. Bioassays on water samples provide a direct functional response that
can relate to the negative effects of a single pesticide and overall toxic proper-
ties of the complex mixture of compounds present in a sample [16].
This study is an overview focused on the application of the main analytical
strategies based on chemical analysis and biological toxicity assays for pesti-
cides, to be used as a combined approach for the evaluation of pesticides in
wastewaters.
2
Chemical Analysis
2.1
Sample Treatment
Due to the predicted and previously detected low concentrations of pesticides

in environmental samples (usually around the nanogram per liter level), a pre-
concentration step of the water samples is necessary prior to measurement. In
this way, a preconcentration factor of several orders of magnitude (200–1,000-
fold) is mandatory to reach the low detection limits necessary for the iden-
tification of pesticides, especially in complex wastewater samples.Also, the use
of surrogate standards (e.g., triphenyl phosphate) added before the extraction
step is a common practice in order to account for possible errors during the
extraction process and for quantitative purposes. The commonly used extrac-
tion methods for polar compounds from water matrices involve isolation using
liquid–liquid extraction (LLE) and solid-phase extraction (SPE), which are com-
mented on below. Other methods such as semipermeable membrane devices
(SPMD) are also mentioned.
2.1.1
Liquid–Liquid Extraction
LLE has been used in the past for the extraction of pesticides from environ-
mental water samples [17]. However, its application in the extraction of waste-
water samples is scarce due to the low efficiency of extraction, especially for
polar analytes. Because of the vast amount of surfactants and natural products
present in wastewater samples, emulsions are formed which complicate the
process of extraction and lead to low extraction recoveries. However, there have
been some useful applications of LLE to wastewater analyses. For example, LLE
was found to be effective for the isolation of herbicide and pesticide organic
compounds from industrial wastewater samples and also from complex ma-
trices [18].
2.1.2
Solid-Phase Extraction
SPE procedures are used not only to extract traces of organic compounds from
environmental samples, but also to remove the interfering components of the
complex matrices in order to obtain a cleaner extract containing the analytes
of interest. In this sense, it is a good sample treatment method for the analysis
of wastewater. In the last few years, there has been a considerable interest in
developing new selective and sensitive methods for extracting and isolating
components from complex environmental matrices. The selectivity is the
degree to which an extraction technique can separate the analyte from inter-
ferences in the original sample.Accordingly, the selectivity of stationary phases
is an important parameter to be taken into account when compounds are to be
extracted from wastewater samples, since the main objective is to remove
interferences and facilitate further analysis by conventional analytical method-
ologies such as gas chromatography (GC) or liquid chromatography (LC).
SPE using C18 or polymeric phases has been used widely for the determi-
nation of pesticides in water samples [19, 20]. These stationary phases are gen-
erally nonselective and can lead to difficulties with interferences coextracted
from the wastewater matrices. Most of the polar pesticides cannot be deter-
mined owing to their coelution with the matrix peak, which is obtained at the
beginning of the chromatogram when wastewater samples are analyzed by
chromatographic techniques [21]. This matrix peak is a coeluting interferent
caused by humic substances present in natural waters. The chromatographic
methodologies used are commonly not selective toward the coextracted com-
pounds present in environmental samples, and consequently it is of primary
importance to use a selective sorbent for the preceding step (SPE) in the entire
analysis. The main goal of the SPE step is to provide a cleaner extract, free
of matrix interferences. This is the first step in the development of a highly
selective and sensitive methodology that can be applied to the determination
of traces of organic contaminants in complex environmental samples. In other
words, the more selective the SPE step, the better the sensitivity achieved. For
these reasons, efforts have been made to develop new selective sorbent mate-
rials for the analysis of wastewater samples.
Modified silica with a C18 reversed-phase sorbent has historically been
the most popular packing material, owing to its greater capacity compared to
other bonded silicas, such as the C8 or CN types [22]. Applications of C18 sor-
bents include the isolation of hydrophobic species from aqueous solutions. The
mechanism of interaction with such sorbents depends on van der Waals forces,
and secondary interactions such as hydrogen bonding and dipole–dipole in-
teractions. Nevertheless, the main drawbacks of such sorbents are their limited
breakthrough volumes for polar analytes, and their narrow pH stability range.
For these reasons, reversed-phase polymeric sorbents are also used frequently
in environmental applications for the trace enrichment of soluble molecules
that are not isolated by reversed-phase sorbents such as C18.
The most widely used polymeric sorbents are the styrene–divinylbenzene
copolymers (SDB), which are among the classical reversed-phase sorbents
introduced in the 1960s [20]. They are currently produced in purified form and
are useful for the isolation of more polar solutes that have low capacities on
the C18 reversed-phase sorbents. Their broader pH-stability range increases the
flexibility of the method since the pH of the wastewater samples is usually in
the high range. Moreover, these kinds of sorbents have a greater surface area per
gram, so they can retain the most water-soluble analytes. Another advantage of
the aromatic sorbents derives from their selective interaction with aromatic
rings in the analytes. Because the styrene–divinylbenzene structures contain
aromatic rings, they have the ability to sorb analytes by specific p–p interac-
tions. More recently, many immunosorbents based on antigen–antibody inter-
actions have been developed for the selective isolation of many pesticides in
water samples [23]. They have proven to be very suitable for the highly selec-
tive preconcentration of organic contaminants from complex environmental
samples, such as sediments and sludges. Since such sorbents are tailor-made for
specific applications, their cost is high compared to conventional sorbents [24].
However, they are very limited for multiresidue applications and therefore only
useful in wastewater analysis for those cases when a conventional sorbent is
not suitable. On the other hand, molecularly imprinted polymers have been
developed as well and are gaining applicability in some environmental areas,
and could be a promisingly useful tool for the trace enrichment of organic con-
taminants in complex mixtures in forthcoming years [25].
2.1.3
Semipermeable Membrane Devices and Other Membrane Processes
SPMD have gained widespread use for sampling hydrophobic chemicals from
water. In these membranes the more hydrophobic compounds are retained and
are further recovered with organic solvents. As an example, SPMD have been
applied to the analysis of pesticides in wastewater samples [26].
Other membrane processes such as microfiltration, ultrafiltration, reverse

osmosis, and colloid-enhanced ultrafiltration have been applied to the separation
of beta-cypermethrin from wastewater samples [27]. In this study, a separation
of above 92% was performed by reverse osmosis by the use of composite mem-
branes and above 80% by colloid-enhanced ultrafiltration by the use of nonionic
surfactants.
2.2
Cleanup Techniques
In environmental analysis, organic compounds are usually present at low con-

centrations and are often masked by complex patterns of interfering compo-
nents. Therefore, cleanup steps are necessary for the analysis of wastewater sam-
ples, especially before analysis by gas or liquid chromatographic techniques.
Florisil and silica phases are the most commonly used cleanup methods for
removing organic acids and humic substances from sample extracts when
analyzing hydrophobic compounds such as organochlorine pesticides by gas
chromatographic techniques. SPE can be easily applied as a cleanup method for
this kind of matrix as well. Accordingly, sequential SPE has been applied as a
preconcentration and cleanup method in the analysis of some pesticides in
wastewater samples before analyses by liquid chromatographic techniques [28].
Other cleanup methods involve the rapid and effective anion-exchange ca-
pacity of the anion-exchange phases to remove humic substances, which are
present in complex water and soil samples [29]. The high selectivity of the SAX
disk for humic substances allows these interferents to be effectively removed
from water samples during trace enrichment of herbicides from complex water
extracts. The concept of layering disks can be used to first remove the humic
impurities on the SAX disk with simultaneous isolation of herbicides on the
lower C18 disk. The concept of stacking adsorbents for trace enrichment was
first introduced in the early 1980s with XAD adsorbents. Both anion-exchange
and reversed-phase methods can then be used to isolate both natural and con-
taminant organic compounds from water. More recently, SPE cartridges have
been introduced with layered adsorbents, which facilitate treatment of the
aqueous samples.
Total or partial ion suppression is a well-known LC–MS effect, which is
induced by coeluting matrix components that can have a dramatic effect on the
intensity of the analyte signal.As can be observed in Fig. 1, analyte suppression
occurs as a consequence of the different matrix interferences present in waste-
water samples, making the identification and/or quantification process difficult
or unfeasible. Even when working under selection ion monitoring (SIM) con-
ditions, these matrix effects can cause ion suppression in the detection of some
analytes that are present at low levels of concentration, as seen in this figure.
Several papers have reported this effect [30–32] and different alternatives to
overcome these problems, such as the inclusion of a size-exclusion step [33] or
sequential SPE [28], have been applied for the determination of pesticides in
Evaluation of Pesticides in Wastewaters
Fig. 1 SPE–LC–ESI-MS analysis (SIM mode) of two wastewater samples, spiked at different levels of concentration. Compounds:
59
(1) ciromazine, (2) oxamil, (3) metomil, (4) carbendazime, (5) thiabendazole, (6) imidacloprid, (7) acetamiprid, (8) thiacloprid
environmental matrices. The advantage of the fractionation of the extract is the

separation of the pollutant mix into different group classes, depending on their
chemical properties, which reduces matrix interferences and makes detection
easier and more reliable.
Solid-phase microextraction (SPME) is also a useful alternative to conven-
tional sample cleanup with LLE or SPE. SPME is based on the enrichment of
analytes by a partitioning process between a polymeric phase coated on a
fused-silica fiber and its surrounding aqueous solution. SPME combines sam-
ple preparation in terms of extraction from a matrix of interfering compounds
with an enrichment process in a single step. A method for the determination
of metazachlor in wastewater samples is described in the literature [34]. In this
study, SPME was shown to be a suitable and simple sample preparation method
for the determination of metazachlor in wastewater by GC–AED.
2.3
Methods of Analysis
A wide range of analytical techniques have been developed in order to identify

the organic contaminants often present at trace levels in complex environ-
mental samples such as wastewaters. These techniques mainly use gas chro-
matography (GC) and liquid chromatography (LC).
Most of the continuously monitored water contaminants are determined
via gas chromatography–mass spectrometry (GC–MS). However, an adequate
separation of polar compounds via GC typically requires derivatization of the
polar moieties (e.g., BSTFA derivatives). In addition to this, as the analyte
groups show different properties concerning the number and kind of func-
tional groups, it is quite difficult to develop a universal derivatization procedure
suitable for all the target analytes. Furthermore, the presence in wastewater of
many other organic compounds requires the use of labeled standards, which
can make application of this method unfeasible [35].
2.3.1
Gas Chromatography
GC is coupled with many detectors for the analysis of pesticides in wastewater.

At the present time the most popular is GC–MS, which will be discussed in
more detail later in this section. The flame ionization detector (FID) is another
nonselective detector that identifies compounds containing carbon but does
not give specific information on chemical structure (but is often used for quan-
tification because of the linear response and sensitivity). Other detectors are
specific and only detect certain species or groups of pesticides. They include
electron capture, nitrogen–phosphorus, thermionic specific, and flame photo-
metric detectors. The electron capture detector (ECD) is very sensitive to chlo-
rinated organic pesticides, such as the organochlorine compounds (OCs, DDT,
dieldrin, etc.). It has a long history of use in many environmental methods,
especially those advocated by the U.S. Environmental Protection Agency. The

next most common is the nitrogen–phosphorus detector (called the NP de-
tector), which is effective for many herbicides and insecticides because they
contain either nitrogen (triazines and acetanilides) or phosphorus (OP in-
secticides). This is an inexpensive detector that has been widely used in the
analysis of pesticides in water. The flame photometric detector (FPD) works on
chemiluminescence and detects pesticides that contain sulfur, phosphorus, and
some metals, such as manganese [36]. The thermionic specific detector (TSD)
and FPD work on pesticides that also contain sulfur, nitrogen, and phosphorus
[37]. The combination of these groups of detectors has value for specific com-
pound identification in complex matrices, where GC–MS may have serious in-
terferences. Wastewater is such an example. A specific detector for measuring
elemental compositions as a percentage is the atomic emission detector (AED),
which can detect all elements, except helium, separately due to its multichannel
ability and selectivity, making it more sensitive than the more commonly used
detectors cited above. This type of detector has been used for the detection of
pesticides in wastewater samples [34].
Finally, gas chromatography coupled to mass spectrometry (GC–MS) is the
most universal technique for the analysis of pesticides in water samples [38].
The high sensitivity and selectivity of modern GC–MS instruments enables low
limits of detection depending on the matrix and in particular on the chemical
structure of the pesticide. With most instruments, full-scan spectra can be
evaluated at the low nanogram level, which means 1 or 10 pg analyte injected
into the GC–MS system with the sample. Spectral averaging and background
subtraction facilities provided by the data system are generally used to remove
contributions from the matrix background or partially resolved contaminants.
However, with very weak spectra, these data processing procedures may lead to
corrected mass spectra of dubious validity. Changing from full spectral scanning
to selected ion monitoring using the reduced number of mass channels leads
to considerably improved detection limits for the specified target compound
ions. The different types of ionization include electron impact (EI) and chem-
ical ionization (CI). One advantage of negative chemical ionization (NCI) is in
the analysis of organochlorine insecticides in complex matrices, because the
background does not ionize and the pesticides are easily detected (see Fig. 2).
The ion suppression and matrix interference effect is clearly shown in this
figure when analyzing wastewater samples in the EI mode, even under SIM con-
ditions. As an example, Fig. 3 shows the analysis of a real wastewater sample in
the NCI mode where three compounds were identified by the corresponding
mass spectra.
Another approach in GC is that of using more power in the separation by
doing GC¥GC. In this approach, a second column is used with a different type
of stationary phase than the primary stationary phase, and fast chromatogra-
phy using TOF-MS as the detector is carried out [39]. This technique uses only
TOF-MS as the detector since it has the most sensitivity for fast-eluting peaks.
The method has been applied to complicated matrix analysis.
62
Fig. 2 GC–MS chromatograms of a spiked wastewater extract with triclosan, endosulfan, and oxifluorfen obtained under EI
(full scan) and EI (SIM) conditions at a concentration level of 625 mg L–1 and NCI (full scan) conditions at 250 mg L–1
M. D. Hernando et al.
Evaluation of Pesticides in Wastewaters
Fig. 3 SPE–GC–NCI-MS chromatogram obtained from a real wastewater sample (influent) where triclosan and endosulfan-a, -b and
sulfate were detected at 4.9 mg L–1, 160 ng L–1, 128 ng L–1, and 15 ng L–1, respectively, under SIM conditions
63
One of the known disadvantages of the use of GC is the need for previous
derivatization of some of the most polar pesticides before analysis can be car-
ried out [40]. These derivatization steps might produce low-efficiency results
in complex wastewater matrices, which make the analysis rather difficult and
cumbersome. However, the reproducibility in retention times when using GC
techniques is so precise, that specific identifications of pesticides can be made
even in complex environmental samples.
Quantification is usually achieved by a standard addition method, use of
labeled internal standards, and/or external calibration curves. In order to allow
for matrix interferences the most reliable method for a correct quantitation of
the analytes is the isotope dilution method, which takes into account intrinsic
matrix responses, using a deuterated internal standard or carbon-13-labeled
internal standard with the same chemistry as the pesticide being analyzed
(i.e., d-5 atrazine for atrazine analysis). Quality analytical parameters are usually
achieved by participation in interlaboratory exercises and/or the analysis of
certified reference materials [21].
2.3.2
Liquid Chromatography–Mass Spectrometry (LC–MS)
Due to the high amount of interferences present in wastewater samples, UV de-

tection is not possible for the identification of pesticides at low levels of con-
centration (see Fig. 4). As can be noted in this figure, the humic and fulvic acid
peak at the beginning of the chromatogram masks the identification of the
most polar pesticides and complicates the identification and quantitation of
the analytes. Furthermore, a higher level of confidence (molecular or fragment
structural information) is necessary for the correct identification of analytes
in such complex matrices. In this sense, liquid chromatography coupled to
mass spectrometric detection is the best choice for the analysis of pesticides
in wastewater samples. Since polar, nonvolatile, thermally unstable or high-
molecular-weight compounds are unsuitable for gas chromatography–mass
spectrometry (GC–MS) analysis, the use of LC–MS has become a robust and
routinely applicable tool in environmental laboratories [41, 42]. Non-GC-
amenable compounds include 15–20% of the present-day pesticides, e.g.,
phenylureas and carbamates, the phenoxyalkanoic acids, and a large majority
of all pesticide transformation products [43]. The performance of LC–MS in
the analysis of polar and thermally labile pesticides that are not amenable to
GC–MS has been well demonstrated in several studies [44, 45]. In the last few
years, interfaces based on atmospheric pressure ionization (API) have resulted
in an increase in the number of applications in environmental determinations.
In this respect, atmospheric pressure chemical ionization (APCI) and electro-
spray ionization (ESI) have recently become the most universal techniques for
environmental analysis due to their high sensitivity, the possibility of detecting
a broad range of analytes, and the useful structural information obtained via
fragmentation similar to collision-induced dissociation (CID) [46]. Compared
Fig. 4 SPE–LC-DAD analysis of a wastewater sample. Peak identification number and peak
retention times (min): (1) azinphosmethy, (11) parathion-methyl, (4) malathion, (3) feni-
trothion, (8) azinphos-ethyl, (6) chlorphenvinphos, (10) parathion-ethyl, (7) diazinon [from
ref. 21]
with older mass spectrometric detection techniques such as TSP and PB, API
techniques offer both structural confirmation and high sensitivity for target
compounds in environmental samples. One of the great advantages of the ESI
interface is its high sensitivity for ionic pesticides such as many herbicide
metabolites containing a sulfonic or a carboxylic group in the chemical struc-
ture [47].
The advent of high-performance liquid chromatography–mass spectrome-
try (HPLC–MS) using quadrupole instruments has made analysis of polar
pesticides in water a common procedure [45, 48]. Many classes of pesticides are
easily analyzed by LC–MS and a more challenging task is to identify the degra-
dation products of pesticides. During the past 5 years many papers have been
published on the analysis of pesticides and their degradation products by
HPLC–quadrupole MS [49]; however, there are several shortcomings yet to be
overcome. For example, often polar pesticides give only a protonated or de-
protonated molecule or a weak fragment ion, especially when the interface is
ESI. The fragmentor or cone voltage is used to enhance CID in the source and
transport regions of the electrospray source, and this fragmentation voltage may
vary substantially among different analytes and sources, which makes frag-
mentation difficult to predict in an analysis of unknown compounds. Second,
there are no universal libraries available for pesticide analysis by HPLC–MS, as
in electron impact GC–MS; this problem makes identification of unknown
pesticides or their degradates nearly impossible by simple HPLC–quadrupole
MS analysis.
These shortcomings may be overcome partially by the application of time-
of-flight mass spectrometry (TOF-MS) and liquid chromatography–quadrupole
ion-trap tandem mass spectrometry (LC–QIT-MS/MS) [50–52]. The LC–QIT-
MS/MS does MS/MS in time rather than in space, which means that ions are
retained in a trap through a set time period. If all the ions are ejected, then the
result is a full-scan spectrum. If the protonated or deprotonated molecule is
retained in the trap and all others are ejected, and this ion is fragmented, the
result is MS/MS. This process may be repeated multiple times, which results
in MSn. In contrast, triple quadrupole MS/MS does the isolation and frag-
mentation in space, which means that the fragmentation is continuous in time,
but the selected ion travels through the flight tube of the mass spectrometer to
the collision chamber where fragmentation occurs, and then on to the third
quadrupole for the mass spectrum.
Two advantages of the ion trap are that it gives excellent sensitivity while
trapping ions in full-scan mode, which then may be selected and fragmented
to yield MS/MS spectra, and second is the ability of the ion trap to do MSn [50].
Typically, three or four isolations and fragmentations are possible before the
sensitivity is too low to record ions in unknown samples. The ability to do mul-
tiple isolation and fragmentation allows one to build a library of spectra using
standard compounds, which give both characteristic fragmentations and di-
agnostic ions that can then be used to identify unknown pesticides or their
degradates. TOF-MS is also useful for identification of synthesized standards to
verify the analysis of QIT-MS/MS when no commercial standards are available
and new standards are synthesized, as well as the identification of degradates in
actual groundwater samples [52].
3
Toxicity Biological Assays
3.1
Bioassays Applied to Evaluate the Toxicity of Pesticides
The toxic effects of pesticides can be diverse and depend on the sensitivity of
organisms to these toxicants, and the pesticide concentration or bioavailability.
Typically, the short- and long-term effects of pesticides have been evaluated
through acute or chronic toxicity bioassays, respectively, using lethality end-
points and sublethal endpoints (e.g., growth and reproduction), particularly
these last in chronic bioassays.
3.1.1
Acute Toxicity Bioassays
Most commonly, bioassays for the evaluation of the acute toxic effects of pesti-
cides are based on single aquatic species selected to be representative of a range
of taxonomic and functional groups, i.e., bacteria, algae, invertebrates or fish
[53, 54]. Generally, toxicity evaluation using a single species is the alternative of
choice rather than the use of multiple species, because extrapolation of effects
to an ecosystem is more difficult and can often lead to incorrect conclusions.
The selection of suitable single species and protocols is not a trivial task and
may be dependent on various factors. Some of these include simplicity, low cost,
or modest material and equipment demand. However, a higher sensitivity than
other species to toxicants may be decisive in this choice in order to serve
as warning systems. Table 1 shows the sensitivity in terms of effective concen-
tration (EC50), which is the toxicity endpoint for the organisms (bacteria,
crustaceans, algae, and fish) selected for the toxicity bioassays. These toxicity
bioassays are usually classified according to the test species involved.
Fish assays have been extensively used for laboratory studies. Among com-
monly used species are Pimphales promelas or Oncorhynchus mykiss. These
species are relatively sensitive and respond to a variety of water constituents
and contaminants including pesticides. P. promelas is a widely distributed
species in the aquatic environment, and its use for whole effluent toxicity
(WET) procedures is also well established [55, 56]. Reported lethal concentra-
tions for pesticides such as chlorotalonil or chlorpyriphos (EC50=22.6 and
381 mg/l, respectively) showed these compounds as “harmful to aquatic organ-
isms” and “not harmful”, respectively, according to toxicity categories [56, 57].
Generally, in addition to the relative sensitivity (Table 1), the use of these bio-
assays presents some disadvantages such as standardization problems, time
consumption or need of specialized equipment [58–60].
Invertebrate species have been widely used in toxicity studies of pesticides
[61]. Zooplankton play a key role in the food chain because they occupy a cen-
tral position. Therefore, their responses to natural and anthropogenic stresses
are intimately linked with other food predator organisms. The most widely
accepted bioassays employ species such as Ceriodaphnia dubia, Daphnia magna,
Artemia salina, or Thamnocephalus platyurus [62–64]. D. magna has been used
for many years as a standard aquatic test species and formally endorsed by the
major international organizations such as the EEC, OECD, and ASTM [65–67].
Its choice is mainly because it represents the zooplankton community and is
a species of worldwide occurrence. In addition, it has a greater sensitivity to
toxicants, particularly pesticides, compared with other aquatic species [61, 68]
(Table 1).
Algae are of vital importance in the primary production of the aquatic
ecosystem because they are primary producers of the food chain. Several species
of green algae are used in toxicity studies of pesticides, especially herbicides
such as Chlorella vulgaris, Chlorella pyrenoidosa, or the standard test microalga
Table 1 Effective concentration (EC50) values of pesticides for bacteria, algae, crustaceans,
and fish
Pesticides EC50 (mg l–1) References
Bacteria Algae Crustaceans Fish
Fenamiphos 35.1a 0.005 [15]

Benomil 0.05 b [71]
Pentachlorlophenol 0.55 [77]
Paraquat 14,800 1 <1.0 2, b [77]1, [68] 2
Deltametrin 0.005g [58]
Dichlorvos 2.10–4,e [63]
Chlorpyriphos 3.21,e 3812, h [68]1, [57]2
Metalaxil 21.1b [71]
Carbendazim 34.6 b [71]
Procymidone 0.74 b [71]
Zineb 0.52 b [71]
Chlorothalonil 0.007 1, c 0.03 2, f 22.6 3, h [14]1, 2, [56] 3
b-Cypermethrinm 0.02 g [59]
Dichlofluanid 0.08 1, a 0.132,c 1.03,f [14]1, 2, 3
Permethrin 0.2 g [60]
1, a
Carbofuran 31.2 0.022,f [15]1, 2
Diuron 0.041, c 8.62,f [14]1, 2
Isoproturon <1.0 d [68]
Atrazine <1.0 d [68]
Formetanate 7.41, a 0.07 2, f [15]1, 2
Pirimiphos-methyl 4.10–4, f [14]
Malathion 1.8·10–3, f [63]
Cyromazine 10.7 f [68]
a
Vibrio fischeri (EC50 at 15 min).
b
Chlorella pyrenoidosa (EC50 at 96 h).
c Selenastrum capricornutum (EC at 72 h).
50
d Chlorella vulgaris (EC at 96 h).
50
e Artemia salina (EC at 48 h).
50
f Daphnia magna (EC at 48 h).
50
g Poecilia reticulata (EC at 48 h).
50
h Oncorhynchus mykiss (EC at 96 h).
50
Selenastrum capricornutum [14, 68, 69]. Herbicides play an important role in

agricultural practices and as a consequence, they can affect nontarget organ-
isms, modifying the structure and function of aquatic communities due to the
alterations of the specie composition species in algal communities. The effects
of new herbicides in agricultural activities have been recently published [68].
Paraquat, diuron, isoproturon or atrazine (see Table 1) are examples of herbi-
cides considered as very toxic according to toxicity categories established in the
Directive 93/67/EEC [57, 68, 70, 71], with toxicity endpoint values expressed as
effective concentration (EC50) less than 1 mg/l.
Bioassays using bacteria as indicators constitute at present one of the most

widely applied screening techniques for identifying the toxicity of substances
and commercial products [72]. The main reason is that, unlike toxicity bioas-
says using more complex organisms such as fish, bacterial bioassays are much
quicker and cheaper.A great number of bioassays based on very different meth-
ods and microorganisms have been described, and include studies of the effects
of toxicants on different parameters such as growth inhibition and enzymatic
activity [73, 74]. Among the bacteria employed, such as Pseudomonas putida
or Escherichia coli as indicators, V. fischeri is the most common standardized
bacteria specie used in toxicity bioassays [14, 72, 75]. The advantages are its sen-
sitivity, reproducibility, and it is a rapid and simple test (Table 1). For toxicity
evaluation of pesticides, there are reported data showing the sensitivity and the
utility of this test [14, 75–77].
The different sensitivity of the species indicates that a single bioassay does
not satisfy the correct evaluation of the wastewater. Thus, normally, various
species are used because toxic substances may produce a specific response in
one species but not in another. Therefore, there is practically generalized con-
sensus on the use of a battery of bioassays involving different trophic levels
of species. The application of this approach is considered an efficient and
essential tool for predicting environmental hazards to the aquatic ecosystems.
According to several authors, the most appropriate way to assess ecotoxicity
is the use of four different test organisms of increasing levels of biological
organization. This system includes the use of bacteria, crustaceans, algae, and
fish to assess the toxicity of chemicals such as pesticides in wastewater, and it
would be performed sequentially, going to the next level when the sample
was found to be nontoxic [14, 76, 78]. However, a general perception is that, for
practical and ethical reasons, the use of fish is not frequently included in these
studies.
3.1.2
Chronic Toxicity Bioassays
Episodic pollution events can adequately be addressed by acute toxicity bioas-

says, however these are not sufficient to investigate the water quality for delayed
toxicity effects of chemicals present. Chronic effects of pesticides can include
carcinogenicity, teratogenicity, mutagenicity, neurotoxicity, and reproductive
effects (endocrine disruption).
Most insecticides, especially the organophosphate group, cause neurotoxi-
city as their major mode of action. Assessment of the neurotoxicity includes
neurochemical endpoints such as cholinesterase (including acetylcholinesterase,
which is the major neurotransmitter in vertebrates such as fish, and other
enzymes such as butyrylcholinesterase) inhibition and behavioral endpoints
such as swimming speed [79]. Studies done in rats show the neurotoxic action
of insecticides such as dimethoate, methyl parathion, dichlorvos, ethyl parathion
or propoxur after a prolonged exposure [80, 81].
Chemical carcinogenicity has been the target of a large list of scientific pub-
lications, because it is one of the toxicological endpoints that poses the high-
est concern. The standard bioassays in rodents used to assess the carcinogenic
potential of chemicals are extremely long and costly and require the sacrifice
of a large number of animals. For these reasons, mutagenicity bioassays
are presented as alternatives to evaluate the DNA-damaging activity [82, 83].
The types of genetic lesions expected can be chromosomal deletion, loss or
translocation, mitotic recombination or base substitution [82]. Therefore,
regular practices to evaluate the possible genetic lesions recommend the use
of a battery of bioassays including a bacterial test for gene mutation, either an
in vitro test for chromosomal aberrations or a mammalian cell mutagenesis
test, and a general test for DNA damage [84, 85]. A great number of studies on
the mutagenic activity of pesticides have been published. Examples of these
show that the chloroacetanilides, classified as herbicides, have a consistent
positive induction for gene mutations [86].
More recently, toxicity studies have shown the importance of noncancer
endpoints in chronic toxicity assessment, with increasing emphasis on end-
points such as endocrine disruption. The endocrine system as a target of pes-
ticide toxicity can manifest reproductive consequences, particularly in terms
of steroid hormone function, resulting in the manifestation of demasculiniza-
tion in fish. The gonad histology and serum vitellogenin (VTG) protein levels
have been widely used as endpoints for screening and testing of potential
endocrine-active compounds and are currently subject to validation by the
OECD and associated scientific groups [87–89]. Some reports have demon-
strated that the presence of organochlorines, such as dieldrin, heptachlor or
aldrin, appears to be closely linked to the induction of VTG synthesis [90, 91].
However, bioassays based on yeast strains are very promising among the test
systems available because of their physiological simplicity, easy handling,
and low costs [92, 93]. In general, they rely on yeast constructs expressing an
estrogen receptor which, upon binding of suitable substrates, acts as a tran-
scriptional enhancer for an estrogen-responsive DNA-element-controlled re-
porter gene, in most cases bacterial b-galactosidase. The activity of this enzyme
can be determined photometrically by using a chromogenic substrate and thus
may serve as a measure of the estrogenic potency of the samples under in-
vestigation. Several active components such herbicides and insecticides (e.g.,
endosulfan, dieldrin or toxaphene) have been reported to possess estrogenic
activity [94, 95].
3.2
Toxicity Studies of Wastewater Containing Pesticides
While reported data on the acute and chronic toxicity of many pesticides is
plentiful, few studies have been published on toxicity bioassays applied to
wastewaters containing pesticides. The application of toxicity bioassays to the
quality control of wastewaters offers several advantages in addition to being a
biological measure able to detect toxic effects. Among these, sensitivity, easy
handling, speed, simplicity, and low costs are the features of choice for routine
purposes. In the last few years, interest in toxicity bioassays for assessing waste-
water has been increasing and recent publications are focused on this approach
[96–98]. Some of these studies proved that there is no correlation between
chemical and ecotoxicological parameters. Control based on global chemical
parameters such as biochemical oxygen demand (BOD), chemical oxygen de-
mand (COD) or total organic carbon (TOC) may be insufficient, even if the
treated effluents meet the threshold concentration levels for discharge. This case
is illustrated in Fig. 5, which shows a monitoring study performed on influent
and effluent wastewaters. Samples corresponding to toxic effluents showed
permissible TOC levels [96].
Wastewater from agricultural areas that arrives at wastewater treatment
plants (WWTPs) is highly variable in nature. Intermittent or accidental episodes
of toxic substances can have a damaging effect on the receiving waters, when the
b
Fig. 5a, b Monitoring study of wastewaters based on chemical and ecotoxicological par-
ameters [from ref. 96]
influent has not been properly treated. Consequently, rapid methods of waste-
water toxicity assessment represent a very useful tool, acting as an early warn-
ing system. The capability of detecting toxic responses in a short time allows
quick decisions to be made regarding the convenience of effluent discharge.
In others words, the capability of detecting toxic effects is one of the best
applications of bioassays in the quality control of wastewaters, because it allows
detecion of unwanted toxicity, potential problems in the treatment station,
and contamination peaks in effluent toxicity before discharging it to receiving
waters [76].
The sensitivity of test species is a decisive feature in the choice of bioassays
to evaluate the toxicity. Despite the diversity of test species available, in many
regulatory schemes the invertebrate species recommended for acute and chronic
testing is the cladoceran D. magna [99, 100]. In the U.S., the Food and Drug
Administration and Office of Pollution Prevention and Toxics (OPPT) of the
Environmental Protection Agency recommend that acute data should be col-
lected with Daphnia species (D. pulex and D. magna) [101]. Presumably, the
focus on D. magna results from its high sensitivity to environmental contam-
inants relative to other species, mainly invertebrate species. The sensitivity of
D. magna to pesticides has been demonstrated in recent publications showing
its capability of detecting toxic responses at concentration levels as low as
nanograms per liter [14]. This means that toxicants at environmentally realis-
tic concentrations can be detected by this bioassay.
However, the detection limit of standardized bioassays may be too high to
detect toxicity and hence pesticide contamination. Therefore, in these cases,
preconcentration of the samples is necessary. Bioassays combined with pre-
concentration of the wastewater have been proved to be a useful strategy for
screening and monitoring in the initial assessment of water pollution by pesti-
cides [102, 103]. Even if bioassays are able to detect toxicity in nonconcentrated
samples, this strategy is a useful approach in order to obtain the toxicity end-
point (e.g., effective concentration EC50) from a full concentration–response
relationship. This combined methodology was applied in a screening study
from an agricultural area where methyl parathion, lambda-cyhalothrin, and
endosulfan are the most commonly used pesticide chemicals. Acute toxicity
was detected in surface water from agricultural areas using standardized bio-
assays with the algae S. capricornutum and crustacean D. magna [104].
Whole effluent toxicity (WET) monitoring offers several advantages because
this toxicity evaluation has to account for the presence of unknown toxicants,
the interactions among multiple toxicants, and the alterations in toxicant
bioavailability caused by the effluent matrix. When evaluating the toxicity
of the complex samples, the detection and identification of regulated or specific
chemicals is a key need for controlling effluent quality. Thus, the identifica-
tion of toxic compounds in complex samples has been the objective of re-
ported studies using toxicity-based procedures. Combined protocols involving
chemical analysis and toxicity evaluation became known collectively as toxic-
ity identification evaluation (TIE), and nowadays they are techniques well
established and developed by the USEPA [104]. TIE methods have been found
to be effective tools for characterizing and identifying toxicants in samples of
effluents, sediments, ambient waters, and other complex mixtures [105, 106].
Regarding the identification of pesticides in wastewaters or ambient waters, few
studies have been published. The use of TIE methods allows the detection of
toxic surface waters and the identification of herbicides (molinate, mefenacet,
symetryn or esprocarb) as major compounds in rivers from agricultural areas
[106]. Recent applications including TIE studies were conducted on influent
and effluent wastewaters from wastewater treatment plants that received
wastewaters from agricultural areas. This approach was used to detect a pos-
sible cause–effect relationship between the plant discharge and the receiving
water quality [107]. The results of this study showed the detection of lindane
and pp¢-DDE in fish, and chemical investigations revealed ammonia and mi-
cropollutants as factors of WWTP effluent impact on receiving waters [108].
As was mentioned above, the interaction among multiple chemicals is one
of the main reasons for the wastewater toxicity. The application of TIE meth-
ods using acute toxicity (D. magna) guided chemical analysis was applied for
water quality evaluation of agricultural land runoff and 11 pesticides widely
applied were used as target compounds. Pesticides such as dymeron, flutolanil,
and mefenacet were detected in concentrations ranging from 6.2 to 29.7 mg/l;
however, these concentrations appeared to be too low to have toxic effects be-
cause their effective toxic concentrations were from 5 to 10 mg/l. Therefore, it
was impossible for the authors to conclude in this study that the observed
daphnia toxicity resulted from a single highly toxic substance. The toxicity was
attributed to the combined effect of the pesticides [109].
The utility of the bioassays to assess the interactions among pesticides
(additive effects, synergism or antagonism) have been demonstrated in differ-
ent studies [14, 15]. It is especially relevant to consider the combined effect
of pollutants because several pesticides and other contaminants can occur in
ambient waters from agricultural areas [110, 111]. The global effect can have a
greater negative impact than the single pollutants.
For a predictive assessment of the aquatic toxicity of pesticide mixtures, two
concepts, concentration addition and independent action, are used. Concen-
tration addition is generally regarded as a reasonable expectation for the joint
toxicity of acting substances [112]. Following this model, the concentration of
each toxicant is expressed as a fraction of its EC50 (toxic unit, TU). In this
model, the EC50 of a mixture is the sum of the single TU and equals unity.
Therefore, when the sum of TU exceeds unity, the combined effect is more than
additive and when it is less than additive, the substances act antagonistically.
Synergism is a common interactive effect among pesticide mixtures. Experi-
ments on pesticide mixtures showed a synergistic effect for 60% of the studied
cases [14]. Table 2 shows the combined effects evaluated by three different
toxicity bioassays. Therefore, it is evident that the consideration of single
pesticides alone is not sufficient for determining the environmental impact
of wastewaters.
Table 2 Combined toxicity effects of pesticides evaluated by three toxicity bioassays
Binary mixtures Bioassays
V. fischeri D. magna S. capricornutum

(15 min) (48 h) (72 h)
1
T.U.M S T.U.i2 1
T.U.M S T.U.i2 1
T.U.M S T.U.i2
Irgarol 1501– 26 5.8 550 183 0.36 0.01

Diruon Synergistic + Synergistic + Synergistic ++
Irgarol 1501– 7.4 333 550 581 5 5
Sea nine 211 Antagonistic ++ Additive Additive
Irgarol 1501– 5.4 0.87 1100 414 0.271 2
Chlorothalonil Synergistic + Synergistic Antagonistic +
Irgarol 1501– 9.4 15.6 132 160 0.152 0.046
Dichlofluanid Additive Additive Synergistic +
Irgarol 1501– 333 25.6 330 151 10 1.08
TCMTB Synergistic ++ Synergistic + Synergistic ++
1 , experimental toxicity.
T.U.M
S T.U.i2, theoretical toxicity.
+=factor≥3.
++=factor≥10.
Acknowledgements This work has been supported by the Project CICYT PPQ2001-1805-
C03-03 from the Ministry of Science and Technology.
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DOI 10.1007/b98608
Fragrance Materials in Wastewater Treatment

Staci L. Simonich (✉)
Oregon State University, Department of Environmental and Molecular Toxicology
and Department of Chemistry, 1141 Agricultural and Life Sciences, Corvallis,
OR 97331-7301, USA
Staci.simonich@orst.edu
1 Introduction to Fragrance Materials . . . . . . . . . . . . . . . . . . . . . . . . 81

1.1 Use and Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
1.2 Chemical Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
1.3 Physical-Chemical Properties and Biodegradability . . . . . . . . . . . . . . . . 84
2 Analytical Chemistry of Fragrance Materials . . . . . . . . . . . . . . . . . . . 86

2.1 Laboratory Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.2 Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
2.3 Aqueous Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
2.4 Solid Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
2.5 Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
3 Sampling Wastewater Treatment Plants for Fragrance Materials . . . . . . . . . 92

3.1 Selection of Wastewater Treatment Plants . . . . . . . . . . . . . . . . . . . . . 92
3.2 Wastewater Treatment Plant Sampling . . . . . . . . . . . . . . . . . . . . . . . 94
4 Mechanisms of Fragrance Material Removal During Wastewater Treatment . . 95

4.1 Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
4.2 Sorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
4.3 Volatilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5 Measurement of Fragrance Materials in Wastewater Treatment . . . . . . . . . 98

5.1 Concentrations in Treatment Plants . . . . . . . . . . . . . . . . . . . . . . . . 98
5.2 Removal During Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6 Predicting Fragrance Material Removal During Wastewater Treatment . . . . . 113

6.1 Framework for Aquatic Risk Assessment . . . . . . . . . . . . . . . . . . . . . . 113
6.2 Simple Treat Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
80 S. L. Simonich
Abstract In recent years, there has been significant interest in understanding the input of
fragrance materials (FMs) to aquatic ecosystems, and this has driven a substantial amount
of research on the removal of FMs during wastewater treatment. Because FMs are semi-
volatile and have a wide range of physical-chemical properties and biodegradabilities,
understanding their removal during the treatment process is complex. The mechanisms of
FM removal from wastewater include biodegradation, sorption, and/or volatilization.A wide
array of analytical methods have been developed to measure FMs in wastewater influent,
primary effluent, final effluent, and solids. Wastewater studies have been conducted in the
U.S. and Europe. Finally, the efficient removal of FMs during wastewater treatment is not only
dependent on the biodegradability and physical-chemical properties of the FM, but is also
highly dependent on plant operation and design.
Keywords Fragrance materials · Wastewater treatment · Polycyclic musks · Nitromusks
Abbreviations
ADBI Celestolide (4-acetyl-1,1-dimethyl-6-tert-butylindene)
AHDI Phantolide (6-acetyl-1,1,2,3,3,5-hexamethyldihydroindene)
AHTN Tonalid (7-acetyl-1,1,3,4,4,6,-hexamethyl-1,2,3,4-tetrahydronaphthalene)
ASE Accelerated solvent extraction
ATII Traseolide (5-acetyl-1,1,2,6-tetramethyl-3-isopropylindene)
CAS Chemical Abstracts Service
DPMI Cashmeran (6,7,-dihydro-1,1,2,3,3-pentamethyl-4(5H)-indanone)
ECD Electron-capture detector
FM Fragrance material
GC–MS Gas chromatography–mass spectrometry
GC–MS/MS Gas chromatography–tandem mass spectrometry
HHCB Galaxolide (1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-
benzopyran)
HRT Hydraulic retention time
Kd Sorption coefficient to activated sludge
Kow Octanol–water partition coefficient
MA Musk ambrette (1-tert-butyl-2,4-dimethyl-6-methoxy-3,5-dinitrobenzene)
MDL Method detection limit
MK Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butylacetophenone)
MM Musk moskene (1,1,3,3,5-pentamethyl-4,6-dinitroindane)
MT Musk tibetene (1-tert-butyl-3,4,5-trimethyl-2,6-dinitrobenzene)
MX Musk xylene [1-(1,1-dimethylethyl)-3,5-dimethyl-2,4,6-trinitrobenzene]
NM Nitromusk
NPD Nitrogen–phosphorus detector
OTNE 1-(1,2,3,4,5,6,7,8-Octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethanone
PCM Polycyclic musk
SOC Semivolatile organic compound
SPME Solid-phase microextraction
SRT Solids retention time
TSS Total suspended solids
Fragrance Materials in Wastewater Treatment 81
1
Introduction to Fragrance Materials
1.1
Use and Disposal
Over 2,000 distinct chemicals are currently available globally for formulation
into fragrances [1]. Although the consumer is most aware of the use of these
chemicals in fine fragrances, by far the largest volume of these chemicals is used
in laundry detergents, fabric softeners, household cleaning products, and air
fresheners [1]. Of these consumer products, fabric softeners and laundry de-
tergents represent the largest volume uses of fragrances and the largest release
to the environment through down-the-drain disposal by consumers following
product use [1].
Fragrance materials (FMs) are added to consumer products to mask mal-
odors and to deliver consumer-preferred odors [2]. Although these chemicals
are used in low concentrations in consumer products, the volume of laundry
detergents and fabric softeners sold throughout the globe can result in sig-
nificant volumes of FMs being released into the environment. Based on a
1995–1996 survey, approximately 90% of these compounds are used globally at
less than 10 metric tons per year [1], with less than 1% being used in volumes
approaching 4,000 metric tons per year [2].
Because the majority of the FM volume enters the environment through
down-the-drain disposal of consumer products, it is important to understand
the removal and fate of these chemicals during municipal wastewater treatment.
These semivolatile organic compounds (SOCs) may undergo a complex combi-
nation of biodegradation, sorption, and/or volatilization during wastewater
treatment. In addition, few SOCs have been studied in wastewater treatment be-
cause few of the conventional SOCs (such as pesticides and products of incom-
plete combustion) enter the environment through down-the-drain disposal and
wastewater treatment. The objective of this chapter is to review the state of the
science in understanding the removal of FMs during municipal wastewater
treatment. Others have reviewed the general environmental fate of FMs, in par-
ticular the polycyclic musks (PCMs) and the nitromusks (NMs) [3–6].
1.2
Chemical Structures
The chemical structures of the majority of FMs that have been studied in
wastewater treatment are given in Figs. 1–3. Figure 1 shows a variety of FM
structures that include alcohols, aldehydes, and ketones, including: benzyl
acetate (phenylmethyl ester acetic acid), methyl salicylate (2-hydroxy-methyl
ester benzoic acid), methyl dihydrojasmonate (3-oxo-2-pentyl-methyl ester
cyclopentaneacetic acid), terpineol (4-trimethyl-3-cyclohexene-1-methanol),
benzyl salicylate (2-hydroxy-phenylmethyl ester benzoic acid), isobornyl acetate
82 S. L. Simonich
Fig. 1 Fragrance materials studied in wastewater treatment [2, 11]
(1,7,7-trimethyl acetate bicyclo[2.2.1]heptan-2-ol), g-methyl ionone [3-methyl-

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one], p-t-bucinal [4-(1,1-
dimethylethyl)-a-methyl-benzenepropanal], hexylcinnamaldehyde [2-(phenyl-
methylene)-octanal], hexyl salicylate (2-hydroxy-hexyl ester benzoic acid),
OTNE [1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)ethan-
one], and acetyl cedrene [3R-(3a,3ab,7b,8aa))-1-(2,3,4,7,8,8a-hexahydro-
3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)ethan-1-one]. Figure 2 shows
the structures of the FMs categorized as nitromusks (nitroaromatic compounds),
including: musk ketone or MK (3,5-dinitro-2,6-dimethyl-4-tert-butylacetophe-
none), musk xylene or MX [1-(1,1-dimethylethyl)-3,5-dimethyl-2,4,6-trinitro-
benzene], musk ambrette or MA (1-tert-butyl-2,4-dimethyl-6-methoxy-3,5-
dinitrobenzene), musk tibetene or MT (1-tert-butyl-3,4,5-trimethyl-2,6-dinitro-
benzene), and musk moskene or MM (1,1,3,3,5-pentamethyl-4,6-dinitroin-
dane). Finally, Fig. 3 shows the structures of FMs categorized as polycyclic
Fig. 2 Nitromusks studied in wastewater treatment
Fig. 3 Polycyclic musks studied in wastewater treatment

84 S. L. Simonich
musks (PCMs), including AHTN or Tonalid (7-acetyl-1,1,3,4,4,6,-hexamethyl-

1,2,3,4-tetrahydronaphthalene), HHCB or Galaxolide (1,3,4,6,7,8-hexahydro-
4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyran),ADBI or Celestolide
(4-acetyl-1,1-dimethyl-6-tert-butylindene), AHDI or Phantolide (6-acetyl-
1,1,2,3,3,5-hexamethyldihydroindene), ATII or Traseolide (5-acetyl-1,1,2,6-
tetramethyl-3-isopropylindene), and DPMI or Cashmeran (6,7,-dihydro-
1,1,2,3,3-pentamethyl-4(5H)-indanone).
The nitromusks (NMs) (shown in Fig. 2) and the polycyclic musks (PCMs)
(shown in Fig. 3) have been studied in wastewater treatment by a variety of re-
search groups because they have been detected in the aquatic environment (see
for example [7–10]), indicating that they escape wastewater treatment to some
degree. The FMs shown in Fig. 1 were studied in wastewater treatment by
Simonich et al. [2, 11] because of their relatively large volumes and wide range
of physical-chemical properties and biodegradability (see below).
It is clear from Figs. 1–3 that FMs have an interesting and wide range of
chemical structures. This results in a wide array of perceived odors, including
musk, wood, fruit, and flower-like odors. The FMs in Figs. 1–3 will be the focus
of this chapter.
1.3
Physical-Chemical Properties and Biodegradability
Table 1 lists the CAS numbers, molecular weight, physical-chemical properties

(including the log of the octanol–water partition coefficient, sorption coeffi-
cient for activated sludge, water solubility, vapor pressure, and Henry’s law con-
stant), and biodegradability of selected FMs [2, 11]. It is clear that the wide
range of FM chemical structures results in a wide range of physical-chemical
properties (some properties ranging over six orders of magnitude). From the
vapor pressures given in Table 1, it is also clear that most FMs can be classified
as SOCs (having vapor pressures less than 1 Pa) [2], and have the potential to
partition into a variety of environmental compartments once released into the
environment.
The properties listed in Table 1 are of interest because they govern FM re-
moval during wastewater treatment and their fate in the environment. The log
octanol–water partition coefficients (log Kow) for the selected FMs range from
2.1 to 5.9. The more hydrophobic FMs (with high octanol–water partition co-
efficients) are desirable in consumer products because they tend to be more
substantive on fabrics and provide a residual fabric odor. Unfortunately, this hy-
drophobicity may also result in bioaccumulation in organisms in the environ-
ment. The sorption coefficient for activated sludge (Kd) is an important prop-
erty to consider for removal of FMs due to sorption onto solids during
wastewater treatment. The Kd value ranges from 132 to 15,400 L kg–1 for the se-
lected FMs. The water solubility and vapor pressure of the FMs listed in Table 1
also vary greatly: from 0.49 to 1,687 mg L–1 for water solubility and from
0.00003 to 21.9 Pa for vapor pressure. Finally, the Henry’s law constant (a mea-
Table 1 Summary of selected FM physical-chemical properties and biodegradability relevant to wastewater treatment [2, 11]
Fragrance material CAS number MW log Kow Kd Water Vapor Henry’s law Biodegradability
(g mol–1) (L kg–1) solubility pressure constant
(mg L–1) (Pa) (Pa m3 mol–1)
Benzyl acetate 140-11-4 150.2 2.1 132 1,265.0 21.9 2.04 Ready
Methyl salicylate 119-36-8 152.2 2.6 247 1,687.0 0.750 0.0607 Ready
Methyl dihydrojasmonate 24851-98-7 226.3 3.0 408 91.72 0.00549 0.135 Ready
Terpineol 98-55-5 154.3 3.3 595 335.7 4.09 0.939 Ready
Benzyl salicylate 118-58-1 228.3 4.3 2,081 24.59 0.000449 0.00416 Inherent
Isobornyl acetate 125-12-2 196.3 4.3 2,081 23.23 10.0 84.4 Ready
g-Methyl ionone 127-51-5 192.3 4.6 3,030 9.0 1.30 89.4 Inherent
p-t-Bucinal 80-54-6 204.3 4.2 1,836 33.0 0.477 12.4 Ready
Musk ketone 81-14-1 294.3 4.3 2,081 1.9 0.00004 0.0061 Not biodegradable
Musk xylene 81-15-2 297.3 4.9 4,412 0.49 0.00003 0.018 Not biodegradable
Hexylcinnamaldehyde 101-86-0 216.3 4.9 4,412 2.75 0.027 5.00 Inherent
Hexyl salicylate 6259-76-3 222.3 5.5 9,355 6.08 0.00325 0.118 Ready
OTNE 54464-57-2 234.4 5.7 12,020 2.68 0.203 31.8 Not biodegradable
Acetyl cedrene 32388-55-9 246.4 5.6-5.9 12,020 1.28 0.058 14.7 Inherent
AHTN 1506-02-1 258.4 5.7 12,020 (10,040) 1.25 0.0608 12.5 Not biodegradable
HHCB 1222-05-5 258.4 5.9 15,400 (12,780) 1.75 0.0727 11.3 Not biodegradable
85
86 S. L. Simonich
sure of air–water partitioning) for the selected FMs in Table 1 ranges from
0.00416 to 89.4 Pa m3 mol–1, indicating that some FMs may undergo volatiliza-
tion during wastewater treatment.
FMs also have a wide range of biodegradabilities (see Table 1). Some FMs
pass the OECD ready biodegradability test criteria, including the 10-day window
[11]. Other FMs pass the OECD inherent biodegradability test or produce CO2
in the OECD ready biodegradability test, but do not meet the 10-day window
[11]. Still other FMs do not biodegrade in standard OECD biodegradation tests
but undergo biotransformation in more realistic tests [11–13].
2
Analytical Chemistry of Fragrance Materials
In order to understand the removal of FMs during wastewater treatment, it is

necessary to measure these compounds throughout the wastewater treatment
process. Because of the complex nature of wastewater matrices and the low
concentration of FMs (0.001–60 mg/L) [11] throughout the treatment plant,
accurate and sensitive analytical methods have been developed by a number of
researchers. Fortunately, the analytical techniques developed to measure tra-
ditional SOCs, such as solvent extraction, extract concentration, and analysis
by gas chromatography–mass spectrometry, in general also apply to FMs.
2.1
Laboratory Quality Control
Because of the ubiquitous nature of FMs in consumer products, it is critical that

any analytical chemistry laboratory measuring these compounds takes extra
precautions to avoid laboratory contamination of samples. Several researchers
[2, 11, 14–17] have pointed out that likely sources of FM contamination in the
modern-day laboratory include the use of consumer products and fine fra-
grances by laboratory workers, fragrances in soaps used to clean glassware and
the laboratory, and laboratory supplies such as gloves.
Before beginning the analysis of FMs at low concentrations, the laboratory
should analyze several laboratory blank samples to assess the degree to which
the laboratory is contaminated. With every set of samples analyzed, the labo-
ratory should also analyze a laboratory and field blank sample. Laboratory
workers should be advised to be aware of their personal use of fragrance-en-
hanced consumer products and the potential for laboratory contamination.
2.2
Standards
As interest in measuring FMs in the environment has increased, researchers have

used a variety of means to obtain FM standards for use in analytical chemistry.
The sources of these standards include FM manufacturers, synthesis by re-

searchers (especially in the case of NM metabolites), and specialty chemical cat-
alogs. When possible, it is preferable to obtain these standards directly from the
FM manufacturers in order to use the authentic material being discharged to
wastewater treatment. In all cases, the purchased, synthesized, or obtained stan-
dards must be extensively analyzed to confirm the chemical structure and purity.
Also of importance is the appropriate use of surrogates and internal stan-
dards for quantification of FMs in wastewater and environmental matrices.
Ideally, stable isotope-labeled analogs (such as stable perdeuterated analogs) of
the FMs are used for this purpose if gas chromatography–mass spectrometry
(GC–MS) is the analysis technique. Simonich et al. [2, 11] synthesized eight
perdeuterated FMs, including d3-benzyl acetate, d3-g-methyl ionone, d3-methyl
dihydrojasmonate, d3-OTNE, d4-acetyl cedrene, d6-musk xylene, d3-AHTN, and
d7-musk ketone, through base-catalyzed exchange of protons with deuterium
and used these as surrogates to quantify 16 FMs in wastewater treatment ma-
trices. d3-Terpineol [2, 11, 18] and d6-HHCB [2, 8, 11] have also been used by
several researchers. Others have used d34-hexadecane [19], 2,4,5-trichlorotoluene
[20], pentachloronitrobenzene [14], 2,2¢-dinitrobiphenyl [14], and perdeuterated
polycyclic aromatic hydrocarbons [14, 18, 19, 21, 22] as surrogates or internal
standards to measure FMs in wastewater treatment.
Finally, the amino metabolites of the NMs have been synthesized by re-
searchers and used as standards. These synthesis methods include reduction
of NMs with hydrogen in the presence of Pd/charcoal to form the amino
metabolites [15, 16, 23] or reaction of NMs with hydrazine hydrate and Raney
nickel [14, 23]. A metabolite of HHCB, HHCB-lactone, has also been synthe-
sized and used as a standard [17].
2.3
Aqueous Matrices
Table 2 lists a variety of analytical methods used to measure FMs in wastewater

treatment. Only three studies [2, 11, 24] have attempted to examine fragrance
materials throughout the wastewater treatment process, including the analysis
of FMs in influent, primary and/or secondary settling, sludge, and/or final
effluent. Others have measured FMs in wastewater treatment influent and/or
effluent [8, 14, 19, 20, 22]. Still others have focused solely on measuring FMs in
sewage sludge and digested sewage sludge [15–18, 21]. Because wastewater
treatment processes consist of solid and aqueous phases, analytical methods
have been developed to measure FMs in both of these matrices.
Although some researchers have chosen to extract aqueous wastewater ma-
trices with traditional methods, such as liquid–liquid extraction [5, 22], other
researchers have used solid-phase extraction (SPE) to exhaustively extract FMs
from these matrices [2, 8, 11, 14, 19, 20]. Simonich et al. [2, 11] used C18 Baker-
bond Speedisks to extract 16 FMs, with a wide range of polarities, from 0.5-L
influent and primary effluent and 1.0-L final effluent samples.Verbruggen et al.
88
Table 2 Summary of analytical methods used to measure FMs in wastewater treatment
Researchers Fragrance materials WWTP matrix Location Analytical method
Simonich 16 FMs, including Influent, primary U.S., United Kingdom, – Extraction of 0.5–1 L influent,
et al. [2, 11] those in Table 1, effluent, activated and The Netherlands primary effluent, and final effluent with C18 SPE
PCMs (AHTN and sludge solids, – Extraction of sludge by accelerated solvent
HHCB), and NMs and final effluent extraction with dichloromethane
(MX and MK) – Silica gel chromatography
– Analysis by stable isotope dilution GC–MS using
nine perdeuterated FMs
– Recovery=97–115%; limit of quantification
=0.5–35 ng/L
Artola- PCMs (AHTN and Influent, primary The Netherlands Freely dissolved
Garicano HHCB) – freely settler, aeration tank, – SPME with GC–MS
et al. [24] dissolved and total secondary effluent, – Limit of quantification=0.1 mg/L
concentrations primary sludge, Total concentration
and waste sludge – Liquid–liquid extraction with cyclohexane
– Silica gel chromatography
– GC–MS
– Recovery=85–106%; limit of quantification
=0.1 mg/L
Kanda PCMs and NMs Influent, effluent United Kingdom – Liquid–liquid extraction with dichloromethane
et al. [22] – GC–MS
– Recovery=69–95%; limit of detection
=3.7–8.5 ng/L
Rimkus NMs and mono- Influent, effluent Germany – Liquid–liquid extraction with hexane
et al. [23] amino metabolites – Silica gel and alumina chromatography
– GC–MS/MS, GC–MS, GC-ECD, and GC-PND
– Limits of quantification=1 ng/L
S. L. Simonich
Table 2 (continued)
Ricking PCMs and NMs Wastewater effluent Canada and Sweden – SPE and filtration and extraction with
et al. [19] n-pentane and dichloromethane
– GC–MS
– Detection limit=0.5 ng/L
Verbruggen PCMs Wastewater effluent The Netherlands – Biomimetic and exhaustive extraction
et al. [20] (AHTN and HHCB) – C18 Empore disks
– GC–MS
– Recovery >95%; detection limit=0.1 ng/L
Osemwengie NMs, PCMs, and Wastewater effluent U.S. – On-site 60-L extraction with NEXUS sorbent
et al. [14] nitromusk – Silica gel and gel-permeation chromatography

metabolites – GC–MS
– Recovery=80–97%; method detection limit
=0.02–0.3 ng/L
Buerge PCMs Wastewater effluent Switzerland – Macroporous polystyrene–divinylbenzene
et al. [8] (AHTN and HHCB) adsorbent
– GC–MS
– Recovery=81–141%; LOD=10 ng/L
Berset NMs and amino Sewage sludge Switzerland – Extraction with hexane by agitation
et al. [16] metabolites – Gel-permeation chromatography and silica gel
chromatography
– GC–MS/MS, GC–MS (EI, NCI, and PCI),
1H and 13C NMR
– Recovery=51–116%; detection limit=50 ng/L

89
90
Table 2 (continued)
Herren NMs, PCMs, and Sewage sludge Switzerland – Extraction with hexane by agitation
et al. [15] amino metabolites – Gel-permeation chromatography and silica gel
chromatography
– GC–MS/MS, GC–MS (EI, NCI, and PCI)
– Recovery=50–118%; detection limit=100 ng/L
Kupper PCMs and Sewage sludge Switzerland – Extraction with hexane and stirring
et al. [17] HHCB-lactone – GC–MS
– Recovery=79–108%; LOD=15–30 umg/kg d.m.;
LOQ=45–90 umg/kg d.m.
Stevens NMs and PCMs Digested sewage United Kingdom – Soxhlet extraction with dichloromethane
et al. [21] sludge – Silica gel and gel-permeation chromatography
– GC–MS
Difrancesco 22 FMs; including Digested sewage U.S. – Accelerated solvent extraction with
et al. [18] PCMs and NMs sludge dichloromethane
– GC–MS
S. L. Simonich
[20] used C18 3 M Empore Disks to exhaustively and biomimetically extract

PCMs (AHTN and HHCB) from 10-L effluent samples. Osemwengie et al. [14]
used 6 g of Nexus sorbent [polystyrene cross-linked with 50% divinylbenzene
and poly(methyl methacrylate)] to extract 60 L of effluent for a variety of NMs,
PCMs, and nitromusk metabolites. Buerge et al. [8] used 10 mL of Bio-Beads
SM-2 (a macroporous polystyrene–divinylbenzene adsorbent) to extract HHCB
and AHTN from 200-mL effluent samples. Finally, Artola-Garicano et al. [24]
used 1-cm lengths of a 100-mm polydimethylsiloxane solid-phase microextrac-
tion (SPME) fiber to measure free concentrations of AHTN and HHCB through-
out wastewater treatment.
Many of the aqueous methods listed in Table 2 utilize adsorption chroma-
tography, such as silica or alumina chromatography, to purify extracts prior to
analysis.
2.4
Solid Matrices
Researchers have chosen to extract sewage sludge for FMs in its wet form using
liquid–liquid extraction with solvent [15–17, 24], or to centrifuge the wet sludge,
decant the water phase, and extract the sludge by Soxhlet extraction with sol-
vent [21] or at elevated temperature and pressure using accelerated solvent
extraction [2, 18]. Herren and Berset [15] and Berset et al. [16] extracted 1 L of
wet sewage sludge for NMs and their metabolites with 600 mL hexane for 2 h
with vigorous agitation. Artola-Garicano et al. [24] measured the free concen-
tration of AHTN and HHCB in 10 mL wet sludge by negligible depletion SPME
and the total concentration by extracting with 6 mL cyclohexane during 2 h of
shaking. Stevens et al. [21] extracted NMs and PCMs in 2.5 g dried, centrifuged,
and digested sludge by Soxhlet extraction for 18 h with 280 mL dichloromethane.
Finally, Simonich et al. [2] and Difrancesco et al. [18] used accelerated solvent
extraction (at 60 °C and 2,000 PSI) with dichloromethane to extract a wide
range of FMs from centrifuged activated sludge solids and digested and de-
watered sludges. In the methods used to extract centrifuged sludges, Na2SO4
was used to remove water from the sample prior to solvent extraction.
Many of the sludge methods listed in Table 2 utilize gel-permeation chroma-
tography (GPC) to remove high molecular weight interferences and/or ad-
sorption chromatography, such as silica or alumina chromatography, to purify
extracts prior to analysis.
2.5
Analysis
Because FMs are semivolatile, they are amenable to analysis by gas chroma-
tography (GC) and gas chromatography–mass spectrometry (GC–MS) without
derivitization. Table 2 shows that all of the analytical methods developed to
measure FMs in wastewater treatment to date utilize GC or GC–MS.
92 S. L. Simonich
In general, FMs can be chromatographically resolved using a 30-m nonpolar

GC column, such as a DB-5 [2, 11, 24] or DB-1701 [18] column. Rimkus et al. [23]
used GC with an electron-capture detector (ECD) and nitrogen–phosphorus
detector (NPD) to analyze for NMs and their metabolites. However, the major-
ity of studies use GC–MS with electron impact ionization to detect and quan-
tify the wide array of FM structures [2, 5, 8, 11, 14, 18–21, 24].An ion-trap mass
spectrometer has been used to analyze for FMs by GC–MS/MS and negative
chemical ionization has been used to improve sensitivity for the NMs [15, 16].
Because of the volatile nature of FMs, care must be taken when evaporating the
extraction solvent to low volumes prior to analysis, and volatile solvents should
be used in order to minimize the loss of FMs during this step [2].
3
Sampling Wastewater Treatment Plants for Fragrance Materials
3.1
Selection of Wastewater Treatment Plants
Because FMs are used in consumer products, it is important that investigations

of their removal during wastewater treatment be conducted at municipal waste-
water treatment plants primarily receiving domestic wastewater (>80% do-
mestic wastewater) [2]. In addition, the operation of these plants should be well
documented and reported, including: plant design, wastewater flow, hydraulic
(HRT) and solids retention times (SRT), and biochemical oxygen demand
(BOD) and total suspended solids (TSS) removal. Specific wastewater treatment
plants should be selected to represent a range of dry and wet climates, geo-
graphic locations (such as the U.S. and Europe), and plant designs (including
primary treatment only, activated sludge, carousel, oxidation ditch, trickling fil-
ter, rotating biological contactor, lagoon, etc.) in order to obtain a comprehen-
sive understanding of FM removal during wastewater treatment. Plant designs
should be selected so that the most prevalent types of wastewater treatment
plant design for that geography are sampled, based on both total wastewater
flow and number of wastewater treatment plants [11]. For example, in the U.S.
approximately 81% of the wastewater flow is treated by activated sludge plants,
7% by trickling filter, 6% by lagoon, 3% by oxidation ditch, 2% by rotating
biological contactor, and 1% by primary treatment [11]. In Europe, activated
sludge, carousel, trickling filter, and oxidation ditch are among the most com-
mon types of wastewater treatment [11].
In a study of the removal of 16 FMs from wastewater, Simonich et al. [11]
conducted their studies at 17 wastewater treatment plants with plant flows of
1.4¥106–1.0¥108 L day–1. Twelve of the plants were located in different states
and regions of the U.S. and five plants were located in Europe. In addition, five
of the 17 plants were activated sludge plants, two were carousel plants, two were
oxidation ditch plants, five were trickling filter plants, one was a rotating bio-
logical contactor plant, and two were lagoons. Artola-Garicano et al. [24] stud-
ied the removal of freely dissolved and total concentrations of AHTN and
HHCB in four wastewater treatment plants located in The Netherlands. These
plants had flow rates ranging from 800 to 4,200 m3/h, HRTs ranging from 4.8 to
8.9 h, and SRTs ranging from 8 to 22 days [24]. Kanda et al. [22] studied PCMs
and NMs in influent and effluent from six wastewater treatment plants in the
U.K. The flow rates of these plants ranged from 103 to 3,198 m3/day and in-
cluded rotating biological contactor with reed beds, submerged aerated filter,
oxidation ditch, biological filter bed, activated sludge, and trickling filter plant
designs. Finally, Buerge et al. [8] measured HHCB and AHTN in effluents from
five wastewater treatment plants in Switzerland with flow rates ranging from
3,177 to 14,250 m3/day–1.
Daily BOD and TSS removal at the plant should be measured and evaluated in
order to judge how well the plant operates during low and high flow conditions.
Measurements of BOD and TSS removal should be done on the days in which FM
monitoring takes place at the plant. This is important, because Simonich et al. [11]
showed that the overall removal of biodegradable, nonsorptive FMs from most
plant designs is positively correlated with plant BOD removal and that the over-
all removal of nonbiodegradable, sorptive FMs is positively correlated with plant
TSS removal (see Fig. 4). This was true for all plant designs except for lagoons,
which had poor BOD and TSS removal (due to aquatic vegetation growing in
lagoons) but had good removal of FMs due to long HRTs and SRTs. BOD and TSS
removal is governed by both plant design and daily operation.
Fig. 4 Correlation of the measured overall removal of terpineol with plant 5-day BOD
removal and the measured overall removal of HHCB with plant TSS removal. Regressions
include all wastewater treatment plants studied by Simonich et al. [11] except for the two
lagoons (see text), and are significant at the 99.9% level; n=15. Dashed lines are the 95%
confidence intervals of the regressions
94 S. L. Simonich
3.2
Wastewater Treatment Plant Sampling
When planning which wastewater compartments to measure and at what fre-

quency, it is important to consider the potential for FM concentrations to vary
throughout the day, within the plant. Simonich et al. [2] measured 16 FMs in the
influent of a U.S. wastewater treatment plant every 2 h over a 24-h period. Their
data indicate that the total FM concentration in influent varies greatly through-
out a 24-h period, with a relative standard deviation in total FM concentration
of 38.9% (see Fig. 5). This variation in influent concentrations has been observed
for other consumer product chemicals, such as surfactants [2], and is a func-
tion of consumer use and disposal of these chemicals and water discharge
volume changing throughout the course of the day. In general, Simonich et al.
[2] measured low FM influent concentrations from 11:30 p.m. to 7:30 a.m. and
high influent concentrations from 9:30 a.m. to 9:30 p.m. These time periods are
consistent with consumer use of these chemicals, considering that sewer trans-
port times to wastewater treatment plants can be on the order of 0.5–2 h in
some locations. Simonich et al. [2] also observed that the total FM concentra-
tion in final effluent varied significantly less than the influent concentrations,
with a relative standard deviation of 8.0% (see Fig. 5). This is most likely due
to the capacity of the treatment process to treat fluctuations in influent con-
centration due to the long residence times within the plant.
Fig. 5 Diurnal fluctuations in total FM concentration in influent and final effluent collected
from an activated sludge wastewater treatment plant [2]. Hourly samples were combined to
represent a 2-h period
Artola-Garicano et al. [24] measured the free and total concentrations of

AHTN and HHCB in the influent of a wastewater treatment plant in The
Netherlands every 2 h over a 24-h period. Their data indicate that the variation
in total concentration of AHTN and HHCB in influent was 19%, while the
variation in free concentration was less than 10% over the 24-h period. These
authors suggested that fluctuations in water volume cause fluctuations in total
concentrations; however, for hydrophobic FMs such as AHTN and HHCB, the
solids act as a reservoir and stabilize the free concentrations.
If the objective of measuring FMs, or other consumer product chemicals for
that matter, in wastewater treatment is to understand FM removal and mech-
anisms of removal across wastewater treatment processes, then it is important
to collect samples at least every 2 h and composite these samples into a single,
flow-based 24-h sample. Otherwise, the results may be significantly over- or
underestimated depending on the time of the day the sample was collected.
However, if the objective is to monitor FMs in only final effluent or sludge, rep-
resentative grab sampling may be sufficient.
4
Mechanisms of Fragrance Material Removal
During Wastewater Treatment
Because of their wide range of physical-chemical properties and biodegrad-

abilities (see Table 1), FMs have the potential to biodegrade, sorb to solids,
and/or volatilize during wastewater treatment. The relative importance of these
removal mechanisms will depend on the specific FM, the plant design, and the
kinetics of each of these processes within the plant. It is also important to
acknowledge that FMs bound to solids are not available for biodegradation or
volatilization, and it is thought that only FMs freely dissolved in the aqueous
phase are available for these processes [24].
4.1
Biodegradation
As shown in Table 1, many FMs meet the biodegradation criteria of a ready or

inherent test. If a FM meets the criteria of a ready test, with or without acclima-
tion, a first-order biodegradation rate of 3 h–1 in activated sludge can be assumed
[1]. For FMs that show extensive biodegradation but fail the ready test criteria,
a first-order rate of 0.3 h–1 can be assumed for activated sludge treatment [1].
Table 1 also indicates that some FMs, including the PCMs and NMs, do not
pass ready or inherent biodegradation tests. However, this does not mean that
these FMs do not undergo biotransformation to polar metabolites under real-
istic conditions. These realistic biodegradation tests may be conducted in vitro,
in bench-top die-away studies, or as continuous activated sludge and porous
pot tests. Ideally, the conditions should include: (1) realistic FM concentrations
96 S. L. Simonich
Table 3 AHTN and HHCB biotransformation rates in activated sludge measured by Federle
et al. [12] and Artola-Garicano et al. [13]
Federle et al. [12] Artola-Garicano et al. [13]
PCM Total kbiodeg Total kbiodeg

concentration (h–1) concentration (h–1)
(mg/L) (mg/L)
AHTN 5 0.015±0.001 5.25 0.023

50 0.008±0.001
HHCB 5 0.010±0.002 10.33 0.071
25 0.021±0.003
(often achieved through the use of radioisotope-labeled FMs), (2) realistic

acclimated sludge concentrations, and (3) realistic exposure times.
Several researchers have studied the biotransformation of HHCB and AHTN
under realistic activated sludge conditions. Federle et al. [12] studied the bio-
transformation of 14C-HHCB in activated sludge die-away tests using realistic
HHCB concentrations (5 and 25 mg/L) and acclimated sludge concentrations
(approximately 2,500 mg/L). The polar biotransformation products of HHCB,
including the lactone and hydroxy acid of HHCB, were identified and their cor-
responding octanol–water partition coefficients estimated by HPLC. The first-
order rate constant for parent HHCB biotransformation in activated sludge was
determined to be 0.010–0.021 h–1, depending on HHCB concentration, in these
tests (see Table 3). Federle et al. [12] also studied the biotransformation of
14C-AHTN in similar activated sludge die-away tests and in a continuous acti-
vated sludge test. Polar biotransformation products of AHTN were identified

and their octanol–water partition coefficients were estimated from HPLC data.
The first-order rate constant for parent AHTN biotransformation in activated
sludge was determined to be 0.008–0.015 h–1, depending on AHTN concentra-
tion in the activated sludge die-away test (see Table 3) [12]. The overall removal
of AHTN (due to biotransformation, sorption, volatilization) in the continuous
activated sludge test was 86.4% and the removal of parent AHTN due to bio-
transformation was estimated to be 37.4%.
Artola-Garicano et al. [13] studied the biodegradation of AHTN and HHCB
in activated sludge by measuring the free concentration, using negligible de-
pletion SPME, and total concentration over time. The first-order rate constant
for parent AHTN biotransformation was determined to be 0.023 h–1, while the
first-order rate constant for parent HHCB biotransformation was 0.071 h–1 (see
Table 3) [13]. These authors also determined that microbial biodegradation ac-
tivity was the rate-limiting step in biotransformation of these compounds and
not desorption from the activated sludge.
Table 3 shows that the first-order rate constants for parent AHTN and HHCB
biotransformation, determined by Federle et al. [12] and Artola-Garicano et al.
[13], are similar even though the techniques used to determine these rate con-
stants were quite different and the sources of activated sludge were different (U.S.
and Europe). Finally, there is empirical evidence of the biotransformation of NMs
and PCMs during wastewater treatment from measurements of the amino
metabolites of the NMs and the lactone of HHCB in sewage sludge [15–17].
4.2
Sorption
For the hydrophobic, nonbiodegradable FMs listed in Table 1, such as the NMs
and PCMs, removal due to sorption on sewage solids is a significant removal
mechanism. Evidence of the significance of this removal mechanism is the mea-
surement of PCMs and NMs in sewage sludge throughout the world [15–18, 21].
Of the FMs listed, Difrancesco et al. [18] measured acetyl cedrene, hexyl salicy-
late, hexylcinnamic aldehyde, AHTN, HHCB, g-methyl ionone, musk ketone,
musk xylene, and OTNE in digested and dewatered sludge samples. The results
indicate that FMs with activated sludge sorption coefficients (Kd) as low as
2,000 L kg–1 have the potential to be removed to a significant degree due to sorp-
tion to sewage sludge.
FM sorption coefficients for sewage sludge have most often been estimated
using the octanol–water partition coefficient of the FM rather than measured
directly. There is general agreement between the Kd values measured for AHTN
and HHCB and the estimates based on their log Kow values (see Table 1) [11].
Artola-Garicano et al. [13] determined the organic carbon normalized sorption
coefficient for activated sludge to be 6,681 L kg–1 for HHCB and 7,018 L kg–1 for
AHTN. Finally, Federle et al. [12] estimated the removal of 14C-AHTN in a con-
tinuous activated sludge test to be 44.7% based on sorption to activated sludge
alone.
4.3
Volatilization
FMs have the potential to volatilize and enter the atmosphere during manufac-
turing and consumer use and disposal. The PCMs and NMs have been detected
in ambient air [9, 25]. However, most FMs have atmospheric lifetimes sufficiently
short that they are unlikely to undergo atmospheric long-range transport [26].
Because some FMs have large Henry’s law constants (Table 1) and some
wastewater treatment plant designs have active aeration and large surface areas
that are exposed to the atmosphere, it is likely that some FMs volatilize during
wastewater treatment. However, the FMs with large Henry’s law constants also
have large Kd values (see Table 1), so that in portions of the treatment process
with active aeration and high solids concentrations (such as activated sludge)
it is not entirely clear whether volatilization or sorption to solids will be the
dominant loss mechanism. Although there are limited experimental data on
FM volatilization during wastewater treatment, volatilization appears to ac-
98 S. L. Simonich
count for <5% of the total FM removal during wastewater treatment. Federle
et al. [12] estimated the removal of 14C-AHTN in a continuous activated sludge
test to be 3.4% based on volatilization alone.
5
Measurement of Fragrance Materials in Wastewater Treatment
5.1
Concentrations in Treatment Plants
Given the variety of analytical methods used to measure FMs in wastewater

treatment and the number of researchers measuring these compounds in
wastewater collected from the U.S. and Europe (see Table 2), it is important to
compare and contrast the concentrations of FMs measured throughout waste-
water treatment by different researchers, in different geographies. In addition,
one might expect that there would be plant-to-plant variation in the FM con-
centration in wastewater treatment because of differences in the volume of FMs
used (per capita FM use) and differences in the per capita water use for a given
geography. These measurements and differences are important to understand
because, ultimately, the concentration of FMs in the final effluent and sewage
sludge are used to develop aquatic and terrestrial environmental risk assess-
ments for these compounds [1].
Simonich et al. [11] compared the concentration of 16 FMs in wastewater in-
fluent collected from 12 U.S. treatment plants and five treatment plants from
the U.K. and The Netherlands (see Table 4). It is important to characterize FMs
in influent because it is a good representation of the relative amounts of FMs
being used by consumers and there is minimal opportunity for biodegradation,
sorption, and/or volatilization in transit to the wastewater treatment plant. In
addition, because the same analytical methods were used by the same labora-
tory to measure U.S. and European influent in the Simonich et al. [11] study, we
can directly compare concentrations between the U.S. and Europe without
questioning differences in laboratory procedures or analytical methodology.
Simonich et al. found that the influent concentrations of 14 of the 16 FMs
measured were statistically similar in both U.S. and European treatment plants
[11]. Only isobornyl acetate and OTNE influent concentrations were statisti-
cally different, with higher concentrations of these FMs in European influent.
The influent concentrations of the 16 FMs are given in Table 4. Figure 6A shows
the normalized relative profile of FMs measured in U.S. and European influent
during the [11] study. In both sets of influent, terpineol dominated the FM pro-
file and had the highest concentrations, while the nitromusks (MX and MK)
had the lowest concentrations. The large standard deviations in the influent
concentrations (Table 4 and Fig. 6A) indicate that there is significant varia-
bility in influent concentrations within both U.S. and European treatment
plants.
Table 4 Summary of FM influent concentrations to wastewater treatment
Researchers Location and Years PCM concentration NM concentration Other FM concentration

number sampled (mg/L) – mean (mg/L) – mean (mg/L)– mean±standard
±standard deviation ±standard deviation deviation and/or range
and/or range and/or range
Simonich U.S.; 1997–1999 AHTN=12.5±7.35; MK=0.640±0.395; Benzyl acetate=3.74±3.46;

et al. [2, 11] n=12 plants HHCB=16.6±10.4 MX=0.386±0.299 methyl salicylate=10.2±9.69;
methyl dihydrojasmonate
=7.21±4.19; terpineol=63.7±36.4;
benzyl salicylate=19.5±10.8;
isobornyl acetate=6.47±8.53;
g-methyl ionone=3.37±2.56;
p-t-bucinal=1.61±0.731;
hexylcinnamaldehyde=15.3±12.1;
hexyl salicylate=5.48±3.56;
OTNE=3.55±1.93;
acetyl cedrene=4.97±2.27
Simonich U.K. and 1999–2000 AHTN=5.97±3.88; MK=0.996±0.741; Benzyl acetate=9.85±10.2;
et al. [11] The Netherlands; HHCB=9.71±5.09 MX=0.248±0.136 methyl salicylate=11.3±13.0;
n=5 plants methyl dihydrojasmonate=11.9±5.31;
terpineol=56.3±33.9;
benzyl salicylate=10.2±4.51;
isobornyl acetate=37.1±28.4;
g-methyl ionone=3.63±1.90;
p-t-bucinal=2.56±1.96;
OTNE=9.00±3.77;
99
100
Table 4 (continued)

Artola-Garicano The Netherlands; 2001 AHTN=0.54±0.05 Not measured Not measured

et al. [24] – total n=4 plants –1.76±0.09;
concentrations HHCB=1.42±0.12
–4.30±0.23
Kanda et al. [22] UK; n=6 plants 2001 AHTN=2.2–8.1; MA=<0.01; Not measured
HHCB=8.4–19.2; MX=<0.01–4.7;
DPMI=<0.01–0.4; MM=<0.01;
ADBI=<0.01–0.44; MT=<0.01;
AHDI=<0.01–0.1; MK=<0.01–2.9
ATII=<0.01–2.9
Rimkus et al. [23] Germany; 1996 Not measured MX=0.150; Not measured
n=1 plant 4-NH2-MX=<0.01;
2-NH2-MX=<0.01;
MK=0.55;
2-NH2-MX=<0.01
S. L. Simonich
Fig. 6A, B Average relative profile and standard deviation of FMs in A influent and B primary
effluent in the U.S. and Europe [11]. The highest concentration FM was normalized to 1. The
highest concentration FM (in mg/L) is in parentheses. The error bars represent the normal-
ized standard deviation of the mean
102 S. L. Simonich
When we compare the PCM and NM influent concentrations measured by

Simonich et al. to those of other researchers (Table 4), we can ascertain some
general trends. AHTN and HHCB are the highest concentration PCMs in both
U.S. and European influent, with concentrations ranging from 1 to 20 mg/L, and
HHCB concentrations being greater than AHTN concentrations. The ratio of
HHCB to AHTN concentration in U.S. and European influent is in the range of
1.3–3.8. In general, the concentration of other PCMs in influent are either be-
low the limit of quantitation or less than 1 mg/L [22].
The NM concentrations in U.S. and European influent are significantly less
than the PCM concentrations and are in the range of 0.2–5 mg/L. MX and MK
are the highest concentration NMs in influent [22]. In general, the amino meta-
bolites of the nitromusks are not detected in influent because there is limited
opportunity for biotransformation in transit to the wastewater treatment plants.
A larger number of researchers have measured FMs in final effluent from
wastewater treatment (Table 5) because this is the most important wastewater
parameter for accessing FM discharge to aquatic ecosystems. The concentra-
tion of FMs in final effluent is a function of the FM concentration in influent
and the efficiency of FM removal across the plant (including plant design and
operation).
In the Simonich et al. [11] study, the concentration of 16 FMs in final efflu-
ent ranged from 0.001–7.6 mg/L in the U.S. to 0.01–4.6 mg/L in Europe (see
Table 5). In addition, terpineol no longer dominated the relative FM profile in
final effluent, except for final effluent collected from two European trickling
filter plants (see Fig. 7) [11]. As the figure indicates, nonbiodegradable and
inherently biodegradable, sorptive FMs dominated the relative FM profile in
final effluent (including HHCB, AHTN, OTNE, and acetyl cedrene).
Of the PCMs, AHTN and HHCB have the highest concentrations in final ef-
fluent (see Table 5) [14, 19, 22]. In the U.S., the concentration of AHTN in final
effluent ranged from 0.024 to 1.7 mg/L, while the concentration of HHCB ranged
from 0.032 to 2.2 mg/L (see Table 5) [11, 14]. In Europe, the final effluent con-
centrations of AHTN ranged from 0.11 to 2.7 mg/L and the concentrations of
HHCB ranged from 0.21 to 6.4 mg/L (see Table 5).
MX and MK are the most prevalent NMs in final effluent and are in the con-
centration range of <1–710 ng/L in European effluents and <MDL–112 ng/L in
U.S. effluents (see Table 5). The amino metabolites of the NMs have been detected
in U.S. and European effluent in the concentration range of <MDL–250 ng/L (see
Table 5).
It is also important to consider the concentration of FMs in sewage sludge
because this is another route in which FMs may be removed during wastewater
treatment and be released to the environment through land application of
sludge solids. Only one study has attempted to measure a large number of FMs
in digested sludge. Difrancesco et al. [18] detected AHTN, HHCB, MK, g-methyl
ionone, hexylcinnamaldehyde, hexyl salicylate, and acetyl cedrene in digested
sludge from U.S. wastewater treatment plants (see Table 6). The other FMs they
studied were not detected.As Table 6 indicates, the concentrations of these FMs
Table 5 Summary of FM final effluent concentrations from wastewater treatment

Simonich U.S.; 1997–1999 AHTN=24–1,710; MK=10–67; Benzyl acetate=2–252;

et al. [2, 11] n=12 plants HHCB=32–2,210 MX=1–112 methyl salicylate=13–693;
methyl dihydrojasmonate=3–456;
terpineol=11–1,079;
benzyl salicylate=5–1,025;
isobornyl acetate=7–112;
g-methyl ionone=7–214;
p-t-bucinal=13–258;
hexylcinnamaldehyde=10–77;
hexyl salicylate=1–243;
OTNE=25–615;
acetyl cedrene=12–1,359
Simonich U.K. and 1999–2000 AHTN=620–2,670; MK=40–770; Benzyl acetate=60–260;
et al. [11] The Netherlands; HHCB=980–4,620 MX=10–170 methyl salicylate=40–220;
n=5 plants methyl dihydrojasmonate=26–1,920;
terpineol=80–15,100;
benzyl salicylate=20–1,960;
isobornyl acetate=10–290;
g-methyl ionone=30–730;
p-t-bucinal=40–180;
hexylcinnamaldehyde=20–910;
hexyl salicylate=10–910;
OTNE=490–3,190;
acetyl cedrene=70–1,430
103
104
Table 5 (continued)

Artola-Garicano The Netherlands; 2001 AHTN=420±60 Not measured Not measured

et al. [24] – total n=4 plants –1,200±180;
concentrations HHCB=1,250±20
–2,220±90
Kanda et al. [22] UK; 2001 AHTN=310–2,700; MA=<10; Not measured
n=6 plants HHCB=1,100–6,400; MX=<10–650;
DPMI=<10–160; MM=<10;
ADBI=<10–91; MT=<10;
AHDI=<10–48; MK=<10–710
ATII=<10–790
Rimkus Germany; 1996 Not measured MX=<3–10; Not measured
et al. [23] n=3 plants 4-NH2-MX=7–34;
2-NH2-MX=<1–10;
MK=6–94;
2-NH2-MK=15–250
Ricking Canada; 2002 AHTN=42–104; MX=<1; MK=<1 Not measured
et al. [19] n=3 plants HHCB=157–423;
DPMI=<1;
ADBI=2–8;
AHDI=2–5;
ATII=<1
S. L. Simonich
Table 5 (continued)

Ricking et al. [19] Sweden; 1999 AHTN=110–520; MX=<1; MK=<1 Not measured
n=5 plants HHCB=205–1,300;
DPMI=<1;
ADBI=4–19;
AHDI=2–6;
ATII=<1
Osemwengie U.S.; Unknown AHTN=26.6–92.2; MX=<MDL–1.3; Not measured

et al. [14] n=2 plants HHCB=35.0–152; MK=<MDL–27.5;
DPMI=<MDL; MA=<MDL;
ADBI=0.3–2.1; MM=<MDL;
AHDI=2.4–5; MT=<MDL;
ATII=<MDL–126 4-NH2-MX=
<MDL–31.5;
2-NH2-MX=
<MDL–0.9;
NH2-MK=<MDL
Buerge et al. [8] Switzerland; 2001 AHTN=310–760; Not measured Not measured
n=5 HHCB=720–1,950
105
Fig. 7 Average relative profile and standard deviation of FMs in final effluent in U.S. and
European plants [11]. The highest concentration FM was normalized to 1. The highest con-
centration FM (in mg/L) is in parentheses. The error bars represent the normalized standard
deviation of the mean
Table 6 Summary of FM concentrations in wastewater sludge
Researchers, Location and Years PCM concentration – NM concentration – Other FM concentration –

sample type, number sampled mean±standard mean±standard mean±standard deviation
and concentration deviation and/or deviation and/or and/or range
units range range
Artola-Garicano The Netherlands; 2001 AHTN=12.38±0.19 Not measured Not measured

et al. [24] – total n=4 plants –82.67±43.09;
concentration HHCB=29.47±10.84
waste sludge in mg/L –234.60±111.86
Berset et al. [16] – Switzerland; Unknown Not measured MA=nd; Not measured
concentration n=10 plants MX=nd–32.5;

in mg/kg MM=nd; MT=nd;
dry weight MK=nd–7.1,
amino metabolites=
nd–49.1
Herren et al. [15] – Switzerland; Unknown AHTN=741–4,161; MA=nd; Not measured
concentration n=12 plants HHCB=2,293–12,157; MX=nd–32.5;
in mg/kg DPMI=38.4–332; MM=nd; MT=nd;
dry weight ADBI=41–330; MK=nd–7.0,
AHDI=64.9–843; amino metabolites=
ATII=n.q. nd–36.2
107
108
Table 6 (continued)
Researchers, Location and Years PCM concentration – NM concentration – Other FM concentration –

sample type, number sampled mean±standard mean±standard mean±standard deviation
and concentration deviation and/or deviation and/or and/or range
units range range
Kupper et al. [17] – Switzerland; 2001 AHTN=2,500–11,200; Not measured Not measured
concentration in n=16 plants HHCB=7,400–3,600;
mg/kg dry weight ADBI=100–1,100;
AHDI=200–1,800;
ATII=200–1,000;
HHCB lactone
=600–3,500
Stevens et al. [21] – U.K.; Unknown AHTN=120–16,000; MA, MX, MM, Not measured
digested sludge n=14 plants HHCB=1,900–81,000; and MT
concentration in ADBI=10–260; not detected
mg/kg dry weight AHDI=32–1,100;
ATII=44–1,100;
DPMI – not detected
Difrancesco et al. U.S.; 2000 AHTN=8,100–51,000; MK=1,300; g-Methyl ionone=1,100–3,800;
[18] – digested n=2 plants and 2002 HHCB=21,800–86,000 MX not detected hexylcinnamaldehyde=4,100;
sludge concentration hexyl salicylate=1,500;
in mg/kg dry weight OTNE=7,300–30,700;
acetyl cedrene=900–31,300;
remaining FMs not detected
S. L. Simonich
ranged from 900 to 86,000 mg/kg dry weight, with AHTN and HHCB having the
highest concentrations (8,100–86,000 mg/kg dry weight) [18].
The remaining studies on sewage sludge were conducted at European waste-
water treatment plants (see Table 6). Of the PCMs, AHTN and HHCB had the
highest concentrations on European sludge (120–81,000 mg/kg dry weight),
however the other PCMs were also detected. In one study, the lactone of HHCB
was also detected on sewage sludge [17]. The NMs and their amino metabo-
lites have been detected on sewage sludge, however their concentrations (nd–
1,300 mg/kg dry weight) are much lower than the PCMs.
5.2
Removal During Treatment
Removal of FMs during wastewater treatment is a function of the tendency for

FMs to biodegrade, sorb, and/or volatilize during treatment, plant design, and
plant operation. FMs may be removed from wastewater during primary and
secondary treatment.
Simonich et al. [11] showed that FMs undergo significant removal during
primary treatment, ranging from 14 to 50% removal (Table 7). Because both
sorptive and nonsorptive FMs are removed, these authors suggested that both
sorption and biodegradation play a role in the removal of FMs during primary
treatment. Further evidence of this is in the comparison of Figs. 6A and 6B, in
which the relative profile of FMs does not change significantly from influent
(Fig. 6A) to primary effluent (Fig. 6B), although the FM concentrations de-
crease. The lack of enhancement of biodegradable, nonsorptive FMs in the
primary effluent relative profile suggests that removal due to biodegradation,
as well as sorption, occurs during primary treatment. The large standard de-
viations in the primary treatment removals measured by Simonich et al. [11]
suggest that there is significant plant-to-plant variability in primary removal
of FMs.
Simonich et al. [2, 11], Artola-Garicano et al. [24], Kanda et al. [22], and
Rimkus et al. [5] studied the removal of FMs following primary and secondary
treatment (see Table 7). Simonich et al. [11] showed that FM removal is de-
pendent on plant design and operation and ranges from 50 to 99.9%. The over-
all removal (primary + secondary treatment) of 16 FMs ranged from 87.8 to
99.9% for activated sludge plants, 58.6 to 99.8% for carousel plants, 88.9 to
99.9% for oxidation ditch plants, 71.3 to 98.6% for trickling filter plants, 80.8 to
99.9% for a rotating biological contactor plant, and 96.7 to 99.9% for lagoons.
Lagoons resulted in the most effective removal of FMs and, in general, the low-
est final effluent concentrations due to long retention times. The relative FM
profiles in final effluent (Fig. 7), broken down by treatment type, gives some
perspective on which plant designs are best at removing biodegradable chem-
icals (significant enhancement of nonbiodegradable, sorptive FMs as in the
case of activated sludge, rotating biological contactor, and oxidation ditch
plants) and those which are not (enhancement of both nonbiodegradable, sorp-
Table 7 Summary of percent removal of FMs during wastewater treatment
110
Researchers Location and Years Percent PCM Percent NM Percent other FM removal
number sampled removal removal
Simonich U.S., UK, 1997 – 2000 Primary removal Primary Primary removal only:
et al. [2, 11] The Netherlands; only: removal only: benzyl acetate=28.2±27.5;
n=17 plants AHTN=28.9±20.1; MX=41.2±21.9; methyl salicylate=40.4±32.2;
HHCB=29.9±23.4 MK=26.6±21.5 methyl dihydrojasmonate=14.6±19.4;
terpineol=15.5±12.0;
Primary+ Primary+ benzyl salicylate=41.8±25.0;
secondary removal: secondary removal: isobornyl acetate=29.0±29.5;
AHTN=50.6–99.9; MX=87.6–99.9; g-methyl ionone=20.8±19.6;
HHCB=63.5–99.7 MK=85.2–96.7 p-t-bucinal=50.6±23.4;
OTNE=28.8±22.7;
Primary+secondary removal:
benzyl acetate=86.4–99.9;
methyl salicylate=92.0–99.9;
methyl dihydrojasmonate=81.9–99.9;
terpineol=95.4–99.9;
benzyl salicylate=90.3–99.9;
isobornyl acetate=84.5–99.9;
g-methyl ionone=83.1–99.8;
p-t-bucinal=84.8–99.3;
hexylcinnamaldehyde=95.3–99.9;
hexyl salicylate=96.4–99.9;
OTNE=51.4–99.4;
acetyl cedrene=71.3–99.9
S. L. Simonich
Table 7 (continued)
Researchers Location and Years Percent PCM Percent NM Percent other FM removal
number sampled removal removal
Artola-Garicano The Netherlands; 2001 Primary+ Not measured Not measured

et al. [24] – total n=4 plants secondary removal:
concentrations AHTN=14.3–56.3;
HHCB=12.0–59.8
Kanda et al. [22] UK; n=6 plants 2001 Primary+ Primary+ Not measured
secondary removal: secondary removal:
AHTN=40.0–96.17; MX=80.3–86.2;
HHCB=39.05–93.49; MK=53.6–64.5
others not given
Rimkus et al. [23] Germany; n=1 1996 Not measured Primary+ Not measured
secondary removal:
MX=93.3; MK=98.9
111
112 S. L. Simonich
tive FMs and some biodegradable, nonsorptive FMs as in the case of trickling
filter). As mentioned previously, Simonich et al. [11] showed that plant opera-
tion, including the efficiency of removal of TSS and BOD, affects the removal
of FMs to a significant degree (see Fig. 4 and earlier discussion) and is likely a
more important variable in insuring efficient removal of FMs during waste-
water treatment than is plant design alone.
The overall removal (primary + secondary treatment) of AHTN and HHCB
has been measured by Simonich et al. [11], Artola-Garicano et al. [24], and
Kanda et al. [22] (see Table 7). Simonich et al. measured the overall removal of
AHTN to be 50.6–99.9% and the removal of HHCB to be 63.5–99.7% in the U.S.
and Europe (UK and The Netherlands), depending on treatment type and TSS
removal. Artola-Garicano et al. measured the overall removal of AHTN to be
14.3–56.3% and the removal of HHCB to be 12.0–59.8% at four treatment plants
in The Netherlands, based on total concentrations. Finally, Kanda et al. mea-
sured the overall removal of AHTN to be 40.0–96.2% and the removal of HHCB
to be 39.1–93.5% at six different treatment plants in the U.K.
The overall removal of AHTN and HHCB measured by Artola-Garicano et
al. [24] appears to be significantly lower than the overall removals measured by
Simonich et al. [11] and Kanda et al. [22]. This may be due, in part, to the col-
lection of grab samples by Artola-Garicano et al. and the collection of flow-
based composite samples by Simonich et al. and Kanda et al., to the relatively
short hydraulic retention times in several of the treatment plants monitored by
Artola-Garicano et al., and/or excessive levels of TSS in effluent from the plants
monitored by Artola-Garicano et al. As previously mentioned, the FM influent
concentrations vary significantly throughout the day (Fig. 5). Artola-Garicano
et al. [24] confirmed that the total AHTN and HHCB concentration in influent
varied throughout the day, while the free FM concentration showed less vari-
ability. Simonich et al. [11] measured relatively low overall removals of AHTN
and HHCB at two carousel treatment plants in The Netherlands (58.6 and 63.5%,
respectively); however, these overall removals were not as low as those reported
by Artola-Garicano et al. [24] for other treatment plants in The Netherlands.
Finally, the range of AHTN and HHCB overall removal measured by Simonich
et al. [11] and Kanda et al. [22] is comparable. Both studies collected flow-com-
posite samples and sampled a variety of different plant designs.
Simonich et al. [11], Kanda et al. [22], and Rimkus et al. [23] measured the
removal of MX and MK during wastewater treatment (see Table 7). Simonich
et al. and Rimkus et al. measured the overall removals of MX and MK in the
range of 85–99.9%. Kanda et al. measured the removal of MX in the range of
80–86% and the removal of MK in the range of 53–65%, however the removal
of these NMs was not reported for all six treatment plants.
6
Predicting Fragrance Material Removal During Wastewater Treatment
6.1
Framework for Aquatic Risk Assessment
Because of the large number of FMs in commerce, a framework document

was developed to prioritize FMs for aquatic risk assessment [1]. An integral
part of this risk assessment is an accurate prediction of FM concentration in
the aquatic environment. In order to do this, the framework document out-
lines some simple calculations to estimate the concentration and removal of
FMs during wastewater treatment. This is done by first using the annual FM
volume of use and the per capita water use in the geographic area to predict
an average influent concentration for the U.S. and Europe. Next, the FM re-
moval during primary treatment (sorption and settling only) is predicted us-
ing the octanol–water partition coefficient. Finally, the FM removal during
secondary treatment and final effluent concentrations are predicted in the
first tier of the framework using the octanol–water partition coefficient to es-
timate sorption. Biodegradation rates are added to sorption in the second tier
of the framework if a more refined exposure assessment is needed. Readily
biodegradable, inherently biodegradable, and nonbiodegradable FMs are as-
sumed to have biodegradation rates of 3, 0.3, and 0 h–1, respectively [1]. This
simple model does not account for volatilization and the assumptions and
equations in the framework document are directly applicable to all primary
treatment, but only to activated sludge secondary treatment. The largest sin-
gle source of error in the model is likely the estimation of annual FM volume
use because of uses outside the fragrance industry and, in some cases, natural
sources [1].
Because of the large number of FMs being evaluated and the wide range
of physical-chemical properties and biodegradabilities they represent, it is im-
portant to determine if calculations outlined in the framework document are
conservative for predicting FM concentration and removal during wastewater
treatment. The Simonich et al. [11] dataset for 16 FMs measured in 17 waste-
water treatment plants was used to evaluate the assumptions made in the
framework document. Figure 8A shows the regression of the measured percent
primary removal (Table 7) with the percent primary removal predicted by the
framework. All of the plants with primary treatment studied by Simonich et
al. are included in this regression. Because of the large variation in measured
primary removal (Table 7), the correlation is not statistically significant. Also,
the framework prediction does not account for potential biodegradation dur-
ing primary treatment, and Simonich et al. measured significant removal of
biodegradable, nonsorptive FMs during primary treatment (Table 7 and
Fig. 6B) [11].
Figure 8B shows the regression of the measured percent overall removal
from activated sludge treatment plants only measured by Simonich et al. with
114 S. L. Simonich
Fig. 8A, B Correlation of A measured primary removal with predicted primary removal and
B measured overall removal with predicted overall removal for activated sludge plants, using
the second tier of the framework model and accounting for sorption and biodegradation [11].
The error bars represent the standard deviation of the mean. The regression for overall removal
(B) is significant at the 98% level, while the regression for primary removal (A) is not statis-
tically significant; n=16. Dashed lines are the 95% confidence intervals for the regressions
the percent overall removal predicted by the second tier of the framework
(accounting for sorption and biodegradation) for activated sludge plants. This
correlation is significant at the 98% level, however the framework significantly
underpredicts overall removal (slope=0.118). This is particularly true for MK,
MX, OTNE, AHTN, and HHCB, which are assumed in the framework to be non-
biodegradable (Table 1) and to have biodegradation rates of 0 h–1. This suggests
that sorption alone does not account for the removals Simonich et al. measured
for MK, MX, OTNE,AHTN, and HHCB and that biotransformation and/or vola-
tilization may be playing a role in the removal of these FMs from activated sludge.
Finally, these data show that the calculations outlined in the framework docu-
ment for estimating FM concentrations in and removal from activated sludge
wastewater treatment are predictive. Finally, Fig. 9 shows that the assumptions
made in the framework document [1] are conservative for predicting final efflu-
ent concentrations, regardless of treatment type and geography (US and Europe).
6.2
Simple Treat Model
Artola-Garicano et al. [27] compared their measured removals of AHTN and

HHCB [24] to the predicted removal of these compounds by the wastewater
treatment plant model Simple Treat 3.0. Simple Treat is a fugacity-based, nine-
box model that breaks the treatment plant process into influent, primary set-
tler, primary sludge, aeration tank, solid/liquid separator, effluent, and waste
sludge and is a steady-state, nonequilibrium model [27]. The model inputs in-
clude information on the emission scenario of the FM, FM physical-chemical
properties, and FM biodegradation rate in activated sludge.
In general, the Simple Treat model predicted the overall removal of total
AHTN and HHCB within a factor of 4 of Artola-Garicano et al.’s measured
removals for three wastewater treatment plants located in The Netherlands.
However, the free AHTN and HHCB concentrations predicted by Simple Treat
were inversely related to the measured free concentrations of these compounds
[27]. As previously mentioned, the overall removal of AHTN and HHCB mea-
sured by Artola-Garicano et al. (14–60%) was significantly less than the overall
removal of these compounds measured by Simonich et al. [11] and Kanda et al.
[22] (39–99.9%).
7
Conclusions
In recent years, there has been significant interest in understanding the input of
FMs to aquatic ecosystems and this has driven the substantial amount of research
that has been conducted on the removal of FMs in wastewater treatment. Because
FMs are semivolatile and have a wide range of physical-chemical properties and
biodegradabilities, understanding their removal during the treatment process is
116 S. L. Simonich
complex. The mechanisms of FM removal from wastewater include biodegrada-

tion, sorption, and/or volatilization. A wide array of analytical methods have
been developed to measure FMs in wastewater influent, primary effluent, final
effluent, and solids, and wastewater studies have been conducted in the U.S. and
Europe. Finally, the efficient removal of FMs during wastewater treatment is not
only dependent on the biodegradability and physical-chemical properties of the
FM, but is also highly dependent on plant operation and design.
Fig. 9A–D Comparison of measured FM concentrations in all plants to predicted FM con-

centrations in the U.S. and Europe [1] for A influent, B primary effluent, C final effluent
using the first tier (sorption only) of the framework, and D final effluent using the second
tier (sorption and biodegradation) of the framework [11]. Points above the line have mea-
sured values greater than predicted by the framework model [1], while those below the line
have measured values less than predicted. The filled circles represent concentrations in the
U.S. and the open circles represent concentrations in Europe
Fig. 9C, D (continued)
References
1. Salvito DT, Senna RJ, Federle TW (2002) Environ Toxicol Chem 21:1301
2. Simonich SL, Begley WM, Debaere G, Eckhoff WS (2000) Environ Sci Technol 34:959
3. Balk F, Ford RA (1999) Toxicol Lett 111:57
4. Tas JW, Balk F, Ford RA, van de Plassche EJ (1997) Chemosphere 35:2973
5. Rimkus GG (1999) Toxicol Lett 111:37
6. Heberer T (2002) Acta Hydrochim Hydrobiol 30:227
7. Oros DR, Jarman WM, Lowe T, David N, Lowe S, Davis JA (2003) Mar Pollut Bull 46:1102
8. Buerge IJ, Buser HR, Muller MD, Poiger T (2003) Environ Sci Technol 37:5636
9. Peck AM, Hornbuckle KC (2004) Environ Sci Technol 38:367
10. Standley LJ, Kaplan LA, Smith D (2000) Environ Sci Technol 34:3124
11. Simonich SL, Federle TW, Eckhoff WS, Rottiers A, Webb S, Sabaliunas D, De Wolf W
118 Fragrance Materials in Wastewater Treatment
12. Federle TW (2004)

13. Artola-Garicano E, Borkent I, Damen K, Jager T, Vaes WHJ (2003) Environ Sci Technol
37:116
14. Osemwengie LI, Steinberg S (2001) J Chromatogr A 932:107
15. Herren D, Berset JD (2000) Chemosphere 40:565
16. Berset JD, Bigler P, Herren D (2000) Anal Chem 72:2124
17. Kupper T, Berset JD, Etter-Holzer R, Furrer R, Tarradellas J (2004) Chemosphere 54:1111
18. DiFrancesco AM, Chiu PC, Standley LJ, Allen HE, Salvito D (2004) Environ Sci Technol
38:194
19. Ricking M, Schwarzbauer J, Hellou J, Svenson A, Zitko V (2003) Mar Pollut Bull 46:410
20. Verbruggen EMJ, Van Loon W, Tonkes M, Van Duijn P, Seinen W, Hermens JLM (1999)
21. Stevens JL, Northcott GL, Stern GA, Tomy GT, Jones KC (2003) Environ Sci Technol 37:462
22. Kanda R, Griffin P, James HA, Fothergill J (2003) J Environ Monit 5:823
23. Rimkus GG, Gatermann R, Huhnerfuss H (1999) Toxicol Lett 111:5
24. Artola-Garicano E, Borkent I, Hermens JLM, Vaes WHJ (2003) Environ Sci Technol
37:3111
25. Kallenborn R, Gatermann R, Planting S, Rimkus GG, Lund M, Schlabach M, Burkow IC
(1999) J Chromatogr A 846:295
26. Aschmann SM, Arey J, Atkinson R, Simonich SL (2001) Environ Sci Technol 35:3595
27. Artola-Garicano E, Hermens JLM, Vaes WHJ (2003) Water Res 37:4377
DOI 10.1007/b98609
Immunochemical Determination
of Industrial Emerging Pollutants
M.-Carmen Estévez · Héctor Font · Mikaela Nichkova · J.-Pablo Salvador ·
Begoña Varela · Francisco Sánchez-Baeza · M.-Pilar Marco (✉)
Department of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona 18–26,
mpmqob@iiqab.csic.es
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
2 Immunochemical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 124

2.1 Antibody-Based Analytical Methods . . . . . . . . . . . . . . . . . . . . . . 137
2.1.1 Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
2.1.1.1 Enzyme-Linked Immunosorbent Assay (ELISA) . . . . . . . . . . . . . . . . 139
2.1.1.2 Enzyme-Multiplied Immunoassay Technique (EMIT) . . . . . . . . . . . . . 140
2.1.1.3 Polarization Fluoroimmunoassay (PFIA) . . . . . . . . . . . . . . . . . . . . 141
2.1.2 Flow-Injection Immunoassay (FIIA) . . . . . . . . . . . . . . . . . . . . . . 141
2.1.3 Immunosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
2.1.4 Immunoaffinity Chromatography (IAC) . . . . . . . . . . . . . . . . . . . . 143
3 Immunochemical Methods for Surfactants . . . . . . . . . . . . . . . . . . 144

3.1 Anionic Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3.2 Nonionic Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.3 Cationic Surfactants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4 Immunochemical Methods for Polychlorinated

and Polybrominated Compounds . . . . . . . . . . . . . . . . . . . . . . . . 153
4.1 PCBs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
4.2 PCDDs and PCDFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
4.3 Chlorophenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
5 Other Industrial Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

5.1 Bisphenol A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
5.2 Phthalate Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
6 General Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Abstract A significant number of immunochemical methods have been described for the
determination of the most important emerging pollutants. The present chapter is a com-
pilation of the information available today regarding immunochemical determination of
industrial residues with a high potential risk of causing negative effects in the environment,
wildlife, and public health. Homogeneous immunoassays, ELISAs, FIIAs, immunosensors, and
selective immunoaffinity sample treatment methods have been reported for the analysis of
an important number of these substances. The bases of these methods are briefly presented.
120 M.-C. Estévez et al.
Immunochemical methods for anionic (LAS), nonionic (APEs and APs), and cationic sur-
factants (BDD12AC and DDAC) are extensively reviewed and the features of these assays
discussed, particularly if examples of their application to environmental samples have been
described. Similarly, a great amount of information has been collected regarding immuno-
chemical determination of organochlorinated substances such as PCBs, PCDDs, PCDFs, and
chlorophenols. On the contrary, immunochemical analysis of organobrominated substances,
such as the BFR agents, seems to be still a goal. Immunochemical methods have also been
reported for bisphenol A and phthalates showing excellent features. The commercial avail-
ability of some of these methods is also presented.
Keywords Immunochemical techniques · Surfactants · Polyhalogenated compounds ·

Bisphenol A · Phthalate esters
Abbreviations
Ab Antibody
ADMBAC Alkyldimethylbenzylammonium compounds
AES Alkyl ether sulfates
Ag Antigen
AP Alkylphenol
APEC Alkylphenol ethoxy carboxylate
APE Alkylphenol polyethoxylate
AS Alcohol sulfates
ATMAC Alkyltrimethylammonium compounds
BBP Butylbenzyl phthalate
BDD12AC Benzyldimethyldodecylammonium chloride
BFR Brominated flame retardants
BMP-IA Bacterial magnetic particle-based immmunoassay
BP Bromophenol
BPA Bisphenol A
BSA Bovine serum albumin
CIA Capillary immunoassay
CLIA Chemiluminescence immunoassay
CP Chlorophenol
CR Cross-reactivity
DADMAC Dialkyldimethylammonium compounds
DBP Dibutyl phthalate
DCP Dichlorophenol
DDAC Didecyldimethylammonium chloride
DDT Dichlorodiphenyltrichloroethane
DEHP Diethylhexyl phthalate
DEQ Diesterquat
DMSO Dimethylsulfoxide
EDC Endocrine disrupter chemical
EIA Enzyme immunoassay
EMIT Enzyme-multiplied immunoassay technique
EO Ethoxylene unit
EPA Environmental Protection Agency
EQ Esterquat
FA Fatty acids
FIA Fluorescence immunoassay
Immunochemical Determination of Industrial Emerging Pollutants 121
FIIA Flow-injection immunoassay

FOH Fatty alcohols
GC–MS Gas chromatography–mass spectrometry
HRP Horseradish peroxidase
HTS High-throughput screening
IA Immunoassay
IAC Immunoaffinity chromatography
IC50 Concentration at 50% of signal inhibition
IgG Immunoglobulin G
LAS Linear alkylbenzenesulfonates
LC–MS Liquid chromatography–mass spectrometry
LDS Linear 4-dodecylbenzenesulfonic acid sodium salt
LIA Liposome immunoaggregation
LIC Liposome immunocompetition
LIF Laser-induced fluorescence
MAb Monoclonal antibody
NP Nonylphenol
NPE Nonylphenol polyethoxylate
OP Octylphenol
OPE Octylphenol polyethoxylate
PAb Polyclonal antibody
PBB Polybrominated biphenyl
PBDD Polybrominated dibenzo-p-dioxin
PBDE Polybrominated diphenyl ethers
PBDF Polybrominated dibenzofurans
PCB Polychlorinated biphenyl
PCDD Polychlorinated dibenzo-p-dioxin
PCDE Polychlorinated diphenyl ethers
PCDF Polychlorinated dibenzofurans
PCP Pentachlorophenol
PFIA Polarization fluoroimmunoassay
QAC Quaternary ammonium compounds
QFIA Quenching fluorescence immunoassay
RAb Recombinant antibody
RIA Radioimmunoassay
SAS Secondary alkyl sulfonates
SDS Sodium dodecyl sulfate
SPC Sulfophenyl carboxylate
STP Sewage treatment plant
TBBPA Tetrabromobisphenol A
TBP Tribromophenol
TCDD Tetrachlorodibenzo-p-dioxin
TCP Trichlorophenol
I-TEF Toxic equivalent factor
I-TEQ Toxic equivalent quotient
TtCP Tetrachlorophenol
1
Introduction
Chemicals and secondary by-products from industry, household chemicals,

personal care products, and pharmaceuticals such as drugs, antibiotics, and
hormones, are some of the substances that have been grouped under the ex-
pression emerging pollutants, making reference to their recent increased use
and release into the environment and to the fact that most of them were not
previously considered as contaminants (see Table 1 for the most important
groups of emerging pollutants). The adverse effects derived from a continu-
ing exposure to these substances are often unknown, and regulations are still
not well established [1, 2]. Much interest has been focused on these com-
pounds not only because of their possible adverse effects, but also for the great
amounts that are produced worldwide. Most of the substances considered as
emerging contaminants are widespread in everyday life and applied in differ-
Table 1 Some of the most important emerging pollutants divided into chemicals with an
industrial origin and pharmaceuticals
Industrial chemicals Pharmaceuticals
Surfactants and their metabolites Antibiotics

Nonionic surfactants Fluoroquinolones
Anionic surfactants Sulfamides
Cationic surfactants Penicillins
Tetracyclines
Organochlorinated substances Macrolides
Polychlorinated biphenyls
Chlorophenols Steroid hormones
Dioxins Estrogens
Androgens
Organobrominated substances Gestagens
Polybrominated biphenyl ethers Corticosteroids
Bromophenols
Dioxin-like compounds Analgesics
Tetrabromobisphenol A Paracetamol
Aspirin
Industrial additives and others Ibuprofen
Phthalate esters Diclofenac
Bisphenol A
Tranquilizers (psychiatric drugs)
Diazepam
Cytostatic agents
Methotrexate
Cyclophosphamide
Ifosfamide
ent fields such as pharmaceuticals (for both animal and human use), drugs,
hormones, and surfactants.
An important number of these substances have an industrial origin. Some
of them, like the pesticides, arrive intentionally in the environment and their
use and release should be theoretically controlled. However, many of them have
not been purposely produced as bioactive substances but more as components
or additives of certain materials. Their significant growth in the chemical in-
dustry has not only been produced as a consequence of the discovery of new
active principles in the pharmaceutical or pesticide area, but also because of the
expansion of new technologies (electronics, containers, textiles, plastics, resins,
foams, etc.), that require the development of new materials and substances with
particular features. Most of these substances enter or are discharged to water and
air sources without regulated controls. Wastewater treatment plants (WWTPs)
are often not yet adapted to completely remove them, and therefore these new
compounds can be found to some extent in wastewater effluents as well as in soil
and sludge.
The release into the environment of this large amount of chemicals has be-
come an increasing concern for the authorities and for the scientific commu-
nity. Although there is positive pressure by governmental bodies and agencies
to push chemical industries toward the development of more environmentally
friendly compounds that are not persistent and are easily biodegradable, the
final metabolites readily formed can be more ubiquitously distributed and/or
present more toxicological effects than the parent compounds. That is the case
for alkylphenol polyethoxylates, a major group of nonionic surfactants used
worldwide, whose major breakdown products are the alkylphenols, which are
considered relevant endocrine disrupters, especially nonylphenol. The same
effects have been reported in the case of certain plastic and polymer additives
such as bisphenol A or some phthalate esters (see BKH report [3]). Other kinds
of substances are not produced intentionally but are generated as by-products
of industrial processes such as combustion or waste incineration. This is the
case for the dioxins or the PCBs. Other polyhalogenated compounds are also
of concern because of their persistence in the environment. This is the case for
the chlorophenols, used for many years as insecticides, and wood and textile
preservatives. Their use is today restricted in most of the developed countries
but residues can still be detected in many environmental compartments. Organo-
brominated substances, used as flame retardant additives, have emerged as a
new generation of polyhalogenated substances whose environmental and toxi-
cological impacts are still not completely determined, although some evidence
suggests an endocrine disrupter action. Some of the substances considered in
this chapter do not have toxicity by themselves, but may affect the permeability
or solubility of other pollutants present in the environment. This is the case for
the anionic surfactants such as LAS, whose presence in the environment is also
a worry due to their high production and use.
The continuous development of more specific and sensitive analytical tech-
niques has allowed the detection of traces of these substances in many com-
partments of the environment. Recently, the US Geological Survey reported the

development of five new analytical methods for the detection of 95 organic
wastewater contaminants, many of them considered as emerging pollutants,
such as several antibiotics, drugs (both prescription and nonprescription),
steroid hormones, phthalates, and nonionic surfactants [4]. As fast as the risk
assessment is being carried out and a regulation is trying to be established for
these substances, more and more rapid, sensitive, and specific methodologies ca-
pable of detecting this huge amount of compounds are continuously demanded.
Immunochemical techniques can fulfill all these requirements because of their
specificity, selectivity, and demonstrated high sample throughput capabilities.
The objective of this chapter has been to collect and to provide information
on the immunochemical techniques available today for the determination of an
important number of the emerging pollutants of industrial origin. The emerg-
ing pollutants considered here have been selected by attending to their toxico-
logical risk, environmental relevance, or their regular use or production (their
generic structure and some reported data regarding their environmental levels
as well as their more probable biodegradation pathways are briefly detailed in
Table 2). For many of these substances there are immunochemical methods
available that have been applied with significant success to the analysis of
environmental samples. For some of these compounds, different antibodies
(both monoclonal and polyclonal) have been produced and manufactured
by different companies either as immunochemical reagents or as immuno-
chemical assay kits. The availability of antibodies, as the key reagents of any
immunochemical technique, opens the possibility of developing a wide variety
of methods, such as those described before, depending on the necessities. How-
ever, in most cases the methods commercially available are ELISA kits in a
variety of formats and supports such as magnetic particles, microtiter plates,
test strips, tubes, etc. (see Table 3 for examples of commercially available im-
munochemical techniques).
2
Immunochemical Techniques
The key component of immunochemical techniques is the antibody (Ab). An-

tibodies are globular proteins generated by the immune system as a defense
against foreign agents (antigens,Ag). Their structure varies depending on their
isotypes. There are five different families of immunoglobulins (IgG, IgM, IgA,
IgD, and IgE) differing in their charge, size, amino acid sequence, and carbo-
hydrates attached. The most abundant class in mammal serum and the most
used in immunochemical applications is the IgG subclass. The IgGs (MW
150 kDa) are composed of four polypeptide chains: two identical “heavy” (H)
chains that carry covalently attached oligosaccharide groups, and two identi-
cal, nonglycosylated, “light” (L) chains. The heavy chains are interlinked by
disulfide bonds, and each light chain is joined by a disulfide bond to a heavy
Table 2 Summarized table with the more important compounds included in the surfactants, polyhalogenated compounds, and other industrial
residues. Their generic chemical structure and the use or origin are shown. Some reported data regarding their environmental occurrence and the
more probably environmental fate are also given
Substance Chemical Structure Uses/Origin Environmental Occurrence Environmental Fate
Nonionic Surfactant
Alkylphenol Ethoxylates (APEs)
NPE (nonylphenol Household and – STPs effluents in Switzerland: Readily biodegradable in
ethoxylates) commercial detergents NP1EO (30–65 mg L–1), NP2EO WWTPs under aerobic and
OPE (octylphenol Emulsifiers (47–77 mg L–1) and in Japan anaerobic conditions [6] to
ethoxyates) Textile and leather NP1EO (0.21–2.96 mg L–1) [5] short chain alkylphenol
industry – SW: usually below 1 mg L–1 ethoxylates and alkylphenols
Pharmaceutical and and maximum peak values Half-life=1 to 4 weeks [7, 8]
R=C8, C9 personal care products around 20 mg L–1[5]
n=1–40 (PPCPs)
Alkylphenols (APs)
NP (Nonylphenol) Major component in – Sewage effluents: More persistent than APE.
OP (Octylphenol) the production of 0.025–330 mg L–1 (NP) Half-life (river waters): 30–58
APEs and breakdown and 0.0022–73 mg L–1 (OP). days (NP) and 7–50 days
product in their Usually below 10 mg L–1 [9] (OP) [5]
degradation. – Found in air [10] and sedi-
R=C8, C9 Plasticizers and ments (up to 14000 mg Kg–1)
Immunochemical Determination of Industrial Emerging Pollutants
stabilizers in plastics [5, 11]

– SW: Usually NP levels below
1 mg L–1. High levels in Spain
(0.15–644 mg L–1) [12] and
England rivers
(0.2–180 mg L–1) [13].
– OP levels below 0.1 mg L–1 [5]
125
DW: drinking water; GW: ground water; SW: surface water; WW: waste water; STP: sewage treatment plant; WWTP: wastewater treatment plant.
Table 2 (continued)
126
Fatty Alcohol Ethoxylates (AE)

Household and – In GW levels: (61–189 ng L–1 Readily biodegradable (>80%
laundry. for the different AEOs in 28 days for linear AE and
R=C9–C15 Pulp and paper C12EO3–9 40% for branched AE) [18].
n=4–14 manufacturing – Total conc. of 710 ng L–1 [14] Slower degradation (AE>20
Textile dyeing – Soil interstitial water: ethoxylene units) [19]
Emulsifiers, spray (48–73 ng L–1 for C12EO3–5
adjuvants (total conc. in the deeper
layers at 194 ng L–1) [14]
– Treated sewage 6.5 mg L–1 [15],
12.5–300 mg L–1 [16]
– Sewage sludge
10–190 mg Kg–1 [17]
Fatty Esters Polyethoxylates
Emulsifiers No data found Easily biodegradable (slower
Textile and leather when it has more than 50 units
industries of ethylene oxide units [20]
R=C12–C18 Component in PPCPs
Fatty Amides Polyethoxylates
Foam stabilization No data found Contradictory data related to
Emulsifier they biodegradability [19].
Solubilizer, antistatic, Readily biodegradable [20, 21]
R=C12–C18 wetting agent in
PPCPs
Hair shampoo, liquid
soaps, shaving creams
M.-C. Estévez et al.
and other PPCPs

Table 2 (continued)
Anionic Surfactants
Linear Alkylbenzene Sulfonates (LAS)
Household and – STPs effluents: 19–71 mg L–1 [15] Readily degradable with a
commercial detergents – WW effluents levels: half-life of 1–87 days. 10–35%
0.09–0.9 mg L–1 [18] adsorbed in the particulate
– WW sludge: <3 mg g–1 [18] are matter [18]
– SW in North Sea
(<0.05–9.4 mg L–1) [22]
m+n=C10–C14 – SW in Brazil (14–155 mg L–1 [23]
– SW in Philippines
(2.2–102 mg L–1) [24]
Sulfophenyl Carboxylates (SPCs)
Major metabolite in – SW and sewage effluents: Found mainly in aquatic
LAS biodegradation 0.5–3.2 mg L–1 [25] compartments.
– Seawater: [26] Shorter alkyl chain SPCs
– Drinking water: (≤C5) are expected to be
1.6–3.7 mg L–1 [23] found as the distance from

– Raw river waters: the discharge point of LAS
m+n=C3–C11 1.8–5 mg L–1 [27] increases
– Up and downstream of
WWTPs: <1–101 mg L–1 [28]
127
128
Table 2 (continued)
Alkyl Sulfonates
Primary Alkyl Liquid detergents No data found Can be adsorbed onto sludge
sulfonates R=C11–C17 (dish washing agents, but under aerobic conditions
cleaning agents, and are readily biodegradable
hair shampoos). (primary degradation in
Secondary Alkyl Commercial products WWTP <90% in 3 days [18]).
Sulfonates (SAS) are almost exclusively Not degraded in anoxic
composed of SAS conditions
R + R1=C12–C18
Alkyl Sulfates (AS)
Laundry detergents – STPs effluents: C12–15 AS Fast biodegradation under
Wool-washing agents, between 1.2 and 12 mg L–1 [15] aerobic and anaerobic
R=C12–C18
soap bars and liquid conditions.
bath soaps, hair Effective removal in WWTPs
shampoos, and [18]
toothpastes
Alkyl Ether Sulfates (AES)
Liquid bath soaps, – Effluents of seven represen- Readily biodegradable in
hair shampoos, and tative STPs: C12–15 AES: WWTPs under both aerobic
R=C10–C14 mechanical 3 and 12 mg L–1 [15] and anaerobic conditions
m=1–4 dishwashing agents. [18, 29]
Ingredient in
industrial cleaning
agents
Table 2 (continued)
Cationic Surfactants
Quaternary Ammonium Compounds (QACs)
ATMAC: Alkyl- Fabric softener Values found for Highly adsorbed onto
trimethyl-ammonium Emulsifying agents ditallowdimethylammonium particulate matter [18].
compounds Biocides, disinfectants chloride (DTDMAC): Short half-life under aerobic
DADMAC: Dialkyl- – STPs-influents: 375–4300 mg L–1 conditions. Poorly anaero-
dimethyl-ammonium R¢=Methyl or – In SW of rivers: 2–34 mg L–1 bically biodegraded [19]
compounds benzyl – Effluent of STPs: 11–55 mg L–1
ADMBAC: Alkyl- X=Cl or Br (reviewed in [30])
dimethylbenzyl- (or Methyl sulfate) – WWTPs influents:
ammonium 340–480 mg L–1 (US);
compounds ≈ 1000 mg L–1 [31]
Quaternary Carboxyalkyl Ammonium Compounds (Esterquats)
Fabric softener No data found Easily biodegraded under
aerobic conditions. It’s also
assumed their degradation in
anoxic conditions [19, 32]
R=C16–C18
Organochlorinated Substances
Polychlorinated biphenyls (PCBs)
Insulating fluid in – SW (up to 100–500 ng L–1) Very persistent in the
electrical equipment [33, 34] environment.
and as hydraulic fluids Low levels in water and air.
m+n =1–10 High levels in soils,
sediments and animals
129
130
Table 2 (continued)
Dioxin Like Substances

Polychlorinated – Marine sediments (Finland): Very persistent in the
diphenylethers (PCDE) <1 pg I-TEQ g–1 dw [35, 36] environment.
Very low levels are found in
m+n =1–10 water and air. High levels in
soils, sediments and animals.
Polychlorinated Are not intentionally – Sediments from polluted areas: Occurrence in the environ-
dibenzo- produced. They are >20 pg I-TEQ g–1 dw [37] ment related to chlorination
p-dioxin (PCDD) formed as byproducts – Effluents from waste degree obtained by photolysis:
m+n=1–8 in several processes incinerator plants (Japan): >chlorination degree>
3.3–120,000 pg L–1. (73.5 TEQ) persistence
Polychlorinated (reviewed in [38])
dibenzofuran (PCDF) – Soil: 0.1–1080 pg I-TEQ g–1 and
sediments: 0.42–8 pg I-TEQ g–1
m+n=1–8 (Spain) [39, 40]
Chlorophenols
Preservatives agents, – GW near sawmills or waste Most of them go into water.
pesticides sites from (0.03 to 91.3 mg L–1) They can be transformed
[41] by photolysis into dioxins
– Drinking water: [48, 49]
m=1–5 0.03–0.7 mg L–1 [42–44]
– SW (several countries):
1.6–26.6 mg L–1 [45, 46]
– Sediments (Canada)
25–10,000 mg Kg–1 [47]
Table 2 (continued)
Organobrominated Substances
Polybrominated biphenyls (PBB)
Flame retardant – Water Pine river (USA): Compounds highly
0.01–3.2 mg L–1 [50, 51] brominated attach strongly
– Sediments: 0.33–0.84 (dw) to sediments. They are slowly
m+n=1–10 mg Kg–1 [52] degraded in the environment
– Water: <0.05 mg L–1 and [53, 54]
sediments: <8 ng g–1 (Japan)
in 1989 [38]
Polybrominated Flame retardant – Sewage sludge: Compounds highly bromi-
diphenylethers 11–28 ng g–1 (Br10) [55] nated attach strongly to
(PBDE) – Water (Japan) in 1988: sediments. They are slowly
m+n =1–10 <0.1 mg L–1 [38] and sediments degraded in the environment
(Japan) in 1996 <25–580 ng g–1
(Br10) [38]
– SW: 0.158 ng L–1; sewage

sludge: 2290–4890 ng g–1; sedi-
ments: 132 ng g–1 (reviewed
in [56])
– Soil (US) <0.1–31.6 ng g–1 [57]
131
132
Table 2 (continued)

Polybrominated Are not intentionally – Sewage sludge from municipal Very persistent in the
dibenzo-p-dioxin produced. They are WWTPs (Germany): environment
(PBDD) formed as byproducts 0.29–3.05 mg Kg–1 (Br1–Br5) [58] Faster degradation than
m+n=1–8 in several processes – Sediments (Japan): PCDDs [59].
0.03–0.37 ng g–1 (Br4–Br6) [38] Very low levels are found in
Polybrominated – River and marine sediments: water and air. High levels in
dibenzofuran (PBDF) 0.03–0.37 mg Kg–1 soils, sediments and animals.
(Br4–Br6) [60] Occurrence in the environ-
m+n=1+8 – Sediments 1.98–17.4 ng g–1 [38] ment related to bromination
degree obtained by photo-
lysis: >bromination degree>
persistence
Bromophenols
Flame retardant – Raw and treated water They can be dehalogenated in
Wood preservative from potable water treatment marine and river sediments
plants (0.6 and 1.3 ng L–1) [61] [62, 63]
– Sediments (Japan)1986:
m=1–5 <0.5–4 ng g–1 [38]
Tetrabromobisphenol A (TBBPA)
Flame Retardant – Sewage sludge TBBPA can be metabolized by
(2.9–76 ng g–1 dw) [55] microbes and broken down by
– Sediments (Japan) 1988: photolysis [64]
2–108 ng g–1 [38]
Table 2 (continued)
Others
Phthalate Esters
Di-n-butyl Plasticizers in PVC – SW levels are near to 10 mg L–1; Fast biodegradation under
phthalate (DBP) production. in rivers, between 0.5–1 mg L–1 aerobic conditions.
Diethyl phthalate Component in the and in sea water between Half-life in water: 1–15 days
(DEP) manufacture of 0.005–0.7 mg L–1 Half-life in soils: 7 days –
Butyl benzyl cosmetics, inks, and – US streams: 2.5 mg L–1 (DEHP) several months [65]
phthalate (BBP) adhesives and 0.25 mg L–1 (DEP) [4]
Di(2-ethylhexyl)
phthalate (DEHP)
Bisphenol A Production of resins – River water mean values: Not persistent in surface water.
(polycarbonate and 0.016 mg L–1 (Europe) and Rapidly biodegraded in aquatic
epoxy resins). 0.5 mg L–1 (US) [66]. environments [68] and removed
Component in flame – SW: <0.001–1 mg L–1 [9] in WWTP.
retardant production – WW effluents mean values: Half-life: 1–4 days [69] in water.
Antioxidant, 1.5 mg L–1 [67] Accumulated in anoxic
preservative sediments [9]
133
134
Table 3 Some representative commercial immunochemical assay kits for the most important emerging pollutants with an industrial origin. The
supplier and the contact web page are also listed
Analyte Immunochemical kit Supplier/manufacturer Contact
Nonionic Surfactants
APEs ELISA kit Japan EnviroChemicals, Ltd. http://www.jechem.co.jp/eco/
AE ELISA kit Takeda Chemical Industries L-EC http://www.takeda.co.jp/index-e.html
AP ELISA kit Japan EnviroChemicals, Ltd. http://www.jechem.co.jp/eco/
Anionic Surfactants
LAS ELISA kit Japan EnviroChemicals, Ltd. http://www.jechem.co.jp/eco/
Organochlorinated substances
PCBs PCB RISc soil test kit EnSys, Inc. (Strategic Diagnostic Inc.) http://www.sdix.com/
PCB RISC liquid waste test system EnSys, Inc. (Strategic Diagnostic Inc.) http://www.sdix.com/
PCB immunoassay kit Hach http://www.hach.com/
EnviroGard PCB in soil Millipore Inc. (Strategic Diagnostic Inc.) http://www.sdix.com/
PCB in soil (tube assay) EnviroLogix http://www.envirologix.com/
D TECH® PCB test kit Strategic Diagnostic Inc. http://www.sdix.com/
DELFIA PCB (soil and food) Hybrizyme http://www.hybrizyme.com/
PCB RaPID Assay® Ohmicron Corp. (Strategic Diagnostics Inc.) http://www.sdix.com/
PCBs ELISA kit, 100T Takeda Chemical Industries L-EC http://www.takeda.co.jp/index-e.html
(magnetic particle)
Table 3 (continued)
Analyte Immunochemical kit Supplier/manufacturer Contact
Organochlorinated substances
PCP PENTA RISc® EnSys Inc. (Strategic Diagnostics Inc.) http://www.sdix.com/
EnviroGardTMin soil Millipore (Strategic Diagnostics Inc) http://www.sdix.com/
D TECH® PCP Test Strategic Diagnostics Inc. http://www.sdix.com/
PCP RaPID Assay® Ohmicron Corp. (Strategic Diagnostics Inc.) http://www.sdix.com/
Dioxins EnSys Dioxin EnSys Inc. (Strategic Diagnostics Inc.) http://www.sdix.com/
High-performance dioxin/furan CAPE Technologies http://www.cape-tech.com/
immunoassay kit insert (IN-DF1)
DELFIA TCDD test kit Hybrizyme http://www.hybrizyme.com/
Others
Bisphenol A ELISA kit Japan EnviroChemicals, Ltd. http://www.jechem.co.jp/eco/
ELISA kit Japan EnviroChemicals, Ltd. http://www.jechem.co.jp/eco/

135
chain. All four chains contain defined constant and variable, locally hyper-
variable, regions within their amino acid sequence (see Fig. 1). The Fc fragment
is the constant (crystallized) region and it is involved in the immune regulation,
whereas the Fab (antibody binding fraction) fragment is the region that con-
tains the variable fraction (Fv) with the specific binding sites that allow inter-
action with the Ag.
Since the publication of the first assay based on the use of antibodies to de-
termine insulin [70], development in this area has undergone rapid growth. The
application of immunochemical methods to detect substances not only in the
clinical field, but also in food and environmental areas has led to the necessity
to obtain more and more specific antibodies in considerable amounts and at
low cost. The important progress made during recent years in the molecular
biology and genetic engineering fields has favored this fact. Currently, we can
speak of three different techniques to obtain antibodies, yielding what we know
as polyclonal (PAb), monoclonal (MAb), and recombinant (RAb) antibodies. In
principle it is possible to obtain antibodies for any kind of substance. In the
case of small molecules (i.e., molecular weight less than 1,000 Da), the design
and synthesis of an appropriate immunizing hapten followed by its covalent
attachment to a carrier molecule [71] has been until now unavoidable; however,
knowledge obtained while engineering new antibody molecules may reduce the
effort necessary in this aspect.
Polyclonal antibodies are obtained directly from the serum of the immu-
nized animals (sometimes a purification step is carried out before their use).
A family of clones is obtained that recognize the global structure of the hapten
immunized, exhibiting each clone to a specific binding to different epitopes
in the molecule. Therefore, the affinity of a PAb will be a combination of the
Fig. 1 Scheme showing the basic H2L2 structure of the immunoglobulins of type G (IgG). It
is formed by two pairs of polypeptide chains interlinked by disulfide bonds. The Fc fragment
is the constant (crystallized) region and it is involved in the immune regulation, whereas the
Fab (antibody binding fraction) fragment is the region that contains the variable fraction (Fv)
with the specific binding sites that allow the interaction with the Ag. Fragments obtained
after papain or pepsin digestion are also shown
activity of each clone for the target analyte. The host animal is usually a rabbit,
but when great amounts of serum are required the use of goats, pigs, or sheep
has been described. The process to finally obtain PAbs is simple once you have
the immunizing hapten but because of the animal variability, a lack of repro-
ducibility can be found from animal to animal. This fact can be a problem when
a constant supply of identical antisera is required.
Monoclonal antibodies are produced by the fusion of antibody-producing
spleen cells from an immunized animal (mouse) with mutant tumor cells
derived from myelomas [72]. In contrast with PAbs, a unique IgG molecule is
obtained from a single cell clone and theoretically this technology provides an
unlimited source of the antibody with identical affinity for the antigen, as long
as the hybridoma line is stable. However, the screening process to isolate the
desired clone is usually tedious and time-consuming, the cost of production of
MAbs is higher than for PAbs, and sometimes they have lower affinities to small
molecules than PAbs.
Whereas in both PAbs and MAbs the specificity and affinity of the final
antibody will be a consequence of the immunizing hapten chosen and the
immunization protocol, in recombinant antibody phage display technology
these problems can be in part solved by the generation of a variety of Ab frag-
ments mimicking the immune response in vitro. The whole process involves
the following steps: (a) the preparation of Ab encoding libraries derived from
different methods (usually by isolation of mRNA from hybridoma, spleen cells,
or lymphocytes of immunized mice); (b) cloning of the genes in a bacterial
plasmid vector; (c) expression in bacteria (E. coli) and coinfection with helper
bacteriophage virus, displaying Ab fragments on its surface as a fusion with
normally occurring coat protein; and (d) screening for antigen specificity and
antigen-driven selection [73, 74]. The ability of this methodology to design the
antibody polypeptidic structure, as well as to modify the existing fragments,
can allow one to improve the affinity of the antibodies or even to change their
selectivity. Although this technology was developed initially for therapeutic
purposes, RAb fragments have also been used in environmental analysis. Re-
combinant antibodies have been produced for insecticides like parathion [75],
for dioxins [76], and for pesticides like triazines [77, 78], although at present
they have not achieved the affinity levels of the corresponding MAbs or PAbs.
2.1
Antibody-Based Analytical Methods
Immunochemical techniques are based on the immunological reaction derived

from the binding of the antibody to the corresponding antigen. This reaction
is reversible and is stabilized by electrostatic forces, hydrogen bonds, and Van
der Waals interactions. The formed complex has an affinity constant (ka) that
can achieve values around the order of 1010 M–1. This great affinity and speci-
ficity between the specific antibody and the antigen (or the analyte) have
turned these techniques into powerful analytical tools to detect and quantify
substances at low concentrations and trace levels. During recent decades many
efforts have been made and important advances have been achieved in this
field. The advances made in microelectronics combined with the availability of
antibodies for a great variety of substances such as proteins, macromolecules,
or low molecular weight molecules such as drugs, metabolites, or environ-
mental, agricultural and food pollutants, have been crucial for this progress.
Despite the robustness and good detectability achieved nowadays by the
chromatographic analytical methods when coupled to sensitive detectors, they
often require expert personnel and expensive equipment. Moreover, precon-
centration and cleanup procedures to remove potential interferences prior to
the analysis are usually mandatory. All these factors lead to an increase of the
final cost of these methodologies and the analysis time. Alternatively, im-
munochemical techniques are simple, fast, and very specific and sensitive.
Nowadays automation and the possibility of development of high-throughput
screening (HTS) have been demonstrated. Overall we can say that they consti-
tute excellent tools to be exploited in monitoring programs where a great num-
ber of samples need to be analyzed. One of their drawbacks is often the fact that
matrix effects should be carefully evaluated beforehand since, in contrast to
other analytical techniques, specific and nonspecific signals are not so easy to
distinguish leading to overestimation or to false positives. Contrariwise, false
negatives are very seldom seen in these techniques. Thus, as effective screen-
ing techniques, immunochemical methods are complementary to the standard
analytical techniques.
Several immunochemical techniques have been developed as analytical tools
or in sample treatment methods to separate an analyte from complex matrices.
Some of the most important or more frequently used are described below.
2.1.1
Immunoassays
Nowadays, immunoassay (IA) is the most extensive immunochemical method-

ology.A great number of IAs have been developed for the detection of pollutants
at trace levels [71, 79–81], such as pesticides and other kinds of industrial
residues, not only in different environmental compartments (water, soils, sedi-
ments, etc.) but also in food and biological matrices. Many of those developed for
pesticides are commercially available and the US EPA (US Environmental Pro-
tection Agency) has validated and included some of them in the SW-846 method
list [82].Wide application has also been found in pharmaceutical, veterinary, and
forensic analysis as well as, more recently, in human exposure assessment to a
variety of industrial chemicals or contaminants such as polyaromatic hydrocar-
bons (PAHs), polychlorinated biphenyls, (PCBs), or pesticides [83–85]. For hu-
man biomonitoring, where large a number of samples are often analyzed, the
possibility of adapting immunoassays to HTS makes them particularly suited to
field studies or large-scale monitoring. In addition, the ability to recognize not
only the target analyte but also other structurally related compounds (Ab cross-
reactivity) may allow both parent molecules and their metabolites to be detected
simultaneously. In this context, the EPA encourages the development of im-
munochemical techniques for human exposure monitoring [84, 85].
In immunoassays the reaction Ab–Ag is quantified by means of labels, un-
der competitive conditions. The general procedure involves a competition step
between a fixed concentration of a labeled derivative and the free analyte for
a limited amount (low concentration) of Ab. The amount of labeled Ag can then
be measured and therefore the amount of free Ag. Several kinds of markers can
be used as labels. The first immunoassay was based on the use of radioisotopes
(radioimmunoassays, RIA) [86], but they have been replaced by more environ-
mentally friendly and less hazardous substances. By using fluorescent (fluores-
cein, rhodamine, etc.), chemiluminescent (i.e., luminol), or bioluminescent
markers, techniques such as fluoroimmunoassay (FIA) and chemiluminescence
immunoassay (CLIA) have been developed, although in FIAs, for instance, the
sensitivity achieved is in many cases not as good as expected, sometimes be-
cause fluorophores are exposed to many interferences that can lead to quench-
ing of the signal. The use of enzyme labels (EIA, enzyme immunoassay) offers
the possibility of increasing detectability, due to the amplification produced
depending on the enzyme turnover, and the option of using a variety of sub-
strates producing colored, fluorescent, or chemiluminescent products. Nowadays
enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP), and
glucose oxidase (GO) are the most frequently used labels in immunoassay.
Immunoassays can be performed in solution (homogeneous format) or by
immobilization of one of the immunoreagents on a solid support (heteroge-
neous format). The solid support can be tubes, nitrocellulose paper, magnetic
particles, microspheres, polystyrene plates, etc. The most used supports are mi-
crotiter plates where up to 96 (or in some cases up to 384) samples can be
processed simultaneously, making use of very small sample volumes. In hetero-
geneous assays a separation between the bound and the free phases is required,
whereas in the homogeneous one, the detection step is carried out in solution,
with both fractions (bound and free) in the immunoreagent mixture. Homoge-
neous assays are faster, simpler, and can be easily adapted to the available auto-
mated analyzers often used in clinical chemistry. However, they are often less
sensitive and are more exposed to matrix interferences, so washing steps to help
remove these interferences must be performed. The common aspect of all these
assays when applied to the analysis of small organic molecules is the fact that the
assay takes place under competitive configurations, as we will see below. In con-
trast, for the determination of large substances, this is not always necessary.
2.1.1.1
Enzyme-Linked Immunosorbent Assay (ELISA)
Among the heterogeneous assays, ELISA is the most common and frequently
used for environmental monitoring. Examples of its wide applicability can be
found in recent reviews [71, 79]. The most usual configurations for the analy-
a Direct Competitive ELISA
b Indirect Competitive ELISA
Fig. 2a, b Scheme of the two ELISA formats most frequently used for the analysis of low
molecular weight analytes. a Direct competitive ELISA. The Ab is coated on the surface and
a competition is established between the analyte and the enzyme tracer. After washing, the
addition of a substrate produces a chromogen product that is easily quantified. b Indirect
competitive ELISA.A coating antigen is immobilized on the solid support and the specific IgG
and the analyte are in solution. After removal of unbound reagents, a secondary IgG labeled
with the enzyme (IgG-enzyme), which specifically recognizes the Ab, is added. After another
washing step the amount bound is also quantified by the addition of the substrate solution
sis of small molecules are shown in Fig. 2. In the direct format (see Fig. 2a), the
Ab is coated onto the solid support (usually a microtiter plate) and an equilib-
rium is established between the Ab, the free analyte, and the enzyme tracer
(both of them in solution).After a washing step, where all the unbound reagents
are removed, the amount of label bound to the Ab is measured, the signal
being inversely proportional to the amount of analyte in the sample. A direct
assay can also be performed by immobilizing an analog of the analyte (coating
antigen) on the plate and performing the competition step with the free ana-
lyte for the labeled Ab in solution.
In the indirect format (see Fig. 2b), the coating antigen is coated on the plate,
but in this case the amount of analyte present in the sample is indirectly mea-
sured by measuring the bound Ab with a second Ab that is conveniently labeled
(AntiIgG-enzyme). Although this format has a step more, it has often proved
to be more robust.
2.1.1.2
Enzyme-Multiplied Immunoassay Technique (EMIT)
EMIT is one of the most common EIAs working under homogeneous condi-
tions [87]. The principle is the competition for the specific antibody between
the analyte and an analog labeled with a particular enzyme (usually glucose-
6-phosphate dehydrogenase, G6P-DH) such that the enzyme activity decreases
upon binding of the labeled antigen to the antibody. In this format the analyte
concentration is directly proportional to the enzyme activity measured. This

kind of assay has been widely applied in the clinical analysis field for the
determination of drugs of abuse in several biological matrices such as urine,
blood, or tissues [88–92], but some examples related to the analysis of pesti-
cides have also been reported [93].
2.1.1.3
Polarization Fluoroimmunoassay (PFIA)
PFIA also works under homogeneous conditions and makes use of fluorescent
labels (usually fluorescein). The principle is the excitation of the sample with
plane polarized light. The free labeled antigen rotates rapidly, emitting light
in many different planes, resulting in a decrease in the intensity of vertical
polarized light. But when it binds to a large molecule like the antibody the
rotation is slower, leading to an increase in the emitted light measured. In the
absence of the analyte, the light measured is thus very small since a great part
of the labeled antigen will be bound to the antibody. The presence of the ana-
lyte is thus evidenced in a direct manner by the increase of the light measured.
As in the case of EMIT this technique has also been used in the clinical area,
sometimes comparing precisely with EMIT in drug analysis, usually as screen-
ing methodology [94–96], but also for environmental pollutants such as pesti-
cides [97–99].
2.1.2
Flow-Injection Immunoassay (FIIA)
In recent years the combination of immunochemical methodologies with au-

tomated devices has grown in importance. In this context, flow-injection sys-
tems coupled to immunoassays (FIIA) [100–102] offer rapid analysis of a great
number of samples, providing rapid results and good levels of sensitivity, and
allowing continuous monitoring. In these devices, a small immunoreactor con-
tinuously receives buffer or reagents through different valves. The technique
can work in homogeneous (usually with fluorophores as label) or heteroge-
neous conditions (usually with the Ab immobilized). The more generic FIIA
works as is shown in Fig. 3. By means of a buffer valve all the solutions are con-
tinuously flowing through the system and the immunoreagents (Ag and labeled
Ag, both together or in sequential steps) are injected in the system. The amount
of labeled Ag is detected downstream and quantified. Subsequently, the system
can be regenerated by passing buffer solutions. Several FIIAs have been devel-
oped for environmental pollutants such as pesticides and also industrial chem-
icals, as will be discussed in following sections [100, 103–106].
Fig. 3 Generic FIIA system. A heterogeneous format is shown. The antibodies are immobi-
lized in the immunoreactor. The analyte and the labeled Ag (in this case with an enzyme)
are passed through the system and the competition step takes place. The flow of the substrate
solution through the system allows the determination of the amount of bound labeled Ag,
which is then detected and measured
2.1.3
Immunosensors
In recent years many efforts have been made to develop immunochemical tech-
niques integrating the recognition elements and the detection components, in
order to obtain small devices with the ability to carry out direct, selective, and
continuous measurements of one or several analytes present in the sample. In
this context biosensors can fulfill these requirements. Biosensors are analytical
devices consisting of a biological component (enzyme, receptor, DNA, cell, Ab,
etc.) in intimate contact with a physical transducer that converts the biorecog-
nition process into a measurable signal (electrical or optical) (see Fig. 4). In
Fig. 4 Schematic representation of a generic biosensor with the essential components (bio-
recognition element, transducer, and electronic part involved in data processing and display)
immunosensors the Ab or the Ag is immobilized on the surface of the transducer

and the formation of the immunocomplex is detected by the transducer.
Although there are many approaches for direct immunosensors (the changes
generated when the antibody binds the analyte), in the case of small organic
molecules the detection usually takes place under competitive configurations
with or without the use of labels. Several transducer principles have been de-
scribed. Electrochemical immunosensors are based on the use of amperometric,
potentiometric, conductimetric, or impedimetric transducers. Optical immuno-
sensors make use of optical fibers, the evanescent wave, or the surface plasmon
resonance principle, among others. Piezoelectric immunosensors are based on
the shift of the resonance frequency of piezoelectric crystals produced after
formation of the immunocomplex. Finally some examples can also be found of
thermometric immunosensors, where the heat of the reaction produced as a con-
sequence of the immunoreaction, usually coupled to an enzyme amplification
system, is detected. When the use of labels is required, the nature of these de-
pends on the transducer principle of the immunosensor. Thus, the use of elec-
troactive, fluorescent, or mass labels among others has been described. Extensive
information on the transducer principles, methods, and examples of all these
types of immunosensors can be found in recent reviews [107–110].Although the
use of biosensors has been mainly reported for clinical purposes [111], nowadays
their application has been extended to different areas, for example the detection
of microorganisms such as viruses and bacteria [112], the detection of drugs,
and also in the control of environmental pollutants [102, 113, 114].
2.1.4
Immunoaffinity Chromatography (IAC)
The application of immunochemical techniques is not only in the development

of analytical detection tools, but also the inherent specificity of the immunore-
action can be exploited to develop selective extraction procedures to be used
prior to the analysis by any other analytical method (either immunochemical or
a conventional one, like chromatography). Immunoaffinity chromatography
(IAC) [115–117] for trace analysis of low molecular weight analytes in complex
matrices has several advantages over other solid phases. Apart from selectivity,
the antibody cross-reactivity allows the extraction of both the analyte and its
metabolites or other structurally related compounds. Preconcentration of the
analyte may assist in increasing the detectability of certain analytical methods.
The essential hydrophilic media necessary when handling biomolecules allow
the purification of polar substances, which can be hindered when other con-
ventional phases are used. Figure 5 shows a scheme of a basic IAC procedure.
The Abs are covalently bound to a solid support and packed in small columns,
which are conditioned and loaded with the sample. After a washing step that
allows removal of the nonspecifically retained compounds, the target analyte
is eluted under appropriate conditions (usually by changing the buffer com-
position). Finally, the column can be regenerated and reused several times.
Fig. 5 Sequential steps involved in an immunoaffinity extraction procedure
Immunoaffinity procedures can be performed either on-line or off-line, and

can be coupled to chromatographic systems [118, 119] or even to immunoas-
says [120]. Many examples can be found in the literature regarding the use
of immunoaffinity extraction of drugs and pharmaceuticals from biological
matrices, as well as of organic pollutants such as pesticides from environmen-
tal samples [115, 121–124].
3
Immunochemical Methods for Surfactants
The use of surfactants in detergent formulations was extended worldwide sev-
eral decades ago. More environmentally friendly compounds with surfactant
properties have gradually replaced the natural soaps. These new substances are
characterized by the presence in the chemical structure of both hydrophilic
(usually charged) and hydrophobic groups (particularly long linear alkyl
chains). This fact imparts unique properties to these compounds as surface-
active agents. Depending on the charge of the hydrophilic part of the molecule
four clear groups can be distinguished: anionic, cationic, nonionic, and am-
photeric surfactants. The production volume of these compounds has increased
considerably since they have been used as substitutes for soap-based detergents.
Thus, in 30 years (between 1940 and 1970) the production of synthetic surfac-
tants in the USA went from 4.5¥103 to 4.5¥106 t [18], whereas that of the
natural surfactants has decreased but not to the same extent. According to the
European Committee of Surfactants and their Organic Intermediates (CESIO),
the total production in western Europe in 2000 was 2.5¥106 metric tons, with
anionic and nonionic surfactants being the most abundant ones at production
volumes near to 1¥106 and 1.2¥106 metric tons, respectively [125]. Cationic
surfactants are produced to a lesser extent in western Europe, constituting
approximately 8% (2¥105 metric tons) of the total production of surfactants.
Fig. 6 General structures of the most important surfactants and metabolites: alkylphenol
polyethoxylate (APE); alkylphenol (AP); alkyl ether (AE); alkylphenol ethoxy carboxylate
(APEC); linear alkylbenzenesulfonates (LAS); alkyltrimethylammonium compounds (ATMAC);
dialkyldimethylammonium compounds (DADMAC); alkyldimethylbenzylammonium com-
pounds (ADMBAC); esterquat (EQ); diesterquats (DEQ). X is usually a chlorine or bromine
atom. DDAC (didecyldimethylammonium chloride) and BDD12AC (benzyldimethyldode-
cylammonium) are the two target analytes with a reported immunochemical technique de-
veloped for their analysis [153, 154]
Contrary to anionic and nonionic agents, they have poor detergency and are
used more in the preparation of germicides, fabric softeners, and emulsifiers.
Amphoteric surfactants are produced in much smaller amounts (5¥104 metric
tons, near to 2% of the total production) [125]; they are biodegradable and their
ecotoxicological importance can be considered low. Their environmental oc-
currence up to know has been just occasional.
Liquid chromatography coupled to mass spectrometry (LC–MS) is the most
usual technique applied for the detection of anionic [126–128], nonionic
146
Table 4 Immunochemical techniques developed for the detection of surfactants. The sensitivity of the method and the matrix considered are shown
Analyte Immunochemical Sensitivity Matrix References

techniquea
LOD IC50
Nonionic surfactants Alkylphenol ethoxylates (APEs)

NP10EO ELISA (direct) 8.9 mg L–1 Buffer/water [144]
FIIA 2.4 mg L–1 24.5 mg L–1 Buffer [144, 145]
OP10EO FIIA 0.5 mg L–1 17.7 mg L–1 Buffer [145]
NPE CIA-GDH biosensor 378 mg L–1 Buffer [146]
ELISA 104 mg L–1 Buffer [146]
Automated BMP-IA 6.6 mg L–1 Buffer [147]
OPE CIA-GDH biosensor 605 mg L–1 Buffer [146]
a CIA-GDH biosensor: capillary immunoassay coupled to a glucose dehydrogenase biosensor; ELISA: enzyme-linked immunosorbent assay;
BMP-IA: bacterial magnetic particle-based immunoassay; FIIA: flow-injection immunoassay; PFIA: polarization fluoroimmunoassay.
b BDD AC: benzyldimethyldodecylammonium chloride; DDAC: didecyldimethylammonium chloride.
12
Table 4 (continued)

techniquea
LOD IC50
Nonionic surfactants Alkylphenols (AP)

NP ELISA 10 mg L–1 Buffer [148, 149]
FIIA 52 mg L–1 1033 mg L–1 Buffer [144, 145]
PFIA 7.9 mg L–1 42 mg L–1 Buffer [144]
PFIA 9 mg L–1 42 mg L–1 Buffer [150]
CIA-GDH biosensor 4481 mg L–1 Buffer [146]
PFIA 9 mg L–1 42 mg L–1 Buffer [150]
OP ELISA 346 mg L–1 Buffer [146]
CIA-GDH biosensor 1560 mg L–1 Buffer [146]
Anionic surfactants Linear alkylbenzene sulfonates (LAS)
ELISA 20 mg L–1 40 mg L–1 Buffer [151]
ELISA 19.8 mg L–1 Buffer [144]
FIIA 19.5 mg L–1 387 mg L–1 Spiked rain/water [144]
PFIA Buffer [144]

PFIA 0.5 mg L–1 9 mg L–1 Buffer [152]
Automated 35 ng L–1 Buffer [147]
BMP-based IA
Cationic surfactants Quaternary ammonium compounds (QACs)b
BDD12AC ELISA 0.043 mg L–1 0.66 mg L–1 Buffer, (milk) [153]
DDAC ELISA 8 mg L–1 29 mg L–1 Buffer [154]
147
[129–138], and cationic surfactants [139], achieving good levels of sensitivity.

Gas chromatography also coupled to mass spectrometry (GC–MS) has been
used more sporadically [140–142]. As is well known, these methodologies can
achieve very low limits of detection (LOD), sometimes in the range of micro-
grams per liter and even nanograms per liter. However, the analytical protocols
employed exhibit important drawbacks derived from the high polarity and
solubility of these compounds in water. This fact makes the necessary prior
extraction/preconcentration step complicated. Solid-phase extraction procedures
are often tedious to ensure the efficient recovery of analytes from water samples
[16, 143]. Moreover, usually they are not single substances but technical mixtures
of several compounds and isomers (see Fig. 6 for chemical structures). Im-
munochemical techniques offer in this case the advantage that the analyses are
generally performed in water and often have sufficient detectability to allow
direct analysis of the sample. Therefore, in principle there is no need to use
organic solvents or to extract the surfactant from the aqueous sample. Table 4
summarizes most of the immunochemical techniques that have been devel-
oped for these compounds and will be briefly commented on in the next few
sections.
3.1
Anionic Surfactants
Linear alkylbenzenesulfonates (LAS) represent more than 40% of all surfactants

used [18] with a production of nearly 8.5¥105 metric tons. Other anionic agents
such as alkyl sulfonates (AS), alkyl ether sulfates (AES), or fatty alcohol sulfates
(FAS) are also produced, with the same final applications but lower consump-
tion (see Fig. 6 for chemical structures).All of them are easily biodegradable and
efficiently removed by the WWTPs (efficiency near 99%) [28], but because of
their large consumption they end up reaching rivers and marine environments.
Both LAS and their metabolites, the sulfophenyl carboxylates (SPCs), are usually
found in surface waters at levels around a few micrograms per liter, although this
level increases in areas where sewage effluents are not connected to municipal
WWTPs [23–25, 27]. They have also been detected in seawater [26] and drink-
ing water (1.6–3.7 mg L–1) [23]. Although LAS cannot be considered as toxic
substances (none of the anionic surfactants mentioned above is included in
the list of dangerous substances of the Council Directive 67/548/EEC), the for-
mation of micelles helps to dissolve and transport many nonpolar organic pol-
lutants, preventing their degradation and enhancing the toxic action of other
substances. Thus, synergistic effects are also observed with pesticides (i.e., DDT
and dieldrin) and heavy metals like Cd or Hg [155].
Although several immunochemical methods have been reported for LAS, few
examples of their application to real environmental matrices have appeared. The
first immunochemical method for LAS was reported by Fujita et al. [151]. It is
a direct ELISA and uses MAbs generated against 5-sulfophenyl valeric acid con-
jugated to BSA through the carboxylic acid, thus preserving the sulfonic group
intact. The LOD achieved is around 20 mg L–1 and the working range between
20 and 500 mg L–1. No cross-reactivity was observed for sodium dodecyl sulfate
(SDS), other sodium fatty acid salts (such as sodium palmitate, laureate, and
stereate), and other surfactants such as nonylphenol polyethoxylates or short-
chain SPCs. Since the long alkyl chains of LAS (between 9 and 13 carbon atoms)
were well recognized, there was a risk that long-chain SPCs would cross-react
in the assay; however, these compounds were not evaluated. The assay was
evaluated in terms of precision and accuracy by measuring spiked river water
samples. Recoveries between 81 and 100% were obtained. Similarly the assay
was also validated with HPLC.
Franek et al. [144] also used several sulfophenyl carboxylic acids with dif-
ferent chain lengths as immunizing haptens to produce a large number of poly-
clonal antibodies. Direct and indirect ELISAs [144], a FIIA [144], and a PFIA
[152] have been developed with these antibodies. For FIIA the tracer was a hap-
ten conjugated to b-galactosidase. The sensitivity reported is quite good and in
the same range for both ELISA and FIIA methods (near 20 mg L–1, see Table 4).
Unfortunately it is referenced to the linear 4-dodecylbenzenesulfonic acid
sodium salt (LDS) instead of to the commercial mixture, which does not give
a real picture of the detectability of these methods. The flow format allowed the
analysis of ten samples per hour. Matrix effects were also studied using spiked
surface and rainwater samples. The latter produced nonspecific interferences
in the assay whereas surface water could be directly measured without prob-
lems. For PFIA [152], several antibodies and fluorescein thiocarbamyl ethyl-
enediamine (EDF) tracers were screened but the sensitivity was worse. The best
combination afforded a LOD of 0.5 mg L–1 and a dynamic range between 3 and
85 mg L–1. These values are about 3 orders of magnitude higher than those
obtained for the ELISA developed with the same antibody [144]; however, this
technique allows higher sample loads since about seven samples can be ana-
lyzed in 10 min. SDS only cross-reacts about 5%.
Recently Matsunaga et al. [147] developed an automated immunoassay sys-
tem based on the immobilization of monoclonal antibodies [151] to magnetic
particles, synthesized by magnetic bacteria (Ab-BMP). The assay is performed
in microtiter plates mounted in the reaction station. This kind of immunoassay
allows the suspension and subsequent easy separation of antibody from the rest
of the immunoreagents via a magnet coupled to the tips used in the automated
pipette. The detection limit obtained in the competitive assays for LAS is very
good (35 ng L–1), similar to those obtained by GC–MS or LC–MS, and shows a
wide working range (between 35 ng L–1 and 35 mg L–1). The assay showed low
cross-reactivity with short alkyl chain alkylbenzenesulfonates and with SDS,
but surprisingly NP is recognized with a 34% cross-reactivity.
Finally, MAbs and an immunoassay kit for LAS have been commercialized
(see Table 3). The working range of the assay is between 20 and 500 mg L–1. The
antibodies are highly specific for LAS with alkyl chains between C8 and C12,
whereas other anionic and nonionic surfactants tested showed no cross-reac-
tivity.
3.2
Nonionic Surfactants
During recent decades the nonionic surfactants used most worldwide have
been the alkylphenol polyethoxylates (APEs). They are widely used in detergent
formulations, both for industrial and domestic applications. They are also used
as plasticizers and stabilizers in plastics. About 5¥105 tons are produced an-
nually [156], nonylphenol ethoxylate (NPE) (and to a lesser extent octylphenol,
OPE) being the most prevalent one (NPEs represent about 80% of APEs used).
This great consumption involves high levels of discharges into the environ-
ment. In the WWTPs they are readily biodegraded under both aerobic and
anaerobic conditions [6, 157] with removal efficiencies of 90–99%, although
it can decrease when high loads of APEs are produced [7]. The mechanism
involves the loss of ethoxylate units and the oxidation of the phenol group to
form alkylphenol ethoxy carboxylates (APECs). The final breakdown products
are APEs with one or two ethoxylate units (AP1EO, AP2EO), APECs, and AP
(see Fig. 6 for chemical structures). All these metabolites have lost their surfac-
tant properties and are much more persistent in the aquatic environment [5, 18].
Several studies have indicated that whereas the parent compounds seem to pre-
sent low toxicity in organisms [19, 158], APE metabolites, particularly APs
(both NP and OP) and short-chain APEs, are highly toxic and their estrogenic
activity is remarkable [3, 159–163].
The most polar APECs are mainly detected in wastewater, effluents, and
rivers, whereas APs, which are more lipophilic, tend to be adsorbed onto soil,
sediments, and sludge. The distribution and fate of this family of compounds
have been widely reported during recent years [4, 5, 9, 164–168]. The levels of
these pollutants in the environment may exceed the predicted no effect con-
centration (PNEC of 0.33 mg L–1) [165] proposed in a risk assessment report
of the EU. As a consequence, their use for household and industrial cleaning
has been banned in several countries of the EU, being substituted by less toxic
and more environmentally friendly nonionic surfactants such as alcohol
ethoxylates (AEs) [156]. In the USA these evidences have also led to regulatory
actions related to the domestic use of APEs. It seems that AEs are rapidly
biodegraded, although it depends on the nature of the alkyl chain, mainly their
branching degree (the degradation decreases when the branching of the chain
increases) [19].
Immunochemical methods have been reported for both APEs and their
metabolites, especially APs.A discussion of the immunochemical methodologies
reported to date, the effect of the immunizing haptens employed, and the fea-
tures of these techniques were recently reviewed [169]. Unfortunately, the de-
tectability achieved is usually far from what is necessary for direct application
to environmental samples. Moreover, the selectivity for APs versus APEs is not
always satisfactory. Thus, Goda et al. [148] developed a direct ELISA for NP with
a LOD of 10 mg L–1 and a working range between 70 and 1,000 mg L–1, but APEs
with one to ten ethoxylate units are also well recognized.
MAbs for AP and APEs have been produced and commercialized by Takeda
Chemical Industries [170] (see Table 3) and have opened up the opportunity to
develop a great variety of assays. FIIAs and ELISAs for NP and NP10EO have
been developed with these antibodies [144]. The detectability achieved is sim-
ilar for both methods, although NP10EO is better recognized than NP. Thus,
while the FIIA dynamic range reported for NP10EO was 10–500 mg L–1, for NP
it was 100–5,000 mg L–1. Using the same antibodies an improved FIIA for APEs
(both NPEs and OPEs) and NP was thoroughly evaluated and validated by
Badea et al., who studied the influence of different factors such as the presence
of organic solvents or heavy metals in the assay media, and its performance in
several water matrices (tap water, surface water, and wastewater) [145]. The
LOD reported for NP was 51 mg L–1 and about 2.5 mg L–1 for NP10EO, although
short NPEO were also highly recognized. The method was applied to the de-
termination of these surfactants in the influent and effluent of WWTPs.
The same MAbs have been immobilized on magnetic particles and used to
develop an automated immunoassay method for APEs (NPEs with 10–20 EO
units) [147]. The wide dynamic range reported (between 6.6 mg L–1 and
66 mg L–1) is related to a low slope assay, which has a direct negative influence
on the immunoassay precision. NPEs with two ethoxylate units and NP are also
recognized in this assay (49 and 31%, respectively). Finally, with the same
antibodies a capillary immunoassay (CIA) coupled to an enzyme biosensor
(glucose dehydrogenase, GDH, biosensor) has been developed for APs (NP and
OP) and APEs (NPE and OPE) [146]. The competitive step takes place off-line
in an antibody-coated plastic capillary and once it has been washed, it is inte-
grated in the flow-injection system with the biosensor as detector unit. The
detectability is much worse than that for the ELISA format and, as with the
other formats, APEs are better recognized than APs.
Franek et al. have also produced polyclonal antibodies for NP [144]. Dif-
ferent antibodies were raised using various para-alkyl hydroxyphenyl com-
pounds with different alkyl chain lengths as immunizing haptens. An indirect
ELISA and PFIA have been developed with these antibodies. The detectability
accomplished is between 1 and 2 orders of magnitude higher for the ELISA for-
mat. While the IC50 of the ELISA was 590 mg L–1, the limit of detection of the
PFIA was around 8 mg L–1. As happened with the LAS PFIA method, the sam-
ple throughput is very high but it is necessary to develop compatible sample
concentration methods in order to apply this technique for environmental
monitoring purposes. Subsequently, Yakovleva et al. [150] also reported the
development of PFIA for NP, obtaining sensitivities in the same order of mag-
nitude (LOD of 9 mg L–1 and a dynamic range between 10 and 177 mg L–1).
Although the cross-reactivity studies showed no recognition of other phenolic
compounds, no APE or other related surfactants were tested, which would have
been interesting in order to determine the capability of the method to dis-
criminate between NP and its parent compounds.
3.3
Cationic Surfactants
The most important cationic surfactants are those derived from quaternary
ammonium salts where one or two hydrophobic groups (usually a long alkyl
chain, between 12 and 18 carbon atoms) are attached to positively charged
nitrogen. The other two groups are short alkyl chains, usually a methyl or a ben-
zyl group. The most used salts in the formulation of commercial products are
quaternary ammonium compounds (QACs). Figure 6 shows a generic structure
of different QACs and their abbreviated names. Alkylquats can be considered
those structures where the hydrophobic alkyl chain is directly linked to the
nitrogen atom (e.g., alkyltrimethylammonium compounds (ATMAC), dialkyl-
dimethylammonium compounds (DADMAC) or alkyldimethylbenzylammo-
nium compounds (ADMBAC)), whereas in the esterquats (EQ) (or diesterquats,
DEQ) the hydrophobic group is linked through an ester bond.As happens with
LAS or APEs, for their preparation several raw substances are obtained from
natural oils and therefore the commercial product is often constituted of a mix-
ture of different alkyl chain lengths. The properties of these surfactants include
not only their surfactant activity but also their biocide effect. They are active
principles in several household products such as fabric softeners or hair condi-
tioners, and are also in disinfectants, biocides, emulsifiers, wetting agents, and
processing additives.After use they are discharged to STPs or directly to surface
waters.Although it seems they are readily biodegraded under aerobic conditions
(a half-life of 2.5 h for octadecyltrimethylammonium chloride in wastewater
[171]), little has been reported concerning anaerobic degradation [172–174]. It
seems that alkylquats are poorly degraded under anoxic conditions since the
presence of O2 is required in the first steps, for the cleavage of the C–N bond,
or for the w-oxidation of the alkyl chain. In contrast, esterquats, whose bio-
degradation mechanism involves in a first step the hydrolysis of the ester bond,
can undergo anaerobic biodegradation [32].
The toxicity of these compounds [173, 175] can be relatively high compared
to other surfactants, but their poor solubility and their tendency to adsorb
to solids or to complex with anionic substances considerably reduce the real
risk and adverse effects for the aquatic environment. [30, 31, 176]. The use of
alkylquats has been substituted by the more easily biodegradable and less
toxic esterquats that are nowadays the cationic surfactants produced in higher
volumes.
A competitive indirect ELISA was developed [154], using PAbs raised against
an immunizing hapten that preserved both long alkyl chains and with one
methyl group substituted by the spacer arm. A LOD of 8 mg L–1 was achieved
when didecyldimethylammonium chloride (DDAC) was used as analyte (see
Fig. 6 for structure). The specificity of the assay was evaluated by testing short-
chain QACs (with methyl or ethyl groups) that were poorly or not recognized,
demonstrating that the methyl group or the charged nitrogen atoms are not the
main epitopes. Fatty acids (FA) or fatty alcohols (FOH) with long alkyl chains
were also tested, and high levels of cross-reactivity were observed for those
with alkyl chain lengths between 10 and 12 carbon units. Shorter and longer
chains were less recognized, which indicates that the main epitope is the decyl
chain. A better detectability has been reported on another ELISA for benzyl-
dimethyldodecylammonium chloride (BDD12AC) [153], a component of the
benzalkonium chloride (BAK) which is a mixture of three alkyldimethylben-
zylammonium chlorides with different alkyl chain lengths (C12, C14, and C16).
BAK is widely used and there is also public concern due to its toxicity. The PAbs
were raised using as immunizing hapten an analog of the analyte where a
methyl group was substituted by the spacer arm. The LOD accomplished was
43 mg L–1 for the bromide analog. No recognition was observed for FAs, FOHs,
tertiary amines with long alkyl chains and benzyl amines, and QACs with short
(methyl) or medium (C6) alkyl chains. Dialkyldimethylammonium compounds
(with C10 and C12 alkyl chains) were slightly recognized, while benzyldimethy-
lalkylammonium compounds (BDACs, with C6–C16 alkyl chains) showed cross-
reactivity values between 42 and 106%. This ELISA has been validated by HPLC
using spiked samples and commercial products.
4
Immunochemical Methods for Polychlorinated
and Polybrominated Compounds
During recent decades much concern has been focused on the adverse health
effects of organochlorinated substances. This group involves several families
of compounds (chlorophenols, CP; polychlorinated biphenyls, PCB; poly-
chlorinated dibenzodioxins, PCDD; polychlorinated diphenyl ethers, PCDE;
polychlorinated dibenzofurans, PCDF etc.) known to be highly persistent and
in some cases with a clearly proven toxicity in organisms. Recently, much more
attention is also being paid to organobrominated compounds because of their
growing use and production. Analog families of the organochlorinated sub-
stances can be found (bromophenols, BP; polybrominated biphenyls, PBB;
polybrominated dioxins, PBDD; polybrominated diphenyl ethers, PBDE etc.).
Each family has a common and generic carbon-based structure and a different
degree of halogen substitution (either Cl or Br), resulting in chemical com-
pounds or commercial products containing mixtures [53]. The structures of the
most common polybrominated and polychlorinated substances are shown in
Fig. 7.
Although the use of organochlorinated substances has been abolished in
most of the developed countries, their extensive use during the past decades
and their persistence in the environment determine their actual widespread
distribution. Moreover, some substances are not used or commercialized but
are still important synthetic intermediates for the preparation of other sub-
stances. Thus, chlorophenols are important intermediates in the production of
pesticides or other chemicals. Other substances such as PCDEs or PCDDs are
Fig. 7 Generic chemical structures of polyhalogenated compounds. X=Cl, Br. (I) Poly-
chlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs); (II) chlorophenols (CPs),
bromophenols (BPs); (III) polychlorinated diphenyl ethers (PCDE), polybrominated diphenyl
ethers (PBDE); (IV) polychlorinated dibenzo-p-dioxin (PCDD), polybrominated dibenzo-p-
dioxin (PBDD); (V) polychlorinated dibenzofuran (PCDF), polybrominated dibenzofuran
(PBDF); (VI) tetrabromobisphenol A (TBBPA)
not intentionally produced, but are generated as undesired by-products in

various industrial activities and all combustion processes [34]. They can be
formed by chemical, photochemical, or thermal reactions from precursors
[177]. Chlorophenols are also unintentionally formed when water with a high
content of organic material is disinfected with chlorine or during wood pulp
bleaching processes. PCBs are used as lubricants, fire retardants, immersion
oils, and dielectric heat transfer fluids. For the latter, there was an estimated
total production of 1.5 million tons in 1992 [178, 179].
The toxicity and the environmental and ecological impact of these substances
have been extensively reviewed, although there are still questions and contro-
versies on the effects after long time exposures. Moreover, the toxicity varies
greatly between families and congeners (for reviews see [180–184]). Thus, the
greatest concern exists around PCDDs, followed by PCDFs, PCDEs, and PCBs.
From the PCDD family, tetrachlorodibenzo-p-dioxin (TCCD) has been identified
as the most toxic congener [182]. Data regarding human dioxin exposure have
been associated with an increased risk of severe skin lesions such as chloracne
and hyperpigmentation, altered liver function and lipid metabolism, general
weakness associated with drastic weight loss, depression of the immune system,
and endocrine- and nervous-system abnormalities. TCDD is considered a potent

carcinogenic, teratogenic, and fetotoxic chemical in certain animals. Human pop-
ulations occupationally or accidentally exposed to chemicals contaminated with
dioxin have increased incidences of soft-tissue sarcoma and non-Hodgkin’s lym-
phoma. On the other hand, some DDT, PCDD, PCDF, and PCB congeners have
been classified as high-concern potential endocrine disrupting substances in the
list of the 66 priority substances according to the BKH report [3].
The use of organobrominated substances has shown extraordinary growth
during the last few years because of their properties as flame retardants (BFRs,
brominated flame retardants) and preservatives for woods, plastics, textiles,
electronic circuitry, and other materials [185]. The most frequently used are
tetrabromobisphenol A, PBB, PBDE (penta-, octa-, deca-brominated diphenyl
ether (oxide) formulations), and hexabromocyclododecane. The production,
application, and potential environmental occurrence of the most important
BFRs have recently been reviewed [186]. The global production (Europe, Asia,
and USA) of BFRs increased from 106,700 to 203,500 tons from 1989 to 1999,
the most spectacular increase being in Asia (from 28,700 to 119,900 tons over
the same period) [186, 187]. Their impact on wildlife and the environment has
been reviewed by authors in different countries [38, 56, 57, 188–191].Although an
important amount of information exists on the potential risk of the organochlo-
rinated substances, more studies have to be done regarding the toxicity of the
polybrominated substances. It is necessary to collect more data on the toxicity
effects in different species and on the metabolism and fate of the BFRs [192, 193].
Some studies indicate that the toxicity of the chlorinated and brominated
analogs could be very similar, but the results are still very much dependent on
the assay used. Thus, toxic effects, including teratogenicity, carcinogenicity, and
neurotoxicity, have been observed for some BFR congeners, in particular the
PBDEs (for recent reviews see [194, 195]). The endocrine-disrupter activity of
the BFRs is being investigated. Hence, PBBs have also been classified as of high
concern in the BKH report [3]; on the other hand, some evidence has been pro-
vided on the disruption of the thyroid hormone system by BFRs, with partic-
ular emphasis on the PBDEs [196].
The application of immunoassays to the analysis of organohalogenated
compounds has not been as frequent as for more water-soluble species [197].
Since immunoassays are typically aqueous-based systems, the low water solu-
bility of these compounds makes the use of immunochemical techniques more
challenging since several facts have to be considered, starting from a careful se-
lection of the immunizing hapten to aspects such as the preparation of sample
extracts or handling of the standards to the last stages in the assay optimiza-
tion. Other problems derive from the tendency of these substances to bind
to the lab ware employed, especially to the plastic ware commonly used for
immunoassays (plates, pipette tips, cuvettes, etc.). In spite of these problems
a variety of immunochemical techniques have been reported for the analysis
of organochlorinated emerging pollutants. Conversely, to our knowledge no
immunochemical techniques have been reported for the determination of
organobrominated substances, although it has been reported that antibodies

raised against chlorophenols have the ability to recognize the corresponding
brominated analogs even more [198, 199]. Moreover, preliminary experiments
performed in our group have shown that the application of the 2,4,6-tri-
chlorophenol immunoassay to the analysis of 2,4,6-tribromophenol from wood
extracts would be feasible [200]. In the following we will describe briefly the
features of some of the immunochemical techniques available today for the
detection PCBs, PCDDs/PCDFs, and chlorophenols (see Table 5).
4.1
PCBs
The first assays for PCBs date from the 1980s [228, 229] and since then several
other attempts have also been carried out (reviewed in [169, 230]). The de-
scribed assays are usually based on the analysis of PCBs such as Aroclors, not
as congeners, and use PAbs produced using an Aroclor or a single PCB as a hap-
ten. RIAs have been developed and used to detect them in biological matrices
such as milk or blood [83, 207, 231], whereas other kinds of immunoassays have
been reported to detect them in environmental samples such as water, soils, and
sediments [201–203, 232, 233]. The detectability reported depends on the hap-
ten used to raise antibodies and also on the congener used to calibrate the as-
say. Simple extraction methods rendering extracts compatible with the aque-
ous media of the immunochemical methods are sometimes reported in order
to be able to perform assays on-site. Usually a water-miscible solvent such
as methanol is present at a certain percentage to improve solubility and to
diminish adsorption of the analyte to the glass containers or plastic surfaces
[205, 210].
a b
Fig. 8a, b a Liposome immunocompetition assay (LIC); R1, liposome/PCB competition zone.
b Liposome immunoaggregation assay (LIA); R2, liposome/antibody aggregation zone. C1,
C2: anti-biotin capture zones. Published with permission of ACS Copyright Office [209]
Table 5 Immunochemical techniques developed for the detection of organochlorinated substances
Analytea Immunochemical Sensitivity Matrix References

techniqueb
LOD IC50
PCBs
Aroclor 1248 ELISA 1.34 mg L–1 22 mg L–1 Soil, sediments [201]
(8.95 ng g–1)
ELISA 1,000 mg L–1 Soil [202]
Aroclor 1242 ELISA 1.57 mg L–1 25 mg L–1 Soil, sediments [201]
(10.5 ng g–1)
ELISA 5–12.9 mg L–1 Soil [203]
FOI 10 mg L–1 River water, soil [204]
Aroclor 1254 EIA magnetic particle 0.2 mg L–1 Water [205]
500 mg Kg–1 Soil [205]
RIA 2 mg L–1 Blood [83]
20 mg L–1 Milk [83]
ELISA 25 mg L–1 217 mg L–1 Buffer [206]
Aroclor 1260 ELISA 38 mg L–1 212 mg L–1 Buffer [206]
RIA 0.013 mg kg–1 Milk [207]
a
DCP: dichlorophenol; PCP: pentachlorophenol; 2,3,7,8-TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCP: trichlorophenol; TMDD: 2,3,7-trichloro-
8-methyldibenzo-p-dioxin.
b EIA magnetic particle: enzyme immunoassay based on magnetic particles; ELISA: enzyme-linked immunosorbent assay; FOI: fiber optic immuno-
sensor; LIA: liposome immunoaggregation assay; LIC: liposome immunocompetition; LIF-microdroplets-QFIA: laser-induced fluorescence de-
tection in microdroplets with quenching- fluorescence immunoassay; RIA: radio immunoassay.
157
158
Table 5 (continued)

techniqueb
LOD IC50
PCBs
Aroclors 1242, 1248, FIIA 1 mg L–1 Buffer [208]
1254, 1260
2-chlorobiphenyl LIA 0.26 pmol Buffer [209]
LIC 0.4 nmol Buffer [209]
3,4,3¢,4¢-tetrachloro- ELISA 0.2 mg L–1 0.9 mg L–1 Buffer [210]
bisphenyl
Dioxins PCDDs/PCDFs
2,3,7,8-TCDD ELISA 4 mg L–1 Buffer [211]
ELISA 200 pg per well Buffer [212]
ELISA 1 ng per well Buffer [213]
ELISA 0.1 mg L–1 Soil [214]
0.025 mg L–1 Water [214]
ELISA 19.5 pg per tube Fly ash [215]
ELISA 10.4 ng mL–1 Buffer [76]
TMDD ELISA 0.01 mg L–1 0.24 mg L–1 Buffer [216]
ELISA 0.004 mg L–1 0.036 mg L–1 Buffer [217]
ELISA 16 ng mL–1 Buffer [218]
Table 5 (continued)

techniqueb
LOD IC50
Chlorophenols
PCP ELISA 0.1 mg L–1 2.9 mg L–1 Water [219]
EIA magnetic particles 0.06 mg L–1 2.2 mg L–1 Buffer [220]
ELISA 500 mg L–1 Soil [221]
ELISA 30–40 mg L–1 Buffer [222]
2,4,6-TCP ELISA 0.2 mg L–1 1.53 mg L–1 Buffer [198]
LIF-micro- 0.04 mg L–1 0.45 mg L–1 Buffer [224]
droplets-QFIA
1.6 mg L–1 Urine
ELISA 0.2 mg L–1 7.8 mg L–1 Water [225]
2,4,5-TCP ELISA 0.053 mg L–1 –1
0.23 mg L Buffer [226]
ELISA 0.07 mg L–1 0.32 mg L–1 Water [227]

0.26 mg L–1 1.28 mg L–1 Urine [227]
0.8 mg L–1 6.46 mg L–1 Serum [227]
2,4-DCP ELISA 2 mg L–1 Buffer [148]
159
Two liposome-based immunomigration techniques have been developed

for PCBs based on a test-strip format for on-site analysis (see Fig. 8). The li-
posome immunocompetition (LIC) assay format measures the competitive
reaction between analyte-tagged liposomes and the sample analyte for im-
mobilized antibodies and can detect 0.4 nmol of PCB in less than 8 min. The
liposome immunoaggregation (LIA) assay is based on the principle of im-
munoaggregation between anti-PCB antibodies and analyte-tagged lipo-
somes, and detects the inhibition of immunospecific liposome aggregation in
solution produced by the presence of the analyte. This last assay can detect
2.6 pmol of PCB in less than 23 min. Both formats utilize capillary action to
transport liposome-containing solutions along strips of nitrocellulose. Mea-
surement of color intensity is then carried out visually or with a desktop scan-
ner [209, 234].
A continuous semiautomated FIIA system has been reported [208, 235]. In
this device the analyte-containing medium is allowed to flow through a column
containing the antibodies immobilized on a support. First, the antibodies are
saturated with a fluorescent dye-labeled analog of the analyte. As the analyte
passes through the immunosorbent, some dye-labeled antigen is displaced and
is then detected in a fluorometer located downstream from the column. The
LOD achieved for the developed system is 1 mg L–1.
Several ELISA formats have been developed for PCB determination. Thus,
MAbs have been developed for coplanar PCBs [210], which are the most toxic
congeners. The ELISA developed is highly selective for PCB 77 and 126, show-
ing IC50 values of 0.9 and 1.2 mg L–1, respectively. Noncoplanar PCBs, PCDDs,
PCDFs, or single-ring halogenated compounds, including chlorinated benzenes
and phenols, do not interfere with this assay. Johnson et al. [201] also produced
PAbs for PCBs and an indirect ELISA has been optimized for the detection of
several Aroclors, giving LODs of about 1.5 mg L–1 that correspond to 9 ng g–1 in
soils and a dynamic range between 50 and 1,333 ng g–1. Potential contaminants
usually found in soil samples such as chlorophenols and chlorobenzenes were
also tested, exhibiting cross-reactivities lower than 3%. A competitive enzyme
immunoassay for the quantification of PCBs in water has been developed us-
ing PAbs covalently attached to amine-terminated superparamagnetic particles
as solid support [205]. The assay detected various Aroclors (1016, 1232, 1242,
1248, 1254, 1260, 1262, and 1268) with detection limits of 0.2 mg L–1 in water and
500 mg kg–1 in soil using Aroclor 1254 as standard. The assay was validated
using the GC-EPA method 8080 in water, demonstrating good performance
and excellent precision and accuracy. This assay is available as a commercial
kit to be applied to different matrices such as soil, wipes, and water. Kim et al.
[206] have used commercial MAbs for development of an immunoassay for
the determination of PCBs in insulating oils. A dynamic range between 30 and
1,000 mg L–1 has been achieved for the assay in methanol and allows analysis of
diluted oils containing >35 mg mL–1 PCBs (neat). The procedure requires a pre-
vious pretreatment of oil samples (either a solid-phase extraction or washing
with KOH-EtOH/sulfuric acid to remove interference).
There are some other firms that also commercialize immunoassay kits for
PCBs based on different formats (see Table 3). They have been mainly validated
for soil and wipe matrices, although the manufacturer can provide optional
protocols for testing sediment or water samples.
A fiber optic immunosensor (FOI) has also been reported for detection of
PCBs in Aroclors [204]. The quartz fiber surface is coated with PAbs against
PCBs and the competitive assay takes place using as fluorescent tracer, an ana-
log of the analyte coupled to 2,4,5-trichlorophenoxybutyrate (TCPB) on the
Ab-coated fiber. The LOD achieved is around 10 mg L–1.
A high-performance immunoaffinity chromatographic (HPIAC) method
has been developed with the aim of improving the analytical methodology of
PCBs [236, 237]. The IAC column prepared using PAbs generated against the
coplanar toxic PCB congeners reduces the cleanup steps, time of analysis, and
the costs because of the ability to selectively retain PCBs. Moreover, this sam-
ple treatment method reduces the use of hazardous organic solvents.
4.2
PCDDs and PCDFs
Several attempts have been made to set up immunochemical techniques for

dioxin analysis (reviewed in [230, 238, 239]). Frequently the detectability and
selectivity accomplished have not been considered appropriate for the direct
analysis of environmental samples. We should notice that due to the poor
solubility of PCDDs and PCDFs in water, the levels of these contaminants in
aqueous samples is very low. For this reason analysts usually prefer the use of
chromatographic and spectrometric methods that perform using organic sol-
vents. However, the speed and high sample throughput that can be accom-
plished with the immunochemical methods have prompted several research
groups and companies to establish immunochemical methods.
The first IA for dioxins was a RIA developed by Albro et al. [240]. It was quite
time-consuming and utilized PAbs showing low specificity. MAbs were devel-
oped later by Kennel et al. [241], but they also lacked sufficient detectability
to analyze dioxins in solution. The first ELISA was developed by Stanker et al.
using MAbs (DD3) generated against 2,3,7,8-TCCD [211, 242]. This congener
is normally used as analyte because of its recognized higher potential toxicity.
The selectivity of the ELISA was very similar to that of the RIA. The optimized
assay had an IC50 around 200 pg per well [212]. Langley et al. [213] also reported
a PAb-based ELISA with an IC50 value of 1 ng of TCDD per well. Several years
later Harrison and Carlson [214] developed a tube test and a microplate test
using the DD3 MAb and the two formats displayed detection limits of 100 pg
per tube and 25 pg per well. Sanborn et al. [218] reported the production of
PAbs using haptens containing an unsaturation in the spacer arm (between the
halogenated dibenzo-p-dioxin ring system and the protein to which it is con-
jugated), which provides a rigid handle structure. The chemical structure of the
hapten was similar to that of TCDD (i.e., 2,3,7,8- or 1,2,3,7,8-) but the polar
groups for hydrogen bonding were lacking. In the haptens synthesized, one
substituent is an alkyl chain (between 3 and 5 carbon atoms) containing at
least one double bond or aromatic ring with a carboxylic group at the end of
the molecule. An ELISA was developed that showed an IC50 of 0.8 ng per well
(16 ng mL–1) when 2,3,7-trichloro-8-methyldibenzo-p-dioxin (TMDD) was
used as analytical surrogate standard in order to avoid using the toxic con-
gener. It was proved that this analyte responded similarly to 2,3,7,8-TCDD.
The same research group was able to improve the assay by introducing several
modifications, such as the chemical structures of the competitor haptens
used as coating (i.e., trans-3-(7,8-dichlorodibenzo-p-dioxin-2-yl)-cis-2-methyl-
propenoic acid coupled to BSA) or the content of DMSO in the assay (up to
37%). The ELISA exhibited an IC50 value of 12 pg per well (240 pg mL–1), with
a working range from 2 to 240 pg per well (40 to 4,800 pg mL–1) [216]. Subse-
quently, the use of a new coating antigen for the indirect assay was optimized
to finally reach a LOD of 4 ng L–1 [217]. Correlation with GC–MS was carried
out, achieving good agreements for soil samples without the necessity for any
additional cleanup step prior to the analysis.
Intensive work in this field has continued in order to improve detectability
and to establish reliable immunochemical protocols, including appropriate
sample treatment methods, for the analysis of PCDDs and PCDFs in real sam-
ples [214, 238, 243–246]. The development of selective extraction procedures as
well as solvent exchange methods to finally achieve immunoassay-compatible
extracts with solvents has been critical. Thus, an immunoaffinity extraction/
cleanup protocol was developed by Harrison et al. [243] and then used on an
assay employing an improved MAb (DF1). Handling of standards and samples
has also been simplified by the use of detergent keeper in the solvent exchange
procedure. Based on an assumption of quantitative recovery, a tenfold concen-
tration of the original sample was accomplished before the immunochemical
measurement. The final analytical method (sample treatment plus immuno-
chemical method) allowed improvement of the sensitivity up to 100 pg L–1,
starting from 2 L of water [238, 244, 246].
Also an attempt has been made to clone and express recombinant Fab anti-
bodies against dioxins [76] using two hybridoma cell lines (DD1 And DD3)
[211]. The option of cloning Fab fragments rather than scFv was chosen because
they are usually more stable, which is important when environmental analysis
of complex matrices is going to be carried out. Using 2,3,7,8-TCDD as standard
an indirect ELISA has been developed. As in the aforementioned work, con-
siderations regarding the use of organic solvent (MeOH) have been taken into
account and an assay with IC50 values around 10.4–14 ng mL–1 was achieved de-
pending on the Fab used. These sensitivities and the results of cross-reactivity
studies are very similar to those obtained with the respective MAbs, showing
their usefulness as analytical reagents.
Several of these assays have become commercially available (see Table 3).
The IA kit in a coated tube method [247], developed by Cape Technologies, has
been made available for the analysis of various types of sample extracts (fly ash,
soil, stack gas, tissue, sediment, water, etc.) after the application of conventional
extraction methods, followed by a solvent exchange step to provide hydro-al-
coholic (methanol–water) extracts compatible with the immunoassay methods.
Also the production of specific Abs for PCDDs/PCDFs has been directed
toward the development of immunoaffinity procedures [248, 249]. Shelver et al.
reported several works regarding the use of IAC to selectively extract and ana-
lyze these compounds from complex matrices such as milk or serum [250–253].
Moreover, a separation of very similar dioxin congeners (i.e., 1,3,7,8-TCDD and
2,3,7,8-TCDD) was also examined [254].
It is also worth mentioning that some authors have tried to demonstrate a
correlation between congener immunochemical recognition and the I-TEF
(toxicity equivalent factor, relative toxicity related to that of 2,3,7,8-TCDD),
which indicates the potential for predicting the I-TEQ (toxic equivalent quo-
tient, total toxicity of a mixture attending to the individual TEF values) [214,
215, 243, 247, 255]. This would provide a method for estimating the I-TEQ value
of a sample by multiplying each mass concentration obtained by GC–MS by the
corresponding immunoassay cross-reactivity value [245]. Harrison and Carl-
son [247] used their immunoassay to correlate PCDD/F congener recognition
profiles with the congener toxicity in order to estimate the TEQ of real samples
[243, 245].
4.3
Chlorophenols
Among all chlorophenols, 2,4,6-trichlorophenol (TCP) and pentachlorophenol

(PCP) are listed as priority pollutants by the US Environmental Protection
Agency (EPA) (IRIS electronic database) and the EU [256]. In particular, PCP
has been classified as a B2 probable carcinogen for humans from animal toxi-
city studies and human clinical data.
There have been various attempts to develop immunoassays for chlorophe-
nols in environmental samples (soil, water). Noguera et al. [219] produced PAbs
for PCP. By using pentachlorophenoxypropionic acid as immunizing hapten
(so that the five chlorine atoms are kept intact in the molecule) a direct ELISA
was developed with a LOD in water of 0.1 mg L–1 and a working range between
0.3 and 30.5 mg L–1. The immunoassay is very specific for PCP since only one
of the compounds tested (2,3,5,6-tetrachlorophenol) shows a significant degree
of cross-reactivity (21.3%). Other CPs and related phenols are not recognized
(CR<0.3%). The assay is applicable to real environmental water with a minimal
sample pretreatment for river and lake samples.
A competitive heterogeneous immunoassay based on the use of magnetic
particles as solid support has also been developed using commercial PAbs for
PCP [220]. Good sensitivities have been achieved with this format (the LOD is
about 60 mg L–1) and it shows low cross-reactivity with related compounds
(only for tetrachlorophenols, TtCPs, is the degree of recognition significant
with CR of 54% for 2,3,5,6-TtCP and 15% for 2,3,4,6-TtCP). The assay allows the
analysis of water samples (e.g., river water, pond water, or groundwater) with-
out sample pretreatment as well as quantification of soil samples including a
simple extraction procedure. It provides results in 60 min and can be easily
adapted to on-site monitoring. The IA has also been compared with GC–MS
and HPLC, obtaining in both cases good correlation levels for spiked water and
soil samples.
Many studies have been carried out in recent years in order to detect PCP in
environmental samples, and most of them are based on the use of commercially
available IAs for PCP in water and urine (shown in Table 3). Thus, several
assays such as PENTA-RISc, PCP RaPID-Assay, or another IA developed by
Westinghouse Bio-Analytic Systems (WBAS) have been evaluated for on-site
screening tests of PCP in soils or aqueous matrices such as surface, drinking, or
ground water [221, 222, 257]. PCP RaPID-Assay was evaluated using certified
wastewater samples, soil samples, and certified reference materials [258, 259].A
critical comparison of the ELISA method with an online liquid–solid extraction
(LSE) method followed by liquid chromatography (LC-UV or LC-MS) analysis
was performed.A good correlation was found between both methods, although
for some samples undesirable matrix effects were also observed.
PAbs have been developed against 2,4,6- and 2,4,5-TCP after careful study of
the chemical structure of these substance using computer-assisted molecular
modeling tools and theoretical calculations [223, 226]. For 2,4,6-TCP both
direct [223] and indirect [198, 199] ELISA formats have been developed. The
direct assay uses a homologous hapten (same chemical structure as the im-
munizing hapten) coupled to horseradish peroxidase as tracer. The microtiter
plate ELISA can be carried out in about 1 h and it has a LOD of 0.2±0.06 mg L–1.
The assay tolerates samples having a wide range of ionic strengths (from 4 to
56 mS cm–1) and pH values (between 5.5 and 9.5). The indirect ELISA format
has a similar detectability but it has proven to be more robust to matrix inter-
ferences. This ELISA uses a heterologous hapten coupled to BSA as coating
antigen and it can be performed in 1.5 h. As with other microtiter plate ELISA
methods, the advantage is that many samples can be processed simultaneously.
Both direct and indirect assays show a similar pattern of selectivity. Thus, the
indirect ELISA [198] recognizes to a much lesser extent other chlorinated phe-
nols, such as 2,3,4,6-tetrachlorophenol (2,3,4,6-TtCP, 21%), 2,4,5-TCP (12%),
recognized than the corresponding chlorinated analogs (ex. 2,4,6-TBP, 710%;
2,4-dibromophenol, 119%). The indirect ELISA formats have been evaluated for
the analysis of several types of water samples [199] and also for urine samples
[198] in order to perform human exposure assessment studies.
Using the same PAbs an optical biosensor system has been developed for
2,4,6-TCP [224]. The principle is the detection of laser-induced fluorescence
(LIF) in single microdroplets by a homogeneous quenching fluorescence im-
munoassay (QFIA). The competitive immunoassay occurs in microdroplets
(d=58.4 mm) produced by a piezoelectric generator system. A continuous Ar
ion laser (488 nm) excites the fluorescent tracer and its fluorescence is detected
by a spectrometer attached to a cooled, charge-coupled device (CCD) camera
Fig. 9 Scheme of the instrumental setup. Microdroplets are generated by a vibrating orifice
aerosol generator (orifice diameter=10 mm) consisting of a syringe pump and a piezoceramic
oscillator. Droplets are illuminated by a laser beam (continuous Ar ion laser, l=488 nm) that
is focused to a laser beam waist of ~200 mm at the trajectory of the droplet stream. The beam
diameter is chosen to be larger than the droplet diameter to ensure that all fluorophores in
the sample are illuminated. A holographic laser band-pass filter eliminates undesirable
plasma lines from the laser source and transmits only the laser line at 488 nm. The fluo-
rescence is collected by a microscope objective lens (N.A. of 0.55) and focused onto the
entrance slit of the imaging spectrometer. Spectra are recorded with a thermoelectrically
cooled camera with a 512¥512 pixel charge coupled device (CCD) detector. A 488-nm holo-
graphic Raman notch filter placed in front of the slit of the spectrometer blocks elastically
scattered laser radiation. Published with permission of ACS Copyright Office [224]
(see Fig. 9). The fluorescence is quenched by specific binding of the TCP PAbs
to the fluorescent tracer (homologous hapten covalently coupled to fluorescein).
The quenching effect is diminished by the presence of the analyte. Therefore an
increase in the signal is produced in a dose-dependent manner when TCP is
present in the sample. The LIF-microdroplet-QFIA method shows a LOD of
0.04 mg L–1 in buffer. The QFIA performed in microtiter plate format using the
same immunoreagents achieved a LOD of 0.36 mg L–1 in buffer. With the LIF-
microdroplet-QFIA system, urine samples can be directly analyzed just after
buffer dilution reaching a LOD of 1.6 mg L–1, which is sufficient detectability for
occupational exposure risk assessment.
PAbs against 2,4,5-TCP have been prepared after theoretical and molecular
modeling chemical studies [226]. Competitive direct and indirect ELISAs have
been developed, but as before the latter format was shown to be more robust.
The indirect immunoassay has an excellent LOD near 0.05 mg L–1. The selec-
tivity of the assay is high in relation to other chlorophenols frequently present
in real samples, but as with the 2,4,6-TCP assay the brominated analogs may
also be recognized. The 2,4,5-TCP immunoassay is stable in media with pH
values ranging from 6.6 to 10.5 and ionic strength values varying within 20 and
80 mS cm–1. It shows a good accuracy and the coefficients of variation within
and between assays are around 12% or lower. The assay has been evaluated for
the analysis of water samples and complex biological matrices, such as serum
and urine. While the water samples could be analyzed without any sample pre-
treatment, the serum and urine samples produced important interferences.
The investigation of simple sample treatment procedures compatible with the
immunochemical method allowed the establishment of reliable analytical pro-
tocols for straightforward immunochemical determination of 2,4,5-TCP in
natural waters, urine, and serum reaching LODs of 0.07, 0.26, and 0.8 mg L–1,
respectively [227].
Whereas the immunoassays for PCP and TCP show a great level of specificity
for these analytes, Noguera et al. [225] have recently developed an ELISA for
screening the total concentration of chlorophenols in environmental samples.
Using 3,5-dichloro-4-hydroxyphenyl propionic acid as immunizing hapten (the
structure contains two chlorine atoms in the ortho position to the phenol group,
i.e., the pattern of 2,6-dichlorophenol), PAbs were obtained and two immuno-
assays developed (in both direct and indirect formats). The most sensitive one
was the direct format and after the optimization of several parameters such
as surfactant concentration, ionic strength, or time of incubation a LOD of
0.2 mg L–1, an IC50 value of 7.8 mg L–1, and a dynamic range between 0.7 and
86.4 mg L–1 were obtained when 2,4,6-TCP was used as standard. The cross-re-
activity studies carried out show high levels of recognition of other CPs. Thus,
the most recognized one was 2,6-dichlorophenol (2,6-DCP; CR=66.7%) since
the pattern of substitution is the same as that in the immunizing hapten,
2,3,5,6-TtCP (CR=34%) and PCP (CR=33%). 2,4,5-TCP, 2,5-DCP, and 2,4-DCP
were recognized to a much lesser extent (<7%) and chlorophenols with just one
or no chlorine in the molecule or other related compounds without the phenol
group didn’t cross-react (CR<0.09%). Owing to the broad degree of recognition
of this assay it has been tested as a potential screening tool in order to estimate
contamination by chlorophenols. Several preliminary studies have been carried
out with spiked water samples by the addition of different amounts of CPs with
CR levels higher than 1%, and the results of the assay, expressed as equivalents
of 2,4,6-TCP, showed good correlation levels when a regression analysis was
performed, with recoveries around 95%.
Goda et al. reported the only attempt carried out regarding the production
of MAbs against CPs [148]. By using a similar hapten to the aforementioned
one (in this case the spacer arm has six carbon atoms instead of three, but the
pattern of 2,6-dichlorophenol is also retained), a direct ELISA using 2,4-DCP as
standard was developed. A LOD of 2 mg L–1 and a working range between 2 and
60 mg L–1 were achieved. This assay also shows a high degree of recognition of
several chlorophenols tested but, unlike the direct assay of Noguera et al [225],
the pattern of recognition is different. Thus, high or moderate cross-reactivity
values were observed for some mono- or dichlorophenols (e.g., 4-chlorophenol
showed a CR=100%), and even for phenol (24%), whereas for more substituted
CPs the recognition was lower (i.e., 2,4,6-TCP, 13%; 2,3,6-TCP, 3%; 2,3,4,6-TtCP,
9%; and PCP only 0.3%). Thus, this assay can be useful for determination of the
overall concentration of CPs instead of the single compound 2,4-DCP, since an
overestimation can be observed in the analysis.
5
Other Industrial Residues
From the wide variety of emerging pollutants of industrial origin that could be
considered here, bisphenol A (BPA) and phthalate esters (PE) are of especial
relevance not only because of the high volumes produced and their widespread
use, but also because of their demonstrated toxicity, particularly as endocrine
disrupters. Both of them have been included in the final report of the European
Commission toward the establishment of a priority list of endocrine disrupter
chemicals, EDCs [3], and have been rated as of high risk of exposure for human
and wildlife populations. Because of their structural characteristics these com-
pounds cannot be included in any of the groups described above, so they will
be described in this section (see Fig. 10).
5.1
Bisphenol A
Bisphenol A (2,2-bis(4-hydroxydiphenyl)propane, BPA) is a man-made chem-

ical mainly used in the manufacture of polycarbonate and epoxy resins. These
plastics are used in the preparation of containers such as food and drink pack-
aging as well as a great variety of products including compact discs, optical
lenses, thermal paper, adhesives, powder paints, or even in dental composite
fillings and sealants. To a minor extent (about 10%), BPA is used in PVC pro-
duction, as an antioxidant and preservative, or as a flame retardant (i.e., tetra-
bromobisphenol A, see above). This wide range of applications has led to high
levels of worldwide production (around 2.6¥106 tonnes in 1999). Besides the
discharge into the environment from their output, another important source of
Fig. 10 Chemical structure of phthalate esters and bisphenol A. DMP: dimethyl phthalate;
DEP: diethyl phthalate; DBP: di-n-butyl phthalate; DOP: dioctyl phthalate; DEHP: diethyl-
hexyl phthalate; BBP: butylbenzyl phthalate
release and human and wildlife exposure comes from leaching from these
products as a result of incomplete polymerization or hydrolysis of the poly-
mers. Therefore BPA has become not only an environmental pollutant but also
a food contaminant. Due to both its industrial and domestic applications it can
be expected to be found in sewage, influent and effluent wastewater, and sewage
sludge. Their discharge into the environment from industrial emitters and
communal wastewater has been monitored [67], with detection of BPA at
concentrations between 35 and 50 mg L–1 in several wastewater samples (i.e.,
hospitals, household areas, and food, chemical, and paper industries). It was
found that almost 90% of the total load was removed, with an effluent mean
concentration of about 1.5 mg L–1. The levels in surface waters are usually lower
[9]. The acute toxicity levels for BPA have been measured for several organisms
such as algae, invertebrates, and fish and range from 1 to 20 mg L–1 [69, 260],
but of most serious concern is their proven estrogenic activity [261, 262].
Several studies have shown that alterations in reproductive organs in female
rats, fish, and mice can be produced [263–267]. BPA is mainly determined us-
ing chromatographic techniques such as GC–MS [141, 142, 168, 268], HPLC-UV
[269], HPLC–MS [142], or HPLC with fluorescence detection [270]. Immuno-
chemical methods can improve monitoring efficiency because of their known
capability to reach low limits of detection and to process many samples.
Specific PAbs and MAbs have been produced against BPA to develop ELISAs
(see Table 6). Due to the evident risk of human exposure to BPA, further ap-
plication of these immunoassays has been carried out to analyze biological
matrices such as serum samples. An indirect ELISA with PAbs was developed
by Zhao et al. [271] with a good LOD of 0.1 mg L–1 and a dynamic range between
1 and 10,000 mg L–1 in water. The PAbs were raised using as immunizing hap-
ten a bisphenolic structure preserving both phenolic groups and replacing one
methyl group by the spacer arm. No important matrix effects were found when
spiked real water samples were analyzed. Other phenolic compounds did not
interfere in this assay. When spiked serum samples were analyzed a dilution
factor of 1:10 was required in order to obtain quantitative recoveries, placing
the LOD still at a good level (around 2 mg L–1). With these antibodies an im-
munoaffinity procedure for the selective extraction of BPA from serum sam-
ples has been developed providing good recovery levels (about 90%) [272].
Ohkuma et al. [273] also prepared PAbs for BPA but in this case one car-
boxyalkyl ether as spacer arm was substituted for one of the phenolic groups.
With these antibodies a direct competitive ELISA was developed, achieving a
LOD of 0.3 mg L–1 and a working range between 0.3 and 100 mg L–1. Accuracy
evaluation was carried out by analyzing spiked human serum samples; recov-
ery values between 82 and 97% were obtained. The interferences caused by the
matrix were negligible. Correlation studies were also done with a conventional
chromatographic technique (GC–MS), obtaining a regression coefficient of
0.990, thus demonstrating the possibility of using this immunochemical
method to directly determine BPA in serum. Phenols and other endocrine
disrupters such as alkylphenols and phthalates were not recognized in this assay.
Table 6 Immunochemical methods developed for the detection and quantification of BPA and phthalate esters

techniquea
LOD IC50
Others Phthalate estersb

DMP (DEP, DBP, BBP TR-FIA 97 ng L–1 Buffer [276]
and DOP)
DBP ELISA 0.2 mg L–1 Buffer [148]
Bisphenol A
–1
ELISA 0.1 mg L Water [271]
2 mg L–1 Serum [271]
ELISA 0.3 mg L–1 Serum [273]
ELISA 0.57 mg L–1 Water [275]
Automated BMP-IA 2.3 ng L–1 Buffer [147]
a
ELISA: enzyme-linked immunosorbent assay; BMP-IA: bacterial magnetic particle-based immunoassay; TR-FIA: time-resolved fluoroimmunoassay.
b
DEP: diethyl phthalate; DBP: di-n-butyl phthalate; BBP: butylbenzyl phthalate; DMP: dimethyl phthalate; DOP: dioctyl phthalate.
169
Other attempts have been made to detect BPA at a low concentration range.
Thus Kodaira et al. [274] analyzed BPA in urine samples with an assay that
showed a working range between 0.5 and 5 mg L–1. The assay was validated by
HPLC. DeMeulenaer et al. [275] developed an indirect competitive ELISA us-
ing PAbs obtained from chicken egg yolk, but the assay achieved an IC50 value
of only 570 mg L–1.
The production and use of monoclonal antibodies against BPA have also
been reported. Nishii et al. [277] developed an ELISA with MAbs selected for
their resistance to organic solvents, achieving a LOD in the order of 1 mg L–1.
Goda et al. [148] also prepared specific MAbs for BPA and developed a direct
ELISA with a LOD of 5 mg L–1 and a dynamic range between 5 and 500 mg L–1.
A high degree of recognition was observed for two bisphenolic compounds,
produced and used to a lesser extent, whereas other related compounds and
BPA metabolites showed no cross-reactivity. Takeda Chemical Industries, Ltd.
have commercialized ELISA kits using these antibodies. The immunoassay
shows a better sensitivity (i.e., the dynamic range is between 0.05 and 10 mg L–1)
and a high specificity toward other related compounds.An immunoassay based
on the immobilization of monoclonal antibodies on the surface of magnetic
particles has been developed [147]. The sensitivities achieved were also very
good but the assay shows a wide dynamic range (between 2.3 ng L–1 and
2.3 mg L–1), indicating a low slope assay as occurred with the assay developed
for APE based on the same principle (see above).
5.2
Phthalate Esters
Phthalate esters are used to manufacture cosmetic products, inks, adhesives,

and solvents, but they are mainly used as additives in PVC production. This
kind of plastic can be found in a wide range of products such as enclosures
for food containers, defoaming agents, soft squeeze toys, and teething rings.
Among the different phthalates that are used for these purposes, diethylhexyl
phthalate (DEHP) represents over 90% of the total phthalate production, near
to 54,000 tonnes per year [278, 279], followed by butylbenzyl phthalate (BBP),
dibutyl phthalate (DBP), and dioctyl phthalate (DOP). Due to this wide range
of applications as well as their high consumption, these compounds can enter
into the environment through different routes, increasing the risk of exposure
of the human population through food and also by inhalation and dermal
contact. For instance, many pipes and bags used in hospitals are made of PVC
and high levels of DEHP in blood have been found in patients treated with he-
modialysis [280–282]. The origin of these levels can be the leaching of phtha-
lates from the tubes or the bags containing blood. These plasticizers are poorly
soluble in water, and therefore it is expected to find them not only in wastewater
but also adsorbed onto sewage sludge and soils. The environmental fate of
phthalates has been extensively reviewed [65] and it can be concluded that their
aerobic degradation occurs rapidly, preventing their accumulation in water and
even in soil. The biodegradation is slower in anaerobic or cold environments,

but several reported experiments indicate that the bioaccumulation of these
compounds is limited by biotransformation.
Regarding their toxicological data, many studies have been done [283–286].
Their effect as testicular toxicants has been proven [287, 288], and great con-
cern has also been focused on their endocrine disrupting effects, which have
been reviewed [289]. Several in vitro studies confirm their estrogenic activity
[161, 290], mainly for BBP and DBP, although it is quite weak compared with
the natural estrogen 17b-estradiol [291]. Other in vivo experiments also show
that DBP and DEHP can produce alterations in male sexual differentiation and
in reproduction [292]. The mechanism of action of these compounds is unclear,
since some studies indicate that they may act as antiestrogens [293].
A high degree of removal efficiency of DEHP in STPs [294] has been found.
Phthalates have been detected in groundwater, rivers, and drinking water [290]
as well as in industrial effluents, sewage sludge, and soils [295]. Their usual
analysis involves the use of both GC and LC coupled to MS detection, although
sometimes a solid-phase extraction is required prior to the analysis [37, 142,
296–298].
To our knowledge, only two attempts have been made to obtain specific
antibodies for phthalate esters (see Table 6), although none of them is capable of
detecting DEHP, the most used phthalate and also the most persistent one
in the environment. The first immunochemical technique is based on the devel-
opment of a time-resolved fluoroimmunoassay (TR-FIA) [276], a heterogeneous
immunoassay that uses europium chelates as labels. Their special fluorescent
properties, such as their longer decay time (over hundreds of milliseconds) com-
pared with other more conventional organic molecules (around nanoseconds),
determine the good detectability of this method. PAbs were produced against an
immunogen derived from dimethyl phthalate (DMP). The assay developed using
these antibodies showed a LOD of 97 ng L–1 and a working range between
97 ng L–1 and 388 mg L–1 for DMP. Other phthalates such as diethyl phthalate
(DEP), DBP, BBP, and DOP were recognized to a similar extent (between 97 and
110%).
MAbs were also produced by Goda et al. [149], and a direct ELISA has been
developed. The LOD accomplished for DBP was 0.2 mg L–1 with a dynamic range
of 0.2–4 mg L–1. DEHP was not recognized (CR<1%). BBP showed a cross-reac-
tivity value of 155% and dipropyl phthalate (DPrP) and dipentyl phthalate
(DPnP) cross-reacted 60 and 51%, respectively.
6
General Summary
It has been widely demonstrated that immunochemical techniques offer a good

alternative to conventional methodologies in many areas due to the high sen-
sitivity and selectivity achieved for the antibodies toward the target analytes.
In clinical and environmental analysis, their use has been broadly spread
because of their sensitivity, specificity, and high sample processing capabilities.
In the case of emerging pollutants of industrial origin, a variety of immuno-
chemical methods and formats have been developed (RIA, ELISA, PFIA, FIIA,
immunosensors, immunoaffinity extraction procedures, etc.). Several surfac-
tants can readily be detected by these methods. However, although attempts have
been made toward the immunochemical determination of NP, under suspicion
because of its potential estrogenic activity, the methodologies reported also rec-
ognize its parent compounds. Immunochemical analytical methods for PCBs
and PCDDs/PCDFs have been reported, showing good detectability levels. Most
of the problems that arise while analyzing these substances are related to their
lack of solubility in the aqueous-based systems of the immunochemical meth-
ods. However, protocols have been developed using organic solvents and water
mixtures. BPA can be efficiently detected with the immunochemical methods
available today, not only in water samples, but also in more complex biological
matrices such as serum, with detection limits in the order of micrograms per
liter or even lower. Regarding phthalates, more efforts must be directed toward
the production of specific antibodies for the main phthalate DEHP, since the im-
munochemical techniques reported up to now do not recognize this congener.
Unfortunately, some of the methods described in this chapter are not yet
being used as regular screening and analytical methods in environmental con-
trol laboratories. A reason for this may lie in the lack of knowledge on the per-
formance of these types of techniques by certain analytical sectors, and also in
the lack of validated protocols for a wide range of sample matrices. Im-
munoassay methods may suffer from undesirable matrix effects that may lead
to false positive or negative results. It is wrong to assume that the selectivity
of the immunochemical method is sufficiently high to overcome nonspecific
interactions of the antibodies with the matrix components. Rigorous evaluation
of the performance of these methods on each sample matrix of interest, and
the consequent establishment of appropriate sample treatment methods, are
required to ensure reliability and to convince control laboratories of the effi-
ciency of these techniques. A close collaboration and interchange of the exper-
tise of analytical chemists and immunochemists are needed to accomplish this
goal and benefit from the advantages of these methods, to assess risk and pro-
tect public health from the adverse effects of these types of pollutants.
Acknowledgements This work has been supported by CICYT (BIO2000-0351-P4-05,

AGL2001-5005-E) and by the EC: nanotechnology and nanosciences, knowledge-based
multifunctional materials, new production processes and devices (contract number NMP-
505485-1).
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DOI 10.1007/b98616
Immunochemical Determination of Pharmaceuticals

and Personal Care Products as Emerging Pollutants
M.-Carmen Estévez · Héctor Font · Mikaela Nichkova · J.-Pablo Salvador ·
Begoña Varela · Francisco Sánchez-Baeza · M.-Pilar Marco (✉)
Department of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona 18–26,
mpmqob@iiqab.csic.es
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
2 Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.1 Penicillins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
2.2 Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
2.3 Tetracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
2.4 Sulfonamides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
2.5 Fluoroquinolones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
2.6 Macrolides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
3 Steroid Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

3.1 Estrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.2 Androgens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
3.3 Gestagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
3.4 Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
4 Other Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

4.1 Analgesics and NSAIDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
4.2 Cytostatic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
5 General Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Abstract A review on immunochemical methods for the analysis of pharmaceuticals is pre-

sented. A broad range of pharmaceutical categories and personal care products may reach
the aquatic environment after excretion through industrial, domestic, and hospital waste-
water. With few exceptions pharmaceuticals for human medicine are not high-production
chemicals and the expected environmental concentrations should be low. However, the use
of some of these chemicals in veterinary medicine increases the probability that the con-
centration values in the aquatic environment might reach higher levels. On the other hand
certain drugs with limited use are of concern because of their high pharmacological potency,
which creates a risk even at trace levels. Attending to these considerations and to the po-
tential human risks, this review focuses on antibiotics, hormones, analgesics, nonsteroidal
anti-inflammatory drugs, and cytostatic agents. Although these procedures have only been
applied to the analysis of environmental samples on a few occasions, immunochemical
methods for several of these substances exist and some of them are commercially available
due to their use in clinical laboratories and forensic medicine.
Keywords Immunochemical techniques · Antibiotics · Steroid hormones · Analgesics ·

Cytostatic agents
Abbreviations
BSA Bovine serum albumin
CAP Chloramphenicol
CE Capillary electrophoresis
COD Codeine
CR Cross-reactivity
E1 Estrone
E2 b-Estradiol
E3 Estriol
EE2 Ethynylestradiol
EIA Enzyme immunoassay
ELIFA Enzyme-linked immunofiltration assay
EMIT Enzyme-multiplied immunoassay technique
ETIA Energy transfer immunoassay
EW Evanescent wave
FPIA Fluorescence polarization immunoassay
HPIAC High-performance immunoaffinity chromatography
IAC Immunoaffinity chromatography
LIF Laser-induced fluorescence
MAb Monoclonal antibody
MECC Micellar electrokinetic capillary chromatography
MIAC Multi-immunoaffinity chromatography
MOR Morphine
MRL Maximum residue level
MTX Methotrexate
NSAID Nonsteroidal anti-inflammatory drug
PAb Polyclonal antibody
PEC Predicted environmental concentration
PFIA Polarization fluoroimmunoassay
PNEC Predicted no effect concentration
PPCPs Pharmaceutical and personal care products
RIA Radioimmunoassay
SPIA Sol particle immunoassay
SPR Surface plasmon resonance
STPs Sewage treatment plants
TIRF Total internal reflection fluorescence immunoassay
WWTPs Wastewater treatment plants
Immunochemical Determination of Pharmaceuticals and Personal Care Products 183
1
Introduction
Pharmaceuticals and personal care products (PPCPs) are a diverse group of

chemicals used internally or externally in the body of humans, domestic ani-
mals, and plants. They include substances such as human and veterinary drugs,
diagnostic agents (i.e., X-ray contrast media), nutraceuticals (bioactive food
supplements), certain feed and food additives, sunscreen agents, fragrances,
cosmetic additives, etc. Their presence in the environment is determined by
their worldwide frequent use by multitudes of individuals or by their use as
veterinary drugs. Thousands of tons of pharmacologically active substances are
used yearly to treat illnesses, to prevent unwanted pregnancy, or to face the
stress of modern life. For example, about 50,000 drugs are registered in Germany
for human use, 2,700 of which are responsible for 90% of the total consumption
and which, in turn, contain about 900 different active substances [1]. In the UK
approximately 3,000 active substances are licensed [2]. In animal farming the
use of antibiotics, feed additives, and hormones has become a usual practice for
certain farmers. Substances such as the UV filters used for sunscreens and cos-
metics, designed to remain on the skin, are generally washed off either during
bathing or swimming, or are transferred to towels or clothes which will be
finally washed. All these are examples of the usual use and release to the envi-
ronment of PPCPs. Several reviews discuss and present real data on the envi-
ronmental occurrence of the most relevant PPCPs [1, 3–7]. We must moreover
remark that their use is continuing and escalating at the same time as new
arrays of more potent chemicals are being introduced onto the market.
Pharmaceuticals are inherently biologically active and remarkably potent
agents. Often they are resistant to biodegradation, as a certain metabolic sta-
bility is necessary for their pharmacological action. PPCPs are released into the
environment unaltered or as still active metabolites. Thus, human and veteri-
nary drugs are frequently excreted as glucuronide or sulfate conjugates that can
easily be hydrolyzed to release again the active parent compound in the envi-
ronment. Certain PPCPs or their metabolites are highly soluble in water. This
parameter combined with a lack of biodegradability limits the removal of
PPCPs in wastewater treatment plants (WWTPs) [4, 8].Additionally, antibiotics
and disinfectants are supposed to disturb the wastewater treatment process and
the microbial ecology in surface waters. Furthermore, resistant bacteria may be
selected in the aeration tanks of STPs (sewage treatment plants) by the antibi-
otic substances present [1]. In these cases the drugs enter the aquatic environ-
ment, contaminating ground and surface waters and eventually also reaching
drinking water. Some other PPCPs are more lipophilic, thus showing a tendency
to bioaccumulate in organisms that fortuitously will be used for human con-
sumption. The consequence is that PPCPs enter the environment resulting in
real exposure of humans and wildlife to these active substances [5, 9–12].
The concern produced within the scientific community, the authorities, and
governmental bodies has prompted the establishment of certain regulations
[13, 14]. Thus, before any new veterinary pharmaceutical product can obtain a
marketing authorization, a review must be carried out by national or European
Union (EU) authorities to ensure its efficacy, quality, and safety to public health
and the environment. The requirements for ecotoxicity testing are regulated by
Directive 2001/82/EC. The European Agency for the Evaluation of Medicinal
Products (EMEA), which coordinates the evaluation and supervision of med-
icinal products for both human and veterinary use, has published guidance
documents on how to perform environmental risk assessment of these products
[15]. The International Cooperation on Harmonization of Technical Require-
ments for Authorization of Veterinary Medicinal Products (VICH) formed by
the EU, USA, and Japan (Australia and New Zealand participate as observers)
are trying to establish uniform risk assessment criteria [16]. The general idea
is to predict the environmental concentration (PEC) of these substances based
on calculations such as number of prescriptions, doses, degradation, environ-
mental models, etc. [12]. The PEC value is then compared to the lowest effec-
tive concentration found, for the parent compound or its metabolites, in certain
ecotoxicity tests performed in soil and/or water to establish what is known as
the predicted no effect concentration (PNEC). The ratio PEC/PNEC should be
lower than 1. Otherwise a risk to the environment is assumed and risk mitiga-
tion measures should be linked to the authorization of the product [17]. The
complete guidance is still being developed, but already some data dealing
with these parameters have been published on the most frequently used phar-
maceuticals (i.e., [18, 19] and personal care products (i.e., [17]). In a study per-
formed in Denmark, taking only pharmaceuticals prescribed for human
medicine into consideration, ibuprofen, acetylsalicylic acid, and paracetamol
exceeded the PEC/PNEC reference value [18]. Similar conclusions were drawn
for paracetamol, amoxicillin, oxytetracycline, and mefenamic acid in a study on
the aquatic environmental assessment of the top 25 English most prescribed
pharmaceuticals [19].
Because of their inherent bioactivity, trace levels of these substances can
have a negative impact on the environment and public health. This fact, to-
gether with the above-mentioned considerations, gives rise to substantial an-
alytical problems. The different formats and benefits that the immunochemi-
cal methods can confer on the analysis of trace contaminants in aqueous-based
samples has been widely demonstrated [47–56] (see also Chap. 5 in this vol-
ume). For this particular case immunochemical techniques offer an additional
benefit derived from the possibility of developing single or class-specific meth-
ods according to necessity. Environmental monitoring of PPCPs requires effi-
cient methodologies able to detect trace levels of contamination caused by both
parent compounds and their metabolites. The present chapter describes some
of the immunochemical methods available today for the analysis of the most
important PPCPs regarding their potential impact on the environment (see
Table 1). The PPCPs treated in this chapter have been selected according to their
production and use. Because of the recent concern about their environmental
impact, the aim of most of the immunochemical techniques reported for ana-
Table 1 Summarized table with the more important PPCPs divided into antibiotics, steroid hormones, and other drugs. Their generic chemical
structures and the use or origin are shown. Some reported data regarding their environmental occurrence and the more probable environmental
fate are also given
Antibiotics
Fluoroquinolones
Ciprofloxacin Human medicine – WW effluents (Switzerland): It’s strongly sorbed onto
249–405 ng L–1 [4] soil.
– Hospital effluents: 3–87 mg L–1 [4, 5] Persistent in the
– High level in hospital WW [4] environment [1]
– US streams: 0.2 mg L–1 [20]
Norfloxacin Human medicine – WW effluents in Switzerland It’s strongly sorbed onto

45–120 ng L–1 in [4] soil.
– high level in hospital WW [4] Persistent in the
– US streams: 0.12 mg L–1 [20] environment [1]
Ofloxacin Human medicine – High levels in hospital WW [4, 5] It’s strongly sorbed onto
soil.
Persistent in the
environment [1]
Immunochemical Determination of Pharmaceuticals and Personal Care Products
DW: drinking water; GW: ground water; SW: surface water; WW: waste water; STP: sewage treatment plant; WWTP: wastewater treatment plant.
185
186
Table 1 (continued)
Sulfonamides
Sulfa- Veterinary medicine – GW: Up to 410 ng L–1 Non-degradable in sewage
methoxazole Aquaculture [4, 21, 22] treatment
Human medicine – STP effluents in Germany:
0.40 mg L–1 [21]
– River waters: 1 mg L–1 [23]
– US streams: 0.15 mg L–1 [20]
Sulfa- Veterinary medicine – US streams: 0.06 mg L–1 [20] Non degradable in sewage
dimethoxine Human medicine – GW of a landfill from waste treatment
pharmaceutical production:
5 mg L–1 (total sulfonamides) [23]
Sulfamethazine/ Veterinary medicine – High concentrations in landfill Non degradable in sewage

Sulfadimidine Aquaculture leaches in Denmark [4, 5] treatment
Human medicine – US streams: 0.22 mg L–1 [20]
– GW in Germany: 10–100 mg L–1
[21]
Sulfathiazole Veterinary medicine – Denmark landfill leaches: Non degradable in sewage
Human medicine 0.04–6.47 mg L–1 [5] treatment
Table 1 (continued)
Penicillins
Penicillin G Veterinary medicine Not detected in the environment Hydrolysis in water of the
Human medicine b-lactam ring
Penicillin V Veterinary medicine – River water: 25 ng L–1 [24] Hydrolysis in water of the
Human medicine – Potable water 10 ng L–1 [24] b-lactam ring
Oxacillin Veterinary medicine Not detected in the environment Hydrolysis in water of the
Ampicillin Veterinary medicine – SW in Germany: 0.26 ng L–1 [25] Hydrolysis in water of the
187
Table 1 (continued)
188
Penicillins
Cloxacillin Veterinary medicine Not detected in the environment Hydrolysis in water of the
Tetracyclines
Oxytetracycline Aquaculture – Detected in molluscs and wild Accumulates in sewage
Veterinary medicine fish in Norway 189–285 mg L–1 in sludges or sediments.
Human medicine sediments in fish farming [26] It forms stable complexes
– River water: 1 mg L–1 [3] with Ca2+.
– US streams: 0.34 mg L–1 [20] Persistent in anoxic
conditions
Tetracycline Aquaculture – River water: 1 mg L–1 [3] Accumulates in sewage
Veterinary medicine – US streams: 0.11 mg L–1 [20] sludges or sediment
Human medicine Degradation rate of
tetracycline in liquid
manure is approximately
50% in 5 months
Photodescomposition
Chlortetracycline Aquaculture – SW in US: 0.42 mg L–1 [20] Accumulates in sewage
Human medicine – River water: 1 mg L–1 [3] sludges or sediment
Growth promoter in After 30 days at 33 °C,
veterinary medicine 44% remained
Table 1 (continued)
Macrolides
Erythromycin Veterinary medicine – GW in Germany: up to Not degradable in the
Human medicine 200 ng L–1 [25] environment [3].
Aquaculture – SW in Germany: 0.15 mg L–1 [23] Labile in acid conditions
– STP effluents in Germany.
2.5 mg L–1 [23]
– River water: 1 mg L–1 [3]
– River Po in Italy: 3.2 ng L–1 [27]
Clarithromycin Human medicine – STP effluents in Germany: More stability than

0.24 mg L–1 [23] erythromycin in acid
– GW in Germany conditions
0.24–0.87 mg L–1 [23]
– River Po in Italy: 1.7 ng L–1 [27]
189
190
Table 1 (continued)
Macrolides
Roxithromycin Human medicine – US streams: 0.05 mg L–1 [20] No data found
Trimethoprim
Veterinary medicine – STP effluents (Germany): Half-life >1 year
Human medicine 0.32 mg L–1 [23]
Mixture with – US streams: 0.15 mg L–1 [20]
sulfonamides
Chloramphenicol
Aquaculture – Sewage and surface level: low Hydrolysis of
Exceptional cases in at mg L–1 [4] chloramphenicol
human medicine – STP in germany: 0.56 mg L–1 [23] glucuronide in
Veterinary use is chloramphenicol
forbidden
Table 1 (continued)
Hormones
Estrogens
Estradiol (E2) Endogenous hormone – Sewage effluent: <0.1–88 ng L–1 Mean Half-life: 2.8 days.
[28, 29] Sorption in the sediments
– SW <0.05–15 ng L–1
Estrone (E1) Metabolite of – Sewage effluent: Mean Half-life: 3.0 days.

estradiol <0.1–220 ng L–1 [28] Main product of
– SW <0.1–17 ng L–1 degradation of estradiol
Estriol (E3) Metabolite of – Sewage effluent: <0.1–42 ng L–1 Average removal

estradiol (different countries) [28] efficiency 96%.
– SW <0.1–3.4 ng L–1 Degradation not reported
Ethynylestradiol Oral contraceptive – Sewage effluent: Mean Half-life: 17 days.

(EE2) <0.053–62 ng L–1 High sorption onto
(different countries) [28, 29] sediments
– SW: <0.053–30.8 ng L–1
191
192
Table 1 (continued)
Estrogens
Mestranol Oral contraceptive – Effluents: <1–8 ng L–1 [30] Degraded in aerobic
(MeEE2) conditions in sludge (80%)
and 7% hydrolyzed to EE2
Androgens
Testosterone (T) Endogenous hormone – Raw sewage in WWTP: 16 to T runs off by leaching of
Growth promoter 700 ng mL–1 aqueous solution from the
Anabolic steroid – GW: 1.0 ng L–1 soil [31]
– Runoff water from manured
fields: 215 ng L–1 [13]
Methyltesto- Growth promoter – Pond Water after treatment with Phelps et al. [32] found
sterone (MT) Anabolic steroid MT food: <5 mg L–1 [32] that MT in the water
returned to the background
levels within one week
after hormone
administration
Trenbolone (Tr) Growth promoter – After 5.5 months in soil Traceable after 8 days of
Anabolic steroid fertilized with liquid manure: fertilization, not detectable
0.16–0.10 mg kg–1 after 40 days [31]
Table 1 (continued)
Gestagens
Progesterone Endogenous hormone – US streams: 0.11 mg L–1 [20] No data found
Norethindrone Oral Contraceptive – River samples (UK) 28% Biodegradation in

17 ng L–1 [33] 6 h after the plant
– Effluents: 8–20 ng L–1 [30] treatment and completely
in 24 h [33]
Corticosteroids
Cortisone Anti-inflammatory No data found Practically insoluble in
agent water [34]
Cortisol Anti-inflammatory No data found Very stable [34]

agent
193
194
Table 1 (continued)
Corticosteroids
Betamethasone Anti-inflammatory No data found Practically insoluble in
agent water.
Growth promoter Very stable [34]
Dexamethasone Anti-inflammatory No data found Practically insoluble in

agent water [34]
Growth promoter
Table 1 (continued)
Others
Analgesics
Paracetamol Mild analgesic – US streams, max: 10 mg L–1 [20] Readily degradable after
(acetomino- Antiphlogistic – STP effluent, max: 6 mg L–1 acclimatization
phen) (Germany) [35]
Aspirin (acetyl- Pain killer – Sewage effluent (England): Readily biodegradable
salicylic acid) Antithrombotic agent 1 ng ml–1 [24]
– STP effluent, max: 1.5 mg L–1
and river and streams water
(Germany): 0.34 mg L–1 [35]
Salicylic acid Metabolite of – Sewage effluents: 13 mg L–1 [36] No data found
acetylsalicylic acid – STP effluent, max: 0.14 mg L–1
and river and streams (Germany):
4.1 mg L–1 [35]
– STP effluent (Berlin):
0.04 mg L–1 [37]
– GW (Berlin) max: 1225 ng L–1 [37]
Gentisic acid Metabolite of – STP effluents: max: 0.59 mg L–1 No data found
acetylsalicylic acid and rivers and streams
(Germany): 1.2 mg L–1 [35]
– GW Berlin max: 540 ng L–1 [37]

195
Table 1 (continued)
196
Analgesics
Ibuprofen Anti-inflammatory – Different German rivers: – Inherently biodegrad-
agent 17–139 ng L–1 [38] able (<95% removed in
Pain killer – STP effluent max: 3.4 mg L–1 and WWTPs) [39]
Antiphlogistic river and streams water
Anti-rheumatic (Germany): 0.53 mg L–1 [35]
– STP effluent (Berlin):
0.1 mg L–1 [37]
– GW (Berlin): max:
200 ng L–1 [37]
– STP influents up to 3 mg L–1 and
STP effluent: 2 ng L–1, rivers
and lakes (Germany): up to
8 ng L–1 [39]
– Italian rivers:
90.6–92.4 ng L–1 [40]
– Sewage effluent: 1.5, 0.87 and
85 mg L–1; SW: 2.7 mg L–1 [36]
Diclofenac Antiphlogistic – Rivers: 15–500 ng mL–1 [38] Readily or inherently
– Effluent: up to 2 ng mL–1 [35, 38] biodegradable; photolytic
– STP influents: 12–560 ng L–1; degradation [4, 42]
STP effluents (Greece):
10–365 ng L–1 [41]
– River Aabach (Switzerland):
11–310 ng L–1 [42]
– Lakes (Switzerland):
<1–12 ng L–1 [42]
– WWTP effluent (Switzerland):
0.99 mg L–1 [43]

Table 1 (continued)
Analgesics
Codeine Analgesic – US streams max: 0.019 mg L–1 Not data found
median value: 0.012 mg L–1 [20]
Cytostatic (antineoplastic) agents

Methotrexate Cancer therapy – River and potable water: Persistent
treatment <6.25 ng L–1 [33]
(chemotherapy)
Cyclophos- Cancer therapy – Treated hospital effluent from Not degradable

phamide treatment STP: 146 ng L–1 [44, 45]
(chemotherapy) – STP effluent (Germany) max:
20 ng L–1 [35]
Ifosfamide Cancer therapy – Treated hospital effluent: No data found

treatment 24 ng L–1 [44]
(chemotherapy) – Oncologic hospital effluent:
mean: 109 ng L–1 [46]

– STP effluent (Germany) max:
2.9 mg L–1 [35]
197
lyzing PPCPs has been the analysis of tissues or body fluids. In this case the
detectability should be in accordance with the MRL (maximum residue level)
established in the legislation (EC Regulation 2377/90). However, considering the
complexity of the biological samples, their application to environmental water
samples should be straightforward. The use of those formats using radioactive
labels, initially developed for biochemical studies, should be avoided whenever
possible due to the problems derived from handling and producing radioactive
waste.
2
Antibiotics
Antibiotics are chemical substances that are able to suppress or kill the growth
of bacteria. They are extensively used in human and veterinary medicine as well
as in aquaculture. They are administered in veterinary medicine for the treat-
ment and control of infectious diseases such as mastitis, enteritis, peritonitis,
and pneumonia. Moreover, certain antibiotic substances have also been used as
growth promoters in food producing animals [57, 58].Antibiotic therapy began
with the clinical use of sulfonamides in 1936 and was followed by the develop-
ment of penicillins (1944), chloramphenicol (1947), tetracyclines (1948), and
fluoroquinolones (1980) (see Fig. 1 for the chemical structures). Since 1950, and
in parallel with the use of antibiotics in human medicine, veterinary use has
provided control of diseases in animal farms. This was followed by their use as
growth promoters in many countries, although at present this practice is be-
coming increasingly controversial.
In Europe about 10,000 tons of antibiotics are consumed each year (FEDESA,
the European Animal Health Association 1998) [59] (see Table 2).According to
these data, 5,000 tons are due to veterinary purposes (3,500 tons prophylaxis
and therapy, and growth promotion about 1,500 tons). The other half of pro-
duction is used in medicine.Among the antibiotics used in veterinary practice,
Table 2 Veterinary consumption of therapeutic antibiotics in Europe
Product group % Share
Penicillins 9
Tetracyclines 66
Macrolides 12
Aminoglycosides 4
Fluoroquinolones 1
Trimethoprim/sulfonamides 2
Others 6
Totala 100
a
Total consumption: 2,494 tonnes of active ingredient at 100% purity.
Fig. 1 Chemical structures of some of the most important antibiotics used nowadays divided
into the most representative families: fluoroquinolones, sulfonamides, penicillins, macrolides,
and tetracyclines. Another important antibiotic, chloramphenicol, is also shown
penicillins and tetracyclines (also applied in aquaculture) and macrolides

are the most frequently administered, whereas in humans fluoroquinolones,
macrolides, and aminoglycosides are the most frequently used. The amount of
antimicrobial agents used in food animals (cattle, chickens, pigs, and turkeys) in
the United States is unknown.At least 17 classes of antimicrobial agents are ap-
proved for growth promotion and feed efficiency in the United States, including
tetracyclines, penicillins, macrolides, lincomycin (analog of clindamycin), and
virginiamycin (analog of quinupristin/dalfopristin). To understand the human
health consequences of the use of antimicrobial agents in food animals, it is
important to evaluate the quantity of antimicrobial agents used in food animals
in the USA. Unfortunately, although reporting systems have recently been im-
plemented in several European countries, no reporting system exists for the
quantity of antimicrobial agents used in food animals in the United States. The
Animal Health Institute, which reportedly represents 80% of the companies
that produce antimicrobial agents for animals in the USA, has estimated that
their member companies produced 18 million pounds of antimicrobial agents
for therapeutic and nontherapeutic (growth promotion and disease prevention)
use in food animals in the USA in 1999 [60]. An alternative report, provided by
the Union of Concerned Scientists in 2001, estimated that 29 million pounds of
antimicrobial agents are used in food animals annually in the USA, of which
25 million pounds are used for nontherapeutic purposes [61]. Though more
precise data on the quantity of antimicrobial agents used in food animals are
needed, these initial estimates provide some perspective on the quantity of an-
timicrobial agents used in food animals in the USA.
The most important impact of the misuse of antibiotics is related to the
development of resistance mechanisms. Antimicrobial resistance may be
viewed as the ability of microorganisms of a certain species to survive or even
to grow in the presence of a concentration of an antimicrobial that is usually
sufficient to inhibit or kill bacteria of the same species. In the presence of an
antimicrobial, organisms with inherent or acquired resistance to the agent will
be selected. The bacterial population then comes to consist largely or entirely
of resistant bacteria, causing failure of the traditional treatments. It has been
reported that more than 70% of bacteria are insensitive against at least one
antibiotic. This situation is causing a serious threat for public health, as more
and more infections can no longer be treated with the presently known anti-
dotes [62–68].
The World Health Organization has recommended that, unless a risk-based
evaluation demonstrates their safety, the growth promotion use in food animals
of antimicrobial agents that belong to the same classes of antimicrobial agents
used in humans should be terminated [69]. Similar recommendations to dis-
continue the use of human antimicrobial agents as growth promoters in food
animals have been made by several independent organizations in the United
States, including the Alliance of Prudent Use of Antibiotics in 2002 [70] and the
distinguished Institute of Medicine of the National Academies in 2003 [71]. For
this reason, the EU has established the principle of using different antibiotics for
humans and animals. Since 1999 the EU has also banned some antibiotics such
as tylosin, spiramycin, virginiamycin, and bacitracin, used as growth promoters
(Council Regulation (EC) No. 2821/98), due to their structural relatedness to
antimicrobial agents used in human medicine.
After administration in humans or animals, these substances pass to the
environment, mainly to the aqueous compartment resulting in some high local
concentrations (e.g., aquaculture, hospital effluents). Several studies have been
carried out in the USA, Germany, Switzerland, and Denmark to investigate the
occurrence and fate of the antibacterial drugs in STPs or surface waters (see
Table 1). Antibiotic resistance causes an important impact on the ecosystem,
water, and soil-dwelling organisms. Moreover, some antibiotics can also pro-
duce adverse effects in animals and plants. For example, sulfadimethoxine and
bacitracin produce loss of weight in roots and leaves in some plants, and oxyte-
tracycline and tetracycline can kill pinto bean plants at a concentration level of
160 mg L–1 [3]. Chloramphenicol can produce pneumonia and sulfamethazine
has been evaluated by the WHO/FAO Expert Committee as a suspected car-
cinogen.
Macrolide antibiotics (clarithromycin, dehydroerythromycin, etc.) and sul-
fonamides (sulfamethoxazole, sulfadimethoxine, sulfamethazine, and sulfathi-
azole) are the most prevalent antibiotics found in the environment with levels
around a few micrograms per liter, whereas fluoroquinolones, tetracyclines, and
penicillins have been detected in fewer cases and usually at low concentrations
(nanograms per liter) [3, 20, 23, 72]. This result is not surprising, since penicillins
are easily hydrolyzed and tetracyclines readily precipitate with cations such as
calcium and are accumulated in sewage sludge or sediments. Several reviews
have reported the environmental occurrence of different antibiotics in aquatic
and soil compartments. Some of these data are detailed in Table 1.
Various techniques based on completely different principles have been used
to detect antibiotic residues. Traditionally, most of the tests used take advantage
of the antibacterial activity of the antibiotics. These growth inhibition tests have
been used in different animal matrices; however, they detect all residue levels
of any antibiotic above the MRL and no conclusion may be drawn about the
identity of the antibiotic or its concentration [73]. On the other hand, HPLC and
GC are highly specific but require extensive sample preparation, sophisticated
equipment, and skilled laboratory personnel. Therefore they cannot be used
for routine screening of a large number of samples [21, 23, 72, 74]. Immuno-
chemical techniques can be excellent tools to assess contamination of the en-
vironment by antibiotics in different matrices due to their high detectability
and specificity. Furthermore, immunoassays are excellent tools for screening
large numbers of samples in short time periods. Table 3 summarizes some of
the immunochemical techniques reported for the detection of several families
of antibiotics.As mentioned in the introduction, usually these techniques have
been developed to determine antibiotic levels in biological matrices, however
their availability opens the door to further applications in analyzing environ-
mental samples.
Table 3 Some immunochemical techniques developed for the detection of antibiotics
202

techniquea
LOD IC50
Fluoroquinolones
Ciprofloxacin ELISA 10 pg mL–1 c Serum [75]
Sarafloxacin ELISA 7.3 mg L–1 Buffer [76]
Norfloxacin ELISA 4.0 g kg–1 Bovine milk [77]
Ovine kidney
Sulfonamides
Sulfathiazole ELISA 1 ng mL–1 6 ng mL–1 Swine liver [78]
ELISA 35.5 mg L–1 Milk [79]
88.0 mg L–1 Honey [79]
Sulfamethazine Biacore Q 10 ng mL–1 Serum [81]
(optical biosensor)
Biacore Q 7.4 ng g–1 Muscle [82]
(optical biosensor)
Biacore 1000 0.023 mg mL–1 0.041 mg mL–1 Biles [83]
RIA 5 mg L–1 Water [84]
IAC–ELISA 0.17 ng mL–1 1.39 ng mL–1 Urine [85]
ELISA 10 ng g–1 Tissues [86]
Biacore 1000 system 0.015 mg mL–1 Porcine bile [87]
a EIA: enzyme immunoassay; ELISA: enzyme-linked immunosorbent assay; ELIFA: enzyme-linked immunofiltration assay; IAC: immunoaffinity
chromatography; SPIA: sol particle immunoassay; SPFIA: solid-phase fluorescence immunoassay; SPR: surface plasmon resonance; RIA: radio-
immunoassay.
Table 3 (continued)

techniquea
LOD IC50
Sulfonamides
Sulfadimethoxine ELISA 1.50 mg L–1 Liver tissue [88]
Sulfadiazine Biacore Q 5.6 ng g–1 Muscle [82]
(optical biosensor)
Biacore 1000 system 0.028 mg mL–1 Porcine bile [87]
Sulfadimidine SPIA 10 ng mL–1 Urine [89]
20 ng mL–1 Milk
Pencillins
Cephalexin Automated flow-through 1 mg mL–1 Raw milk [90]
amperometric IA
Ampicillin Biacore SPR 5.9 mg L–1 48.8 g L–1 Buffer [91]
12.5 mg L–1 71.6 mg L–1 Milk [91]
SPFIA (Parallux kit) 50 mg L–1 Bovine and [92]
porcine kidney
ELISA (LacTek kit) Plasma [93]
qualitative
Penicillin G Biacore SPR 5–7 mg L–1 Milk [91]
Biacore SPR 2.6 mg kg–1 Milk [94]
RIA 2 mg L–1 Water [84]

porcine kidney
Penicillin M Biacore SPR 30 mg L–1 Milk [91]
203
204
Table 3 (continued)

techniquea
LOD IC50
Tetracyclines
Chlortetracycline RIA 1 mg L–1 Water [84]
porcine kidney
Oxytetracycline SPFIA (Parallux kit) 300 mg L–1 Bovine and [92]
porcine kidney
ELISA (TC Microwell 100 mg kg–1 Muscle tissue [95]
test kit)
Tetracyclines SPFIA (Parallux kit) 300 mg L–1 Bovine and [92]
porcine kidney
EIA 20 mg kg–1 Honey [96]
ELISA (qualitative) Pork meat [95]
RIA (Charm II RIA test) 1 mg L–1 Water [97]
ELISA 0.1 ng mL–1 Milk [98]
Macrolides
Macrolides ELISA 0.3 ng mL–1 8 ng mL–1 Buffer [99]
Erythromycin RIA 10 mg L–1 Water [84]
ELISA 0.4 ng mL–1 Bovine muscle [100]
ELISA 0.3 ng mL–1 8 ng mL–1 Buffer [99]
Tylosin ELISA 4 ng mL–1 Bovine muscle [100]
Table 3 (continued)

techniquea
LOD IC50
Chloramphenicol
Chloramphenicol RIA (Charm II assay) 5 ng g–1 Tissue [101]
20 ng mL–1 Urine [101]
EIA (RIDASCREEN) 0.5 ng g–1 Tissue [101]
0.3 ng mL–1 Urine [101]
ELISA 3 ng mL–1 Swine muscle tissue [102]
ELISA 3 ng mL–1 Muscle tissue [102]
ELIFA 0.7 ng mL–1 Milk [103]
Dipstick EIA 17 ng mL–1 Milk [103]
ELISA (Le Carte Test) 2 mg kg–1 Meat [104]
EIA kit (5091CAP1p) 0.1 mg kg–1 Shrimp tissue [105]
205
2.1
Penicillins
Penicillins are one of the most important families of antibiotics used in vet-
erinary and human medicine. But due to their rapid transformation in envi-
ronmental media (easy hydrolysis of the b-lactam ring), their persistence in en-
vironmental samples should be low. Thus, some works aimed at detecting
antibiotic residues in water samples point out the absence of penicillin residues
in spite of this drug being widely used [24, 25].
Several immunochemical methodologies have been developed for the detec-
tion of penicillins in food samples of animal origin [91, 93, 94, 106] (see Table 3).
Most of them are based on the use of the commercial surface plasmon resonance
(SPR) biosensor Biacore.A SPR is an evanescent electromagnetic field generated
at the surface of a metal conductor (usually Ag or Au) when excited by the im-
pact of light of an appropriate wavelength at a particular angle (qp). Surface
plasmons are generated by electrons at the metal surfaces that behave differently
from those in the bulk of the metal. These electrons are excited by the incident
light, producing an oscillation (resonance) of different frequency from that in the
bulk of the metal film. The absorption of light energy by the surface plasmons
during resonance is observed as a sharp minimum in light reflectance when the
varying angle of incidence reaches the critical value. The critical angle depends
not only on the wavelength and polarization state of the incident light, but also
on the dielectric properties of the medium adjacent to the metal surface and
therefore is affected by analytes binding to that surface (see Fig. 2). Thus, when
the immunocomplex is formed or dissociated a shift of the SPR angle is observed.
The Biacore sensor was applied by Gaudin et al. to detect different penicillins
in milk using commercial monoclonal antibodies (MAb) against ampicillin
[91]. These MAbs had a higher affinity for the open b-lactam ring compounds
than for those with the closed ring. Therefore, the analyses had to be performed
by carrying out pretreatment of the samples in order to open the b-lactam ring
Fig. 2 Surface plasmon resonance (SPR) principle. Surface plasmons are excited by the light
energy at a critical angle (q) causing an oscillation and the generation of an evanescent wave.
Under this condition a decrease in the reflected light intensity is observed. The angle q depends
on the dielectric medium close to the metal surface and therefore is strongly affected by mol-
ecules directly adsorbed on the metal surface. This principle allows the direct detection of the
interaction of the analyte and the antibody
of the penicillins and accomplish acceptable limits of detection (for ampicillin:

5.9 mg L–1 in buffer and 12.5 mg L–1 in milk).With these antibodies a high cross-
reactivity (CR) was observed with penicillin G, penicillin V, amoxicillin, and
cloxacillin. Gustavsson et al. [94] tried to improve the procedure of the Biacore
using carboxypeptidase and antibodies against a hydrolyzed peptide generated
by an enzymatic reaction. This method had the advantage of detecting only the
intact b-lactam structure. Penicillins inhibit this enzyme and therefore the
amount of penicillin present in the sample can be measured by a decrease in
the concentration of the hydrolyzed peptide. This method could be applied to
the analysis of penicillins in milk with limits of detection around few micro-
grams per liter.
Moreover, several ELISA (enzyme-linked immunosorbent assay)-type im-
munoassays have also been described for analyzing penicillins in different ma-
trices (see Table 3). Thus, the determination of penicillins in plasma samples
from cattle by ELISA has been reported [93]. Monoclonal antibodies against
ampicillin have been used to develop a direct ELISA and a multi-immuno-
affinity chromatography method (MIAC) for penicillins [106]. Moreover, many
immunoassay kits have become commercially available (see Table 4). Thus, the
LacTek ELISA was established as a qualitative rapid prediction test of amoxi-
cillin and ampicillin residues in tissues and, applying a modified methodology,
also in milk. The test is performed in test tubes and takes 7 min to complete one
analysis. Analyte and an enzyme tracer compete for an antibody coated on the
tube wall. After washing, a color developer is added to visualize the surface-
bound complex. The color intensity is measured in a spectrophotometer and
compared with a penicillin standard indicating positive or negative results. At
the moment there are also LacTek tests available to detect tetracyclines, sul-
fonamides, and chloramphenicol.
The Charm II 6600/7600 is a semiquantitative radioimmunoassay (RIA) de-
veloped to analyze b-lactam antibiotics in multiple food matrices (tissue, urine,
milk, honey, etc). It is based on the use of 1H- and 14C-tagged drug tracers.
After a step of competition between tracer antibiotic and sample residues for
the antibody, the bound tracer–antibody complex is separated by centrifuga-
tion from the unbound tracer. The complex is analyzed in a scintillation
counter for 1 min to obtain a resultant count. Samples with high-count results
are considered negative while samples with low count are considered positive.
The assay is very fast (about 10 min per sample) and the detection limit varies
depending on the antimicrobial drug and the matrix. For example, penicillin is
detected in milk at levels around 3.5 mg L–1 whereas, in the case of animal tissues,
the sensitivity reach levels of 50 mg L–1. This test has also been applied to the
analysis of other kinds of antibiotics such as tetracyclines, chloramphenicol,
sulfonamides, and macrolides [84].
The Delvo X-Press and SNAP tests are cost-effective, rapid b-lactam im-
munoreceptor assays developed for the screening of cow’s milk before milk
intake at the laboratories of the receiving stations. The Beta Screen Test employs
a fluorescence endpoint and takes about 10 min per assay, showing a high speci-
208
Table 4 Some representative commercial immunochemical assay kits for the most important PPCPs. The supplier and the contact web page are also
listed
Analyte IA kit Supplier/manufacturer Contact
Antibiotics Fluoroquinolones
Enrofloxacin Charm ROSA Charm Sciences Inc. http://www.charm.com
(Strip ELISA)
Biacore prototype Biacore http://www.biacore.com
5101ERFX1p Euro-Diagnostica http://www.elisa-tek.com
Sulfonamides
Sulfonamides Charm II 6600/7600 Charm Sciences Inc. http://www.charm.com
Biacore X Biacore http://www.biacore.com
Parallux IDEEX Laboratories Inc. http://www.Idexx.com
Sulfamethazine LacTek IDEEX Laboratories Inc. http://www.Idexx.com
5101SUL1p Euro-Diagnostica http://www.elisa-tek.com
Sulfadiazine 5101SUDA1p Euro-Diagnostica http://www.elisa-tek.com
Penicillins
SNAP test IDEXX Laboratories Inc. http://www.Idexx.com
Biacore X Biacore http://www.biacore.com
LacTek IDEXX Laboratories Inc. http://www.Idexx.com
Delvo X-Press Gist-Brocades BV http://www.dsm.com/
Beta Screen Test Advanced Instruments Inc. http://www.aitests.com/
Parallux IDEXX Laboratories Inc http://www.idexx.com
Charm II 6600/7600 Charm Sciences Inc. http://www.charm.com
Table 4 (continued)
Antibiotics Tetracyclines
RIDASCREEN Biopharm AG http://www.r-biopharm.com
LacTek IDEXX Laboratories Inc. http://www.idexx.com
TC Microwell Idetek Inc.
Macrolides
Other antibiotics
Chloramphenicol Charm II 6600/7600 Charm Sciences Inc. http://www.charm.com
LacTek IDEXX laboratories Inc. http://www.idexx.com
RIDASCREEN Biopharm AG http://www.r-biopharm.com
ELISA kit In Vitro Biologics Ltd. http://www.invitrobiologics.com
5091CAP1p Euro-Diagnostica http://www.elisa-tek.com
Veratox Neogen Corporation http://www.neogen.com
Multianalyte
Sulfamethazine/ Parallux IDEXX Laboratories Inc. http://www.idexx.com
Penicillins
Tetracycline/ Parallux IDEXX Laboratories Inc. http://www.idexx.com
Penicillins
Sulfamethazine/ Parallux IDEXX Laboratories Inc. http://www.idexx.com
Tetracyclines
Sulfonamides/ Parallux IDEXX Laboratories Inc. http://www.idexx.com

Tetracyclines/
Penicillins
209
210
Table 4 (continued)
Hormones Estrogens
Estradiol (E2) Estradiol EIA kit Immunometric Ltd.
17b-Estradiol ELISA kit Japan EnviroChemicals, Ltd.
17b-Estradiol EIA kit Assay Designs Inc.
E2 EIA kit Biosense Laboratories
Estradiol EIA kit Atlas Link http://www.atlaslink-inc.com
Estradiol ELISA kit Oxford Biomedical Research http://www.oxfordbiomed.com/
Estriol (E3) Estriol EIA Assay Designs Inc.
immunoassay kit
Estriol ELISA kit Oxford Biomedical Research
Estriol EIA kit Atlas Link http://www.atlaslink-inc.com
Estrone (E1) E1 EIA kit Biosense laboratories http://www.biosense.com/
Ethynylestradiol EE2 ELISA kit Japan EnviroChemicals, Ltd
(EE2)
EE2 EIA kit Biosense Laboratories http://www.biosense.com/
Estrogens Estrogen ELISA kit Japan EnviroChemicals, Ltd
(E1+E2+E3) ES EIA kit Biosense Laboratories http://www.biosense.com/
Table 4 (continued)
Hormones Androgens
Testosterone Testosterone EIA kit Immunometric Ltd.
Salivary assay test Salimetric
Colorimetric EIA kit Assay Designs Inc.
Testosterone ELISA kit Oxford Biomedical Research http://www.oxfordbiomed.com/
Androstenedione Androstenedione Oxford Biomedical Research http://www.oxfordbiomed.com/
ELISA kit
Trenbolone Trenbolone ELISA InVitro Biologics Ltd http://www.invitrobiologics.com/
test kit
Nortestosterone Nortestosterone InVitro Biologics Ltd http://www.invitrobiologics.com/
ELISA test kit
Stanozolol Kit for stanozolol, Tecna
ELISA
Stanozolol ELISA In Vitro Biologics Ltd http://www.invitrobiologics.com
test kit
211
212
Table 4 (continued)
Hormones Corticosteroids
Cortisol Cortisol EIA kit Assay Designs Inc.
Cortisol EIA test kit Atlas Link http://www.atlaslink-inc.com
Cortisol EIA kit Oxford Biomedical Research http://www.oxfordbiomed.com
Corticosterone Corticosteone EIA kit Assay Designs Inc. http://www.assaydesigns.com
Dexamethasone ELISA test kit InVitro Biologics Ltd http://www.invitrobiologics.com
Corticosteroids Bio-X corticosteroids Bio-X Diagnostics http://www.biox.com/
(family) ELISA kit
Gestagens
Progesterone Progesterone EIA kit Assay Designs Inc.
Progesterone Oxford Biomedical http://www.oxfordbiomed.com
ELISA kit Research
Progesterone Atlas Link http://www.atlaslink-inc.com
EIA kit
17a-OH EIA kit Assay Designs Inc. http://www.assaydesigns.com
Progesterone
Table 4 (continued)
Others Analgesics
Fenantyl Single-step ELISA Diagnostix, Inc. http://www.diagnostix.ca
Microplate EIA Cozart Bioscience http://www.cozart.co.uk
Microplate EIA Ora-Sure/STC http://www.stech.com
Technologies, Inc.
Opiates (morphine, EMIT Syva Corporation http://syva.com
codeine, cocaine, etc.)
Microplate EIA Cozart Bioscience http://www.cozart.co.uk
ELISA Neogen http://neogen.com
Salicylate FPIA (Adx, TDxFLx, Abbott Laboratoires http://abbott.com
AxSYM Systems)
Cytostatics
Methotrexate FPIA (Adx, TDxFLx, Abbott Laboratoires http://abbott.com
AxSYM Systems)
EMIT Syva Corporation http://syva.com
213
ficity for penicillin. Finally, the Parallux, a solid-phase fluorescence immunoas-

say (SPFIA) intended for use as a rapid detection method (it takes only 4 min)
in raw bovine milk, can detect all six major b-lactam antibiotics in one test. This
system has been developed as a multianalyte method to detect simultaneously
the presence of a variety of antibiotics from different families (see Table 4). The
kit is based on the use of antibodies immobilized on four glass capillary tubes
presented in the form of a disposable cartridge. Two different kinds of cartridges
are included: one consists of four tubes each containing the same range of
antibodies (the “cillins” multicartridge), so that four different samples can be
screened simultaneously. The other one also includes four tubes but each con-
taining different antibodies (the “individual” cartridge), so that a positive sam-
ple can be further identified. The sample is mixed with dried reagents, and
antibiotic competitively binds to the coated tube; after the tube is washed and
the cartridge centrifuged, the fluorescence signal is measured with a limit of
detection (LOD) for penicillin of 50 mg L–1. This test has also been applied to the
detection of tetracyclines, sulfonamides, cephapirin, and ceftiofur.
To our knowledge only one work has been reported on the use of a com-
mercial immunochemical test to detect penicillins (penicillin G, penicillin V,
ampicillin, cloxacillin, and oxacillin) in several environmental compartments.
Thus, Campagnolo et al. [84] measured penicillin using the Charm II RIA test
in water samples proximal to a US farm. The LOD of the technique was 2 mg L–1.
2.2
Chloramphenicol
Chloramphenicol (CAP) is a broad-spectrum antibiotic that was widely used in

veterinary medicine. Since 1994 the use of CAP is banned in the EU because
of certain toxicological problems (i.e., aplastic anemia and the “grey baby
syndrome”) observed in its administration to humans [107] that have prompted
the establishment of a zero tolerance for the presence of these residues in meat
and animal products.As a consequence, many efforts have been made to develop
sensitive methodologies capable of detecting CAP residues or its metabolites.
Several qualitative and quantitative immunochemical methods for CAP
analysis in biological matrices of animal origin have been described [101, 102,
104, 105] (see Table 3).Van de Water et al. [102] described an ELISA that detected
CAP in swine muscle tissue with an IC50 value of 3 ng mL–1. This immunoassay
was improved and subsequently optimized incorporating the streptavidin–
biotin amplification system. There are also several commercially available test
kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the
quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter
plate. The measurement is made photometrically, obtaining a LOD of 100 ng L–1
in meat and eggs and 150 ng L–1 in milk. The test has been also applied to the
analysis of tetracyclines.
On the other hand, the 5091CAP1p test is a direct competitive enzyme im-
munoassay for the quantitative analysis of these residues in milk, eggs, meat,
urine, tissue, and honey. In this test in a 96-well microtiter plate is coated with
antibodies. The LOD is 0.2 ng mL–1 in urine and milk, with a cross-reactivity
of 65% with chloramphenicol glucuronide. Veratox is a semiquantitative CAP
ELISA test designed to detect the presence of CAP in farm-raised shrimp. The
test format provides a 48-well plate operating on the basis of competition
between the enzyme conjugate and the antibiotic in the sample for the anti-
body-coated wells.After the addition of a colored substrate, a semiquantitative
determination can be made of the level of drug present. Results are obtained
in approximately 90 min showing low cross-reactivity to other antibiotics and
a sensitivity of 2 ng g–1.
Lynas et al. [101] compared the use of two immunochemical methods,
Charm II RIA and the RIDASCREEN EIA (enzyme immunoassay), in tissues
and fluids of treated cattle. Charm II reached LODs of 5 ng g–1 and 20 ng mL–1
in tissues and urine, respectively, whereas RIDASCREEN improved these values
to 0.5 ng g–1 for tissues and 0.3 ng mL–1 in urine. One of the advantages of these
methods is that the CAP metabolites are also detected. On the other hand,
Keukens et al. [104] used the Le Carte test kit, a polyclonal antibody (PAb)-
based assay for the regulatory control of this antibiotic in meat with a LOD of
20 mg kg–1. The assay was also applied to urine samples with a detectability of
5 mg L–1. Moreover, Impens et al. [105] used the 5091CAP1p test for the screen-
ing of CAP in shrimp tissues and Van de Water et al. [108] developed a MAb-
based cleanup procedure, prior to HPLC, for the analysis of CAP in eggs and
milk. At present we have not found examples of the application of any of these
immunochemical methods to environmental samples, but as mentioned in the
introduction, it can be assumed that application to wastewater samples should
produce fewer matrix effects than those produced by the biological samples.
2.3
Tetracyclines
Tetracyclines are broad-spectrum antibiotics with activity against Gram-pos-

itive and Gram-negative bacteria that have been widely used for the treatment
of infectious diseases in veterinary and human medicine, as well as additives
in animal foodstuffs. Normally tetracyclines are not found at high levels in the
environment because they readily precipitate with cations such as calcium and
are accumulated in sewage sludge or sediments [3, 20, 26].
Immunochemical methods have been developed and placed on the market
to analyze tetracycline residues (see Table 4). Thus, a qualitative EIA has been
developed and used to analyze tetracyclines in honey samples with a detection
level of 20 µg/kg–1 [96].A microplate-based indirect ELISA has been developed
to analyze tetracyclines using polyclonal antibodies. The assay could measure
tetracycline in the range between 0.1 and 6 ng mL–1. Other tetracycline antibi-
otics such as chlortetracycline, rolitetracycline, or minocycline are also highly
recognized in this assay [98]. Several immunoassay kits are commercially avail-
able for the analysis of tetracyclines although, to our knowledge, none of them
has been applied to the analysis of environmental samples (see Table 4). The
RIDASCREEN EIA (see above for CAP) recognizes four tetracyclines (tetra-
cycline, chlortetracycline, minocycline, and rolitetracycline). It has been used
to analyze milk samples with a LOD around 1.5 mg L–1 for tetracycline and
15.5 mg L–1 for oxytetracycline. This kit can also be applied to the analysis of
more complex matrices such as meat and honey. The Parallux system (see above
for penicillins) has also become available to detect tetracyclines and
sulfonamides (see below). This responds to the presence of tetracycline, chlorte-
tracycline, and oxytetracycline with sensitivities of 100, 125, and 100 mg L–1,
respectively. Recently, Okerman et al. [92] described the use of this kit on bovine
and porcine kidneys with a sensitivity of 300 mg L–1. Moreover, De Wasch et al.
[95] reported the use of the TC Microwell test kit to analyze oxytetracycline in
pork meat samples, reaching sensitivity levels of around 100 mg kg–1.
The use of the Charm II RIA test to analyze tetracycline antibiotics in water
(both surface and groundwater) has been reported [84, 97]. This RIA, which
was initially developed to analyze tetracycline in serum, urine, and milk, was
subsequently adapted to analyze water samples at concentration levels around
1 mg L–1. Thus, samples from hog lagoons, surface water samples, and ground-
water samples were tested using the RIA method and the results confirmed by
LC–MS.
2.4
Sulfonamides
Sulfonamides are antibiotics widely used in animal husbandry and as feed ad-
ditives. A large number of immunoassay screening methods for sulfonamides
in foods and other related complex matrices have been reported in the litera-
ture [78, 81, 85, 88, 89, 96, 109] (see Table 3 for immunochemical methods.).
Lee et al. [78] described the development of ELISAs with a series of MAbs that
can detect sulfathiazole in animal tissues with IC50 values ranging from 6 to
21 ng mL–1 in swine liver samples. Verheijen et al. [89] described the develop-
ment of a sol particle immunoassay (SPIA) for the detection of sulfadimidine
residues at the qualitative level. This kind of immunoassay is based on the use
of dyed colloidal particles as labels (i.e., gold, carbon, or latex). The device here
reported is a one-step strip test where the antigen is immobilized on the mem-
brane, and is based on the use of affinity-purified PAbs anti-sulfadimidine-
labeled with colloidal gold particles in the mobile phase. The use of colored
particles as labels allows their use as a direct detector reagent. This test has
been applied to the analysis of urine and milk samples, giving positive results
at concentrations above 10 and 20 ng mL–1, respectively. Situ et al. [87] evalu-
ated the performance of a high-throughput SPR to simultaneously analyze
eight samples and also to detect sulfamethazine and sulfadiazine in porcine bile
in an online system.
Most of the antibodies developed (both monoclonal and polyclonal) only
detect individual sulfonamides. However, due to the widespread use of sulfon-
amides and the variety of congeners that can potentially be used, several at-
tempts have been made to produce antibodies showing broader specificity
[80, 110–112]. Thus, Spinks et al. [110] carried out molecular modeling studies
of the hapten structure in order to accomplish this aim, although no conclusive
results were obtained. Korpimäki et al. [80] studied the use of protein engi-
neering to modify MAbs so that they would recognize a wider range of sulfon-
amides with similar affinities, and have achieved a significant improvement in
this aspect when compared to the wild-type antibody.
As occurred with the other antibiotics, commercial immunoassay formats,
also available as kits for tetracyclines and penicillins such as the Parallux, the
LacTek, or the Charm II, have also been placed on the market for the analysis
of sulfonamides (see Table 4). Thus, the Parallux detects sulfamethazine and
sulfadimethoxine in raw milk with a LOD of 10 mg L–1. The Charm II detects
almost all sulfonamides in honey and milk with a LOD in the range from 1 to
10 mg L–1, whereas LacTek is able to detect sulfamethazine. Moreover, the
5101SUL1p and 5101SUDA1p tests reach LOD values for sulfamethazine and
sulfadiazine of around 0.2 mg L–1 and they have been applied to the analysis
of urine, milk, and plasma. These tests have proved to be efficient as a point of
care for “on-site” applications on farms. Moreover, commercially available
antibodies can be found from several sources such as Silver Lake Research, US
Biological, Cortex Biochem. Inc., Accurate Chemical Scientific, Fitzgerald In-
dustries International Inc., and Biotrend Chemikalien GmbH.
We have found only one attempt to use immunoassays to detect sulfon-
amides in environmental samples.As in the case of penicillins and tetracyclines
and also for fluoroquinolones (see below), Campagnolo et al. [84] measured
sulfonamides in water samples proximal to a farm in Iowa using a commercial
Charm II RIA test, accomplishing a LOD of 5 mg L–1 for sulfamethazine.
2.5
Fluoroquinolones
Fluoroquinolones have mainly been used in human medicine but more recently
some fluoroquinolones (enrofloxacin, sarafloxacin, ciprofloxacin) are also being
employed in veterinary medicine. Conventional methods like spectrofluoro-
metric assays and HPLC have normally been used to detect fluoroquinolones in
human samples, although some immunoassays have also been described [75–77,
113] (see Table 3). Thus, the production of MAb and the development of an in-
direct ELISA against sarafloxacin have been reported [76, 113]. The IC50 ranged
from 7.3 to 48 mg L–1 depending on the MAb used. The antibodies obtained for
sarafloxacin were also able to recognize enrofloxacin, difloxacin, norfloxacin,
and trovafloxacin. With the same MAbs a high-performance immunoaffinity
chromatography (HPIAC) was developed that consisted of the extraction of
the analyte by using the immunoaffinity capture columns coupled online to a
reversed-phase liquid chromatography system. The optimized procedure was
applied to the detection of fluoroquinolones in serum [114], chicken liver [115],
and milk [116]. The relative affinity of these immobilized MAbs toward the
different fluoroquinolones allowed the gradual elution, separation, and indi-
vidual quantification of two fluoroquinolones [117].
Snitkoff et al. [75] reported the development of an EIA for the detection of
ciprofloxacin in serum, which was sensitive at picogram per milliliter levels of
the antibiotic and no cross-reaction with its metabolites was observed. Gobbo
et al. [118] recently described the production of PAb for ciprofloxacin with the
aim of detecting fluoroquinolones in Brazilian livestock. On the other hand,
Bucknall et al. [77] produced antibodies for quinolones and fluoroquinolones
with the aim of developing both generic and specific immunoassays. ELISAs for
ciprofloxacin, enrofloxacin, flumequine, and nalidixic acid were developed with
sensitivity values around 4 mg kg–1 (on both the generic and specific assays) in
bovine milk and ovine kidney.
Regarding commercially available immunochemical kits, we could mention
the Charm ROSA Enrofloxacin Test that detects ciprofloxacin and enrofloxacin
equally (see Table 4) and the 5101ERFX1p test. This last one is a direct compet-
itive ELISA, which uses MAbs and has a LOD of 3 ng g–1 in tissues. Some other
companies do have antibodies available as reagents for different applications
such as Biodesign International and QED Bioscience Inc.
We have only found one example of the application of an immunoassay
kit to the analysis of fluoroquinolones in environmental samples [84]. The as-
say is able to detect enrofloxcin as standard analyte with sensitivity levels of
5 mg L–1.
2.6
Macrolides
Macrolide antibiotics are macrocyclic lactones widely used in veterinary med-

icine to treat diseases and infections and also as feed additives to promote an-
imal growth. Some immunochemical methods have been developed to analyze
macrolides, such as a RIA for erythromycin A and its chemical by-products
[119] or an ELISA with a broad range of recognition for macrolide antibiotics
[99] (see Table 3). As mentioned earlier, the RIA has the disadvantage of han-
dling and producing radioactive residues. The ELISA developed by Yao et al.
uses PAbs and has a LOD as low as 0.3 ng mL–1 with an IC50 value of 8 ng mL–1.
Macrolides with 12-, 14-, or 16-carbon rings possessing amino-substituted sugar
moieties are well recognized by these antibodies, regardless of the presence of
neutral sugar residues. In contrast, little or no cross-reactivity was observed with
the acrocyclic lactone ring structure (tylactone) or macrolides containing only
neutral sugar. Also a Japanese patent [120] reported the production of anti-
bodies against 16-membered-ring macrolide antibiotics such as ricamycin,
midecamycin, josamycin, leucomycin A7, and leucomycin V.
Regarding sensors, Draisci et al. [100] reported the development of an elec-
trochemical competitive ELISA for the detection of erythromycin and tylosin in
bovine muscle. They used MAbs against these two macrolides and the activity
of an enzyme label was electrochemically measured with an electroactive sub-

strate. The detection limits were 0.4 and 4 ng mL–1 for erythromycin and ty-
losin, respectively. The specificity of the assay showed only cross-reactivity with
roxithromycin, a macrolide derived from erythromycin.
The Charm II 6600/7600 system is the only commercial immunoassay test
available for the detection of several macrolides in different matrices (see
Table 4). The LOD of erythromycin in milk is 40 mg L–1 and 100 mg L–1 in tissues.
Several common methods for the detection of these compounds in environ-
mental media have been proposed such as microbiological assay and conven-
tional chromatographic methods. However, only one example of the application
of immunochemical methods to the analysis of environmental samples has been
reported [84]. In this case, erythromycin could be measured at concentrations
higher than 10 mg L–1 using the Charm II 6600/7600 assay in water samples prox-
imal to livestock farms.
3
Steroid Hormones
Steroid hormones are a group of biologically active compounds controlling

human body functions related to the endocrine system and the immune sys-
tem. Steroids are synthesized from cholesterol and have in common a cyclo-
pentan-o-perhydrophenanthrene ring. Natural steroids are secreted by the
adrenal cortex, testis, ovary, and placenta in humans and animals, and include
progestagens, corticoids, androgens, and estrogens [121]. Estrogens (estradiol,
estrone, and estriol) are predominantly female hormones which are important
for maintaining the health of the reproductive tissues, breast, skin, and brain,
are secreted from the ovary, and work to make an ovulatory phase of menstrual
cycles. Gestagens (progesterone) act as hormone balancers, particularly of
estrogens; they are excreted from the ovary and act during the luteinizing phase
of the menstrual cycle and are involved in maturation of the endometrium of
the uterus. Androgens (testosterone, dehydroepiandrosterone, and androstene-
dione) play an important role in tissue regeneration, especially of the skin,
bones, and muscles. Glucocorticoids (cortisol) are produced by the adrenal
glands in response to stressors such as emotional upheaval, exercise, surgery,
illness, and starvation [122]. All the steroid hormones cause their action by
passing through the plasma membrane and binding to intracellular receptors.
Besides these endogenous hormones, many synthetic steroids have been pro-
duced in order to take advantage of their high bioactivity. Table 5 summarizes
both natural and the most important synthetic steroids for each group as well
as their more usual applications either in veterinary or human medicine; their
chemical structures can be seen in Fig. 3.
As well as the endogenous steroids, the xenosteroids and their metabolites
are excreted by humans and animals in the form of glucuronide or sulfates [31].
These steroids end up in the environment through sewage discharge and ani-
Table 5 Most important natural and synthetic steroid hormones
Endogenous Synthetic Use

hormones hormones
Androgens Testosterone Boldenone Growth promoter

Nandrolone in livestock
Methyltestosterone
Trenbolone
Stanozolol
Estrogens Estradiol Ethynylestradiol Contraceptive
Estriol Mestranol
Estrone
Progestagens Progesterone Norgestrel Contraceptive
17a-Hydroxy- Norethindrone
progesterone Melengestrol
Medroxyprogesterone
Corticosteroids Cortisone Dexamethasone Growth promoter
Cortisol Betamethasone Anti-inflammatory
Deoxymethasone
Prednisone
Prednisolone
Flumethasone
mal waste disposal. All of these compounds have been detected in effluents of
STPs and surface waters. The water solubility of steroid conjugates is higher
than that of the free forms and they are also more easily metabolized by de-
grading bacteria. The octanol/water partition coefficient of steroids is high and
therefore adsorption on sediments and suspended soils and accumulation take
place. These compounds have a half-life of between 2 and 6 days in the envi-
ronment depending on the environmental conditions and the climate [28]. The
fate and occurrence in the environment of the different steroid hormones are
shown in Table 1.
The unconjugated compounds are the active forms responsible for a po-
tential environmental impact, especially in the aquatic compartment, at very
low concentration levels [123]. Compounds like the estrogens have an en-
docrine disrupting activity, causing adverse effects such as the induction of
vitellogenesis (plasma vitellogenin induction) and feminization of male fish
[28]. For instance, concentrations between 4.7 and 7.9 ng L–1 of estradiol led
to induction of vitellogenin in juvenile rainbow trout [124]. Therefore, vitel-
logenin induction in male or juvenile fish has become a useful biomarker
for identifying estrogenic contamination of the aquatic environment [125].
Regarding androgens, they can induce a masculinization of the female sexual
organs. The administration of androgens could be made in the free form or as
esters (mainly acetate) [126]. After administration, the ester compound is hy-
Fig. 3 General structures of the most important natural and synthetic steroid hormones
drolyzed to the free form under phase I metabolism, leading to the active com-
pound.
As a result of the continuous growth of the population and of livestock
farming, the level of endogenous hormones excreted into the environment has
gradually increased. However, the nonethical human and veterinary practices
related to the use of the natural and synthetic sex hormones as anabolic sub-
stances and growth promoters are more worrying. For instance, it has been
reported that about 33 tons of estrogens, 7.1 tons of androgens, and 322 tons of
gestagens are excreted per year by livestock in the EU [31]. These data don’t
include the synthetic steroids, such as ethynylestradiol or norethindrone, which
are commonly used as oral contraceptives. The use of hormones, both natural
and synthetic, to enhance growth and as reproductive aids for synchronization
of the ovarian cycle, has been regulated for animal drugs because they alter the
structure or function of the animal. For these reasons, the EU has banned the
use of these compounds as growth promoters in food-production animals (Di-
rective 85/649/EEC replaced by Directive 88/146/EEC). In order to detect the
use of legal or illegal natural hormones or xenobiotic drugs, and to prevent the
inappropriate use of therapeutic drugs, veterinary and public health control
laboratories require efficient screening methods. Directive 96/23/EC and the
Directive 2377/90/EC regulate the MRLs and the analytical methods to detect
them. Hormone implants are widely used in the USA, Australia, and Canada
where their use is allowed. The use of progesterone, testosterone, estradiol,
zeranol, and trenbolone acetate for animal food production has been regulated
by the US Food and Drug Administration (FDA) and by the Food and Agri-
culture Organization of the World Health Organization (FAO/WHO).
The number of analytical methodologies currently available for determi-
nation of steroids in water samples is limited. The methodologies are based
on either biological or chromatographic techniques [30, 127, 128], which are
usually accurate but time-consuming methodologies. Immunoassays can be
extremely sensitive and often can be directly applied to the analysis of water
samples. Many examples can be found in the literature regarding immuno-
chemical determination of steroid residues in biological matrices. In contrast,
their application to environmental samples, and particularly wastewater, has
rarely been reported. However, as mentioned before their application to less
complex matrices such as aqueous samples can open the possibility to perform
more and more efficient controls of the contamination of the environment
by these groups of substances. We will briefly describe the most important
immunochemical methods reported for the most relevant steroids that can be
detected in the environment (see Table 1).
Although usually the antibodies and immunochemical methods developed
can recognize different congeners of the same family, few examples of real
multianalyte (several families screened simultaneously) procedures have been
described. It is only worth mentioning the case of immunoaffinity methods of
extraction. Thus, some of these columns have been prepared not only to selec-
tively extract a single family of steroids, but also to extract a larger number of
analytes using a multi-immunoaffinity chromatographic column (MIAC). Dif-
ferent antibodies are linked to the solid support, allowing extraction of several
steroids at once and therefore multianalyte-screening procedures using im-
munochemical or conventional analytical methods. The convenience of these
procedures for biological and environmental monitoring programs of vet-
erinary drug residues is that often, animal treatments are performed using
cocktails of different substances (i.e., anabolic steroids with corticosteroids, or
estrogens with gestagens). Dubois et al. [129] used MIAC combined with
GC–MS detection for the screening of different anabolic steroids in urine and
feces from bovine specimens. Information about 12 different compounds could
be obtained in just one run. The MIAC gel columns were prepared by mixing
individual gels prepared with different specific antibodies (against methyl-
testosterone, nortestosterone, fluoxymesterone, zeranol, clostebol, ethynylestra-
diol, diethylstilbestrol, and trenbolone). The fractions selectively eluted were
evaporated to dryness, dissolved in ethanol, derivatized, and injected into the
GC–MS system. The MIAC gel could be regenerated with mixtures of methanol/
water, water, and finally PBS and stored at 4 °C. More examples can be found in
the literature regarding the use of multi-immunoaffinity columns, for example
against different anabolic steroids [129–131].
3.1
Estrogens
Due to their endocrine disrupting activity estrogens have been the most stud-
ied regarding residues in the environment. Table 6 shows some examples of the
large number of different immunological screening methods reported for es-
trogens.A first RIA was described in 1985 for the analysis of ethynylestradiol in
water [33], and also a RIA was employed for the detection of 17b-estradiol in
wastewater [132]. This method allows rapid, sensitive, and inexpensive screen-
ing of a large number of samples. However, as already mentioned, the major
disadvantage of RIA is that it requires radioisotopes and scintillation fluids.
Huang et al. [159] used an ELISA to quantify estrogenic hormones in waste-
water effluents and surface water. The LODs accomplished levels around
0.1 ng L–1 in wastewater effluents and 0.05 ng L–1 in surface waters. Results in-
dicated that the concentrations of the estrogenic hormones 17b-estradiol and
17a-ethinylestradiol discharged by WWTPs were comparable to those that
induce vitellogenesis in fish. Some hormones appear to be removed by effluent
filtration, of which >95% of estrogenic hormones are removed by reverse
osmosis. Compared to GC–MS/MS, the ELISA method had lower detection
limits and was less susceptible to matrix interference. This produced a certain
discrepancy in the results obtained by both methods that was attributed to the
fact that the concentrations measured were near to the LOD of the GC–MS/MS
method. Another technique that has been applied to detect 17b-estradiol in
wastewater is an electrochemical ELISA [133]. The activity of the label enzyme
(horseradish peroxidase) was measured electrochemically using 3,3¢,5,5¢-tetra-
methylbenzidine (TMB) as electrochemical substrate, accomplishing a LOD of
5 pg mL–1. The interday and intraday precision (RSD) ranged from 1 to 3% and
from 3 to 6%, respectively. Analysis of wastewater from three different treat-
ment plants demonstrated the absence of matrix effects if an extraction with
diethyl ether–water was performed or the samples were just diluted 1:1 with
buffer. Validation of the method was performed by analyzing 36 samples and
comparing the results with those obtained by LC–ESI-MS/MS (liquid chro-
matography–electrospray ionization-tandem mass spectrometry). The results
correlated very well with an R2 of 0.960.
Commonly the development of these techniques has been directed toward
the detection of the estrogen family instead of an individual compound. For
instance, Goda et al. [136] developed different ELISAs for several hormone
disrupting chemicals (HDCs) and one of them, based on commercial MAbs, is
addressed to the detection of total natural estrogens (ES): estrone (E1), estra-
diol (E2), and estriol (E3). Depending on the MAb used and considering E2 as
standard analyte, several assays were developed with working ranges within
224
Table 6 Immunochemical techniques developed for the detection of steroid hormones

techniquea
LOD IC50
Estrogens
Estradiol ELISA 5 pg mL–1 Wastewater [133]
TIRF-IA 0.16 mg L–1 1.84 mg L–1 Buffer [134]
ETIA 0.85 mg L–1 1.2 mg L–1 Buffer [134]
ELISA 0.1 ng L–1 Wastewater effluent [135]
0.05 ng L–1 Surface water [135]
Estriol ELISA 12 pg per well Saliva [137]
Chemiluminescence 10 pg per well Buffer [138]
ELISA
Estrone TIRF-IA 0.07 mg L–1 0.51 mg L–1 Buffer [134]
Estrogens ELISA 0.1 mg L–1 Buffer [136]
Ethynylestradiol ELISA 0.1 ng L–1 Wastewater effluent [135]
0.05 ng L–1 Surface water [135]
TIRF-IA 0.07 mg L–1 1.07 kg L–1 Buffer [134]
RIA 5 ng L–1 Water samples [33]
a
EIA: enzyme immunoassay; ELIFA: enzyme-linked immunofiltration assay; ELISA: enzyme-linked immunosorbent assay; ETIA energy transfer
immunoassay; RIA: radioimmunoassay;TIRF-IA: total internal reflection fluorescence immunoassay.
Table 6 (continued)

techniquea
LOD IC50
Androgens
Testosterone ELISA 3.9 pg mL–1 Human serum [139]
ELISA 2.5 pg per well Human serum [140]
ELISA 10 pg per well Human serum [141]
Boldenone ELISA 26 pg per well Urine [142]
0.1 ng g–1 Faeces [142]
Trenbolone ELISA 0.1 mg L–1 Meat samples [143]
ELISA 0.1 ng mL–1 Urine [144]
0.02 ng g–1 Muscle tissue [144]
Nandrolone ELISA 1 ng mL–1 Equine urine [145]
ELISA >2 ng mL–1 Bovine bile [146]
Gestagens
Progesterone ELISA 0.5 nmol L–1 Plasma [147]
ELISA 3.8 pg per tube Human serum [148]
RIA 5 ng L–1 Water [33]
Norethindrone EIA 10 ng L–1 Water [33]
225
226
Table 6 (continued)

techniquea
LOD IC50
Corticosteroids
Cortisone Direct luminescence 2 nmol L–1 Human blood plasma [149]
EIA
Cortisol ELISA 1.4 nmol L–1 Saliva [150]
ELISA 2.8 ng mL–1 Human serum [151]
Prednisolone ELISA 0.1 ng g–1 Faeces [152]
Betamethasone ELISA 12.5 ng mL–1 Equine urine [153]
Dexamethasone ELISA 3.1 ng mL–1 Equine urine [153]
EIA 0.51 ng mL–1 Plasma [154]
ELIFA 390 ng mL–1 Equine urine [155]
ELISA 4 ng mL–1 Urine [156]
ELISA 0.01 ng mL–1 Urine and blood [157]
ELISA 2 ng mL–1 Equine urine [158]
Flumethasone ELISA 2.5 ng mL–1 Equine urine [153]
0.1–5 mg L–1. The cross-reactivity measurements were 100% for E2, 87% for E1,
and 55% for E3. The authors claim the possibility of obtaining a global value for
estrogens in a particular environmental sample. Coille et al. [134] described the
use of two fluorescence immunochemical methods to detect different estro-
genic compounds in wastewater: TIRF (total internal reflection fluorescence
immunoassay) and ETIA (energy transfer immunoassay). The first is an im-
munosensor technique based on the evanescent wave (EW) phenomenon using
fluorescence detection. The antigen is bound to the transducer surface, which
usually is an optical fiber inside a flow cell, and interacts with a fluorescent
compound-labeled antibody. Light travels by the fiber optic by total internal re-
flection and the associated EW interacts with the immobilized antigen. As a
consequence of the biorecognition reaction with the labeled antibody, a change
is produced in the features of the light that is traveling (see Fig. 4). The ETIA
works under homogeneous conditions. In this format the antibody is labeled
with a donor fluorescent dye, whereas the Ag is labeled with an acceptor dye via
a BSA molecule. When they form the complex a quenching of the fluorescence
of the labeled antibody by energy transfer is observed. In the presence of the
analyte an increase in the fluorescence is produced. The LODs obtained by
TIRF for estrone, estradiol, and ethynylestradiol were 0.07, 0.16, and 0.07 mg L–1,
respectively, whereas using ETIA the corresponding detectability accomplished
was 0.5, 0.85, and 0.01 mg L–1.
Immunoaffinity purification procedures have also been described for es-
trogens and applied to environmental and biological samples [160–163]. Fer-
guson et al. [160] described a method, based on immunoaffinity extraction
coupled to LC–ESI-MS, for the determination of the steroid estrogens b-estra-
diol (E2), estrone (E1), and a-ethynylestradiol (EE2) in wastewater. The use of
highly selective immunosorbents for sample preparation prior to the analysis
allowed the removal of interfering sample matrix components present in the
wastewater extracts that would otherwise cause severe ionization suppression
of the estrogens during the electrospray process. The authors claim that the use
of immunoextraction removed much of the isobaric noise from the selected ion
monitoring chromatograms, increasing the signal-to-noise ratios and improv-
Fig. 4 Waveguide evanescent wave (EW) principle. Light is propagated through the wave-
guide (n1) and an electromagnetic field (called EW) is generated in the external medium
(n2). The EW interacts with immobilized molecules that absorb energy, leading to attenua-
tion in the reflected light of the waveguide
ing the detectability of the analytical method (0.18 and 0.07 ng L–1 for E2 and
E1, respectively). The optimized method was applied to the analysis of estro-
gens in two wastewater effluents. Recoveries of E2 and E1 were excellent
(>90%), while EE2 was not retained (recovery <2%) from effluent extracts due
to its structural differences with the immunizing hapten. The precision of the
method was high, with relative standard deviations below 5%. The concentra-
tions of E2 found in wastewater were 0.77–6.4 ng L–1, while levels of E1 were
higher (1.6–18 ng L–1). Farjam et al. [163] developed an immunoaffinity precol-
umn (immuno-precolumn) immobilizing antibodies directed against estrogen
steroids on Sepharose. They evaluated different desorbing techniques, suitable
for online coupling to an HPLC-UV system. The most effective approach used
95:5 methanol–water mixtures, although the use of cross-reactants to elute the
target was also considered. The final system consisted of a column-switching
unit allowing preconcentration of the samples on an immunoaffinity sorbent.
After elution the analytes were concentrated on a C18-bonded silica precol-
umn, and then separated on a C18-bonded silica analytical column. The min-
imum concentration of estrogens detected in urine using this system was
around 200 ng L–1 with a repeatability of 6–8%. The total analysis time was
45 min, which gave an estimation of about 30 analyses per day in this auto-
mated unattended method [164].
Besides these works, there are a lot of commercially available immunoas-
says. Their main application is directed toward clinical analysis and food qual-
ity. Table 4 shows the most representative kits that can be found nowadays on
the market.
3.2
Androgens
The main areas of application of immunochemical techniques for androgens

is doping control in athletes, forensic chemistry, farm animals for human con-
sumption, and food analysis [165–167]. Immunochemical methods for andro-
gen detection have been applied to a great variety of matrices (see Table 6);
however, to our knowledge, their application in the environment field has not
yet been recorded. One of the anabolic steroids widely used is 19-nortestos-
terone. Different ELISAs have been developed for the analysis of this compound
in feeds, food from animal origin, and for doping control [145, 146]. Similarly,
ELISA methods have also been developed for other anabolic steroids such as
boldenone and trenbolone, used as growth promoters [142, 144]. In the case of
testosterone, several immunochemical studies have been addressed to establish
the real physiological levels of this hormone in different animals.A highly sen-
sitive microplate-based direct ELISA was developed to analyze testosterone
levels in human serum [139]. The specificity and accuracy of the assay were
established, demonstrating negligible cross-reactivity with other related steroids.
Previously other ELISA methods had been reported to analyze testosterone in
human plasma [140, 141]. Thus, Rassie et al. [140] developed a PAb-based direct
immunoassay performed in microplates with a sensitivity of 2.5 pg per well. The

assay was very specific for testosterone and did not show any cross-reaction with
other related C19 steroids tested. Replacement of immunoassay plates by poly-
propylene tubes raised the detection limits to 25 pg per tube, but improved the
range of testosterone that could be measured up to 10,000 pg. Similarly, Rao
et al. [141] developed a direct immunoassay on microplates using PAbs and
penicillinase as tracer. The LOD was 10 pg of testosterone with a dynamic range
between 15 and 1,000 pg. No interference was produced by other common an-
drogens, estradiol, or progesterone, whereas a low level of cross-reactivity with
5a-dihydrotestosterone (6.2%) and 11b-hydroxytestosterone (1%) was observed.
Specific antibodies for androgen compounds have also been used to develop
immunoaffinity columns in order to include a purification step prior to the
analysis of androgens such as stanozolol [168], methyltestosterone [169], testos-
terone, trenbolone, and nortestosterone [170]. In the case of methyltestosterone,
a comparison made between XAD solid-phase extraction and immunoaffinity
procedures showed that immunoaffinity could be more efficient in isolating and
concentrating anabolic steroids from complex matrices such as urine and serum
[169].An immunoaffinity precolumn packed with Sepharose-immobilized PAbs
against 17b-19-nortestosterone (b-19-NT) was used for the selective online
pretreatment of raw extracts of urine, bile, and tissue samples followed by
HPLC-UV detection (247 nm) [171]. b-19-NT and its metabolite 17a-19-
nortestosterone (a-19-NT) could be detected in these biological samples with
detection limits around 0.05 mg kg–1. Using the same immunosorbent 17b- and
17a-trenbolone were also detected. By percolating high sample volumes it was
possible to confirm these results by GC–MS. Farjam et al. [172] also evaluated
an immunoaffinity purification procedure coupled to GC. The immunosorbent
contained antibodies raised against the synthetic steroid hormone b-19-NT. The
online connection between the immunoaffinity precolumn and the capillary GC
was performed with an interface that consisted of a 10¥2-mm reversed-phase
precolumn and a diphenyltetramethyldisilazane-deactivated GC retention gap.
After preconcentration on the immunoaffinity precolumn the analytes were
eluted and reconcentrated on the reversed-phase precolumn. Subsequently, this
precolumn was desorbed with 75 mL of ethyl acetate, which was directly intro-
duced into the retention gap by using partially concurrent solvent evaporation.
The system allowed the automated pretreatment and GC analysis of 19-nor-
steroids at the nanogram per liter level.
A variety of immunoassay test kits for androgens are available from com-
mercial sources. Table 4 indicates some of the most important companies com-
mercializing these assays.
3.3
Gestagens
As with estrogens and androgens, several commercial immunoassay kits are

nowadays available for the analysis of gestagens [173–175] (see Table 4). Sim-
ilarly, different research groups have also made efforts to develop assays and to
demonstrate the performance of those assays in a variety of sample matrices
(see Table 6). Thus, Aherne et al. described different immunoassays for detect-
ing natural and synthetic steroids in water [33]. Norethindrone and proges-
terone were detected at concentrations above 10 and 5 ng L–1 using an EIA and
a RIA, respectively. Results below the level of detection were obtained in all the
samples examined (eight river samples and six potable supply samples), except
for two river samples that contained norethindrone (17 ng L–1) and one river
sample and one potable water sample that were positive for progesterone
(6 ng L–1). They concluded that the presence of norethindrone in river water
was caused by the low biodegradation of norethindrone in the usual 6-h water
treatment processes. Thanks to these studies the authors found that a 24-h
treatment of the sludge system was necessary.
3.4
Corticosteroids
Corticosteroids are synthetic glucocorticoids that produce a strong anti-in-

flammatory effect. Corticosteroids such as dexamethasone are commonly used
in veterinary practice for the treatment of illnesses such as respiratory and
gastrointestinal disorders. However, corticosteroids are also used illegally as
growth promoters in animal feed. The misuse of glucocorticoids in livestock
production occurs, sometimes in combination with b-adrenergics in mixtures
or cocktails to achieve growth promotion of food-producing farm animals. The
aim is to reduce meat fat, to increase the appetite of the animals, and to increase
the efficiency of the use of b-agonists. For consumer health and safety, the use
of these compounds for this purpose is banned within the EU (Council Direc-
tive 86/469/EEC). Their therapeutic use is also regulated by the MRLs that have
been established for dexamethasone, betamethasone, prednisolone, and
methylprednisolone in different tissues (EC Regulation 2377/90).
Owing to their high potency they are very effective in low doses, which re-
sults in low residue levels in biological matrices. Therefore, there is a require-
ment for sensitive analytical methods for the quantification and confirmation
of these compounds in biological samples. Previously GC–MS methods have
been used for the analysis of corticosteroids. However, recent developments
in liquid chromatography–mass spectrometry technology have led to reports
on the use of LC-based approaches for analysis of these compounds. The use
of LC–MS reduces the analysis time due to elimination of the lengthy deriva-
tization or oxidation procedures necessary for GC–MS methods. Efficient
screening procedures based on ELISA methods have been described for the
most important corticosteroids (see Table 6) [153, 176]. Rodriguez et al. [153]
reported an ELISA using PAbs raised against flumethasone that also recognized
several synthetic corticosteroids such as dexamethasone (CR=80%) and be-
tamethasone (CR=20%), while endogenous corticosteroids such as cortisol
gave very low cross-reactivity (<0.5%). This assay can be directly applied to
1:10 diluted urine samples without hydrolysis of glucuronide or sulfate conju-

gates or any other treatment of samples. The PAbs were obtained by immuniz-
ing sheep with a flumethasone derivative linked to human serum albumin.
Flumethasone, dexamethasone, and betamethasone could be detected at levels
around 2.5, 3.1, and 12.5 ng mL–1, respectively. Similar results were obtained by
Roberts et al. [158] with antibodies raised against dexamethasone using 21-
hemisuccinate dexamethasone coupled to BSA as immunizing hapten (Fig. 5).
These Abs also recognized flumethasone, betamethasone, and deoxymethasone
(see Table 7 for CR values). The immunizing hapten chemical structure strongly
determines the specificity of the resulting antibodies. However, considering the
structural similarities, the presence of common epitopes determines the high
cross-reactivity observed in these assays (see Fig. 5 for chemical structures).
A direct enzyme immunoassay for screening of synthetic glucocorticoids in
biological samples was also developed by Meyer et al. [154], by raising antibod-
ies against dexamethasone using the same immunizing hapten as Roberts et al.
(21-hemisuccinate-BSA) and using prednisolone-21-hemisuccinate horseradish
peroxidase as tracer. The system had an analog cross-reactivity pattern with
similar CR values: dexamethasone (I) (100%), flumethasone (103%), betametha-
sone (45%), triamcinolone (18%), and prednisolone (17%). The natural gluco-
corticoids cortisone and cortisol, the gestagens progesterone and pregnenolone,
and the androgen testosterone were not recognized (CR<0.4%). Recently, there
Fig. 5 Chemical structure of the hapten conjugated to BSA used as immunogen by Roberts
et al. [158] and Meyer et al. [154] to raise antibodies against dexamethasone. Structures of
other corticosteroids subsequently tested by Creaser et al. [180] in order to evaluate the
selectivity of the ELISA obtained are also shown
has also been an increasing interest in the use of saliva to detect drugs. In this
context, Anfossi et al. [150] described the use of an ELISA for cortisol that
achieves a LOD of 1.4 nmol L–1 in this matrix and recoveries from spiked sam-
ples of between 80 and 120%.
An ELIFA (enzyme-linked immunofiltration assay) method has also been
reported [155] for the rapid detection and semiquantification of dexametha-
sone in equine urine samples. The assay consists of an indirect competitive
ELISA in which dexamethasone in standards or samples competes for the an-
tibody binding with a dexamethasone–protein conjugate immobilized as a spot
on the surface of a cellulose nitrate filter. The sheep anti-dexamethasone anti-
bodies are complexed with an alkaline phosphatase-labeled second antibody.
The filtration system allows rapid washing and incubation steps, so the signal
could be visualized in just 15 min by an insoluble colored dye as a spot on the
filter at the site of the immobilized drug–protein conjugate. The assay has a
LOD of 390 ng mL–1 for a visual endpoint in which the color intensity of spots
developed in the presence of samples is compared with those of standards.
Twelve filters can be processed in a single batch consisting of two standards and
ten samples.
Immunoaffinity procedures have also been developed to selectively extract
corticosteroids from different sample matrices. Thus, Seymour et al. demon-
strated the higher efficiency of the immunoaffinity methods compared with the
conventional extraction procedures using organic solvents [177]. Immuno-
sorbents have also been used for online procedures followed by HLPC-UV [178,
179], HPLC–APCI-MS [179, 180], GC–MS [176, 181], or capillary electrophore-
sis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support
material for the anti-dexamethasone antibodies used in IAC. The online IAC–
HPLC–MS allowed determination of dexamethasone and flumethasone in
equine urine with LODs in the range 3–4 ng mL–1 [180]. The cross-reactivity
values obtained in the ELISA and the recoveries of an IAC–HPLC procedure are
presented in Table 7. Bagnati et al. developed an immunoaffinity extraction
Table 7 Cross-reactivities for selected corticosteroids using IAC–HPLC (reproduced from

[180] with editor permission)
Corticosteroid Relative cross-reactivity (%)
IAC–HPLC ELISA
Dexamethasone 100 100

Flumethasone 96 96
Betamethasone 30 37
Deoxymethasone 27 21
Cortisol 0 1
Prednisolone 0 4
Antiserum batch: AD60.

method for dexamethasone and betamethasone in bovine urine, followed by

HPLC fractionation and GC–MS detection [181]. The immunoaffinity cartridge
was inserted in an automatic HPLC system for online extraction and purifica-
tion. The purified collected fractions containing the analytes of interest were
derivatized to yield the tetra-trimethylsilyl derivatives of the three corticos-
teroids, which were analyzed by gas chromatography–selected ion monitoring
mass spectrometry. The method allowed a detection limit of 0.1 ng mL–1 for
dexamethasone and 0.2 ng mL–1 for betamethasone.
4
Other Drugs
This section is dedicated to providing information on the immunochemical

methods available today to determine drugs that do not belong to the above
groups, but that years of unrestricted emission to the environment require to
be considered [183]. From the broad range of pharmaceuticals that can reach
the environment, drugs such as analgesics and nonsteroidal anti-inflamatory
drugs (NSAIDs) are regularly employed, often even without prescription. On
the other hand, cytostatic agents are of concern not because of their production
volume but for their high pharmacological potency.
In Germany for instance the total quantities of acetylsalicylic acid sold per
year have been estimated to be greater than 500 tons, 75 tons for diclofenac, and
180 tons for ibuprofen [35]. The same occurs in other EU countries where com-
mon drugs such as paracetamol or aspirin are sold in quantities comparable to
high production volume materials – close to or exceeding 1,000 tons per year
[184]. Ibuprofen, which is in the top ten list of pharmaceuticals used in Den-
mark in 1995, is used in yearly amounts of 33 tons and analgesics 28 tons [3].
Psychiatric drugs were used in a yearly amount of 7.4 tons in Denmark in 1995
[35]. Antineoplastics (cytostatic agents) differ from the other groups by the fact
that they are mainly utilized in the hospital sector and by their intrinsic muta-
genic action.About 13–14 kg of cyclophosphamide is used in hospitals per year
[10]. In addition, 5,969 kg is prescribed for sale at private pharmacies.
Considering all aspects, sex hormones, antibacterials, and antineoplastic
agents were identified by Christensen as the three most relevant groups of
chemicals concerning their potential human risk as a consequence of drug ex-
posure via the environment [10]. Immunochemical methods for hormones and
antibiotics have already been discussed above. In this section we will describe
methods based on the use of antibodies for the analysis of analgesics, NSAIDs,
and cytostatic agents.
As occurs with other groups, after administration these drugs are excreted
into wastewater, enter the aquatic environment, and eventually can reach drink-
ing water if they are not biodegradable or eliminated during sewage treatment.
Data on the environmental occurrence of the pharmaceuticals treated in this
section are found in Table 1.
4.1
Analgesics and NSAIDs
Acetylsalicylic acid (aspirin) is still the most widely used analgesic, anti-in-
flammatory, and antipyretic agent followed by paracetamol. Ibuprofen [2-(4-
isobutylphenyl)propionic acid] and diclofenac (diclofenac-Na) from the
NSAID group are used extensively for the treatment of rheumatic disorders,
arthritis, pain, and fever. Fentanyl is a very strong opioid with analgesic prop-
erties 80 times stronger than those of morphine. The narcotics law therefore
regulates its use. It is used in major surgery and in the treatment of pain in
tumor patients [185]. Other opioids, including codeine (COD), morphine
(MOR), and heroin have been used therapeutically and/or consumed illicitly
for many years.
Most of these substances have been shown to be readily or inherently bio-
degradable [11, 18, 39, 42]. Photodegradation was identified as the main elimi-
nation process of diclofenac in lake water [4, 42].With a relatively high sorption
coefficient to particles, ibuprofen might be eliminated by sedimentation [43].
In contrast to diclofenac, ibuprofen and its metabolites are efficiently degraded
(>95%) during treatment in WWTPs [39].Acetylsalicylic acid and its metabolite
(salicylic acid) were detected in 22 and 33, respectively, of the 49 STP effluents
analyzed by Richarson et al. [24]. The same authors reported that they found
these substances in rivers and streams at levels between 0.2 and 0.5 mg L–1.
Several other analgesics and NSAIDs such as aminophenazone, fenoprofen,
indomethazine, ketoprofen, mefenamic acid, naproxen, and phenazone have
also been detected in sewage, and surface and groundwater samples (i.e.,
[21, 36]).
Immunochemical methods have been reported for the determination of
these substances in body fluids (see Table 8) in clinical and forensic analyses.
In the case of illicit use of opioid drugs, methods have also been reported for
the control of drug abuse and assessment of intoxication using body fluids,
tissue extracts, post-mortem specimens, and seizure samples. For this reason
there are several commercially available immunochemical methods (see Table 4).
Some research groups have used commercial immunoreagents (antibodies,
tracers, and other conjugates) to develop new immunochemical methods. Thus,
capillary zone electrophoresis or micellar electrokinetic capillary chromatog-
raphy-based immunoassays with laser-induced fluorescence detection have
been used for the determination of salicylate and gentisic acid in urine [187].
Similarly, Wey et al. [196] developed two rapid, competitive binding, elec-
trokinetic capillary-based immunoassays recognizing urinary opioids (COD,
codeine-6-glucuronide, dihydrocodeine (DHC), dihydrocodeine-6-glucuronide,
MOR, morphine-3-glucuronide, and ethylmorphine (EMOR)).Aliquots of urine
and immunoreagents of a commercial, broadly cross-reacting polarization
fluoroimmunoassay (PFIA) for opiates were combined and analyzed by capil-
lary zone electrophoresis or micellar electrokinetic capillary chromatography
with laser-induced fluorescence detection. Assay sensitivities for COD and
Table 8 Some representative immunochemical techniques developed for the detection of analgesics, NSAIDs, and cytostatic agents

techniquea
LOD IC50
Analgesics
Paracetamol MECC-IA <20 mg mL–1 Serum [186]
(acetaminophen)
Salicylate MECC-IA Serum [186]
Salicylic acid (SA) CE-IA-LIF 10 mg mL–1 (SA) Urine [187]
Gentisic acid (GA) CE-IA-LIF 5 mg mL–1 (GA) Urine [187]
Salicylic acid ELISA 0.39 mmol L–1 Plants [188]
Ibuprofen ELISA 3.62 ng mL–1 Buffer [189]
ELISA 100 pg per assay Buffer [190]
Diclofenac ELISA 6 ng L–1 60 ng L–1 Pure, tap, and surface [191]
water; wastewater
ELISA (chemilumi- 0.0048 ng mL–1 Umbilical cord, [192]
nescence detection) maternal plasma
ELISA (spectropho- 0.045 ng mL–1 Umbilical cord, [192]
tometric detection) maternal plasma
ELISA 0.25 ng mL–1 Human urine [193]
ELISA 0.5 ng mL–1 Human urine [194]
a
CE-IA-LIF: capillary electrophoresis-based immunoassay with laser-induced fluorescence detection; EIA: enzyme immunoassay; ELISA: enzyme-
linked immunosorbent assay; EMIT: enzyme-multiplied immunoassay technique; MECC-IA: micellar electrokinetic capillary chromatography-
based immunoassay; RIA: radioimmunoassay.
235
236
Table 8 (continued)

techniquea
LOD IC50
Analgesics
Codeine ELISA 1 ng mL–1 Buffer [195]
CE-IA-LIF (com- 10 ng mL–1 Human urine [196]
mercial reagents)
Morphine EMIT 0.020 mg L–1 Blood [197]
0.200 mg L–1 Bile [197]
0.100 mg L–1 Tissue [197]
ELISA 400 pg mL–1 Equine blood, urine [198]
ELISA 100 pg mL–1 Buffer [199]
ELISA 100 pg mL–1 Urine [200]
Cytostatic (antineoplastic)
Methotrexate RIA 6.25 ng L–1 Water samples [33]
EIA 50 pg mL–1 Serum [201]
ELISA 5¥10–12 g mL–1 Buffer [202]
CE-IA-LIF 5 pg Buffer [203]
MOR were comparable (10 ng mL–1), whereas those for DHC and EMOR were
about fourfold lower. Furthermore, glucuronides were shown to react like the
corresponding free opioids.Validation with real urine samples was performed
with identification of the peaks by capillary electrophoresis–ion-trap mass
spectrometry (CE–MS) after solid-phase extraction.
A PFIA, commercialized by Abbot, is one of the most common immuno-
chemical methods used in clinical laboratories to analyze salicylic acid (SA)
in serum. The assay also recognizes gentisic acid (GA) but is insensitive to
salicylamide, salicyluric acid, and conjugates of SA and of its metabolites [187].
With the aim of investigating the mechanisms involved in the hypersensi-
tivity reactions, an enantioselective immunoaffinity extraction method has
been developed that specifically isolates peptide fragments that have been
modified with optically active ibuprofen. The antibodies were obtained by
immunizing rabbits with (S)-ibuprofen coupled to BSA through a b-alanine
group. The elicited antibody strongly recognizes the asymmetric center and the
isobutylphenyl moiety of (S)-ibuprofen and its conjugates.A 0.5-mL aliquot of
the immunosorbent (11.5 mg of IgG per mL gel) prepared by immobilization
of the antibody was capable of retaining up to 1 mg of (S)-ibuprofen [190].
The immunochemical methods available today for the analysis of analgesics
or NSAIDs should be easily adaptable to the analysis of environmental samples,
although few examples have been reported. In this context, an indirect ELISA
has been developed and applied to the determination of diclofenac in tap
water, surface water, and wastewater samples [191]. The authors used a diclo-
fenac-BSA conjugate as immunogen to produce antisera. The ELISA showed a
LOD of 6 ng L–1 in buffer. A greater recognition for the glucuronide conjugates
was observed. In order to validate the assay the results obtained were compared
with those from GC–MS.
4.2
Cytostatic Agents
Cytostatic drugs are frequently used in chemotherapeutic treatments. Residues

of these substances should exclusively occur in hospital sewage at low concen-
trations. Among the cytostatic agents more frequently employed we should
consider: methotrexate (MTX) (4-amino-10-methylfolic acid), a folic acid an-
tagonist; the alkylating antineoplastic drug cyclophosphamide is one of the old-
est known cytostatics and is one of the most frequently used agents in cancer
chemotherapy; and ifosfamide is a widely used antitumor agent. Cytostatic
agents fall far below the quantitative importance of other drugs. However, seen
from the potential ecotoxicological impact viewpoint, they are an important
group of drugs with a high potential risk for humans and wildlife. Although
their effects against higher aquatic organisms such as fish or algae have only
been seldom investigated [1], their carcinogenic, mutagenic, and embryotoxic
effects are clearly demonstrated [204]. Most of the active substances investigated
proved to have a low biodegradability. Therefore, the active substances are ex-
pected to pass unchanged through municipal STP and thus reach surface waters
[1] when they are not eliminated by adsorption onto sewage sludge. Steger-Hart-
mann et al. [44] did not observe a significant reduction in a laboratory-scale STP.
In four out of 16 effluent samples from German STPs, cyclophosphamide was
detected at maximum concentrations of 20 ng L–1. Ifosfamide was detected
in only two samples, but in one of those it reached a value of 2.9 mg L–1 [35]. To
our knowledge cytostatics have not been detected in surface waters but they
have an estimated PEC of 0.8 ng L–1 [1, 7, 46].
Few examples of immunochemical methods for cytostatic agents have been
reported (Table 8).Within the context of work performed by Aherne et al. [33],
on the use of immunochemical methods in the analysis of microcontaminants
in water samples, was reported the use of a RIA for the detection of methotrex-
ate with a LOD of 6.25 ng mL–1. With the exception of a hospital effluent
(concentration of 1 ng mL–1 of methotrexate was found), all samples (river and
potable water) were negative.
Ferrua et al. [201] developed an EIA with enzyme-labeled Ab and an analog
antigen of MTX bound to polystyrene spheres through a methylated bovine
albumin carrier. Serum samples of treated patients were analyzed, and good
agreements with other methodologies developed to measure MTX were ob-
tained. Recently, the use of commercial antibodies for MTX for the development
of an immunoassay by capillary electrophoresis with laser-induced fluorescence
detection has also been described [203], achieving good sensitivities. The pro-
cedure includes an initial competition step between the immunoreagents and
the analyte followed by the separation of the species and detection, both steps
carried out simultaneously with CE. The rapid separation allows the reduction
of the time per assay to a few minutes.
5
General Summary
Immunochemical methods have been developed or are commercially available

for the analysis of the most important groups of pharmaceuticals with an in-
creased human risk when in contact with the environment. However, additional
work is necessary in order to expand the number of families of compounds
that can be detected using these methods, and especially to adapt them to the
analysis of environmental samples. Considering the complexity of the bio-
logical matrices, there is great promise regarding their potential application to
the analysis of water samples. Within the advantages of implementing these
methods for environmental monitoring purposes are their simplicity and the
high sample processing capabilities. However, we must consider the low con-
centrations expected in the environment even though, due to their high bioac-
tivity, these levels may be sufficient to cause adverse effects on the ecosystem.
Acknowledgements This work has been supported by CICYT (BIO2000-0351-P4-05,AGL2001-

5005-E) and by the EC: nanotechnology and nanosciences, knowledge-based multifunctional
materials, new production processes and devices (contract number NMP-505485–1).
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DOI 10.1007/b98617
Wastewater Quality Monitoring:

On-Line/On-Site Measurement
Olivier Thomas1 (✉) · Marie-Florence Pouet1
1
Environment and Sustainable Development Institute, Université de Sherbrooke,
QC, Canada
olivier.thomas@usherbrooke.ca; marie-florence.pouet@usherbrooke.ca
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
2 Measuring, Why and How? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

2.1 The Main Practical Objectives of Measuring . . . . . . . . . . . . . . . . . . . 247
2.2 Sampling Versus On-Site Measurement . . . . . . . . . . . . . . . . . . . . . 248
2.3 Types of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
2.4 Analytical Characteristics and Measurement Objectives . . . . . . . . . . . . 250
2.5 Parameters and Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
3 On-Site Measurement: From Needs to Solutions . . . . . . . . . . . . . . . . 251

3.1 Minimizing the Measurement Error . . . . . . . . . . . . . . . . . . . . . . . 251
3.2 Taking Account of Variability . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
3.3 Preventing Sample Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
3.4 On-Site Measurement Constraints and Solutions . . . . . . . . . . . . . . . . 254
4 Parameters and Substances Monitored by On-Line Systems . . . . . . . . . . 257

4.1 On-Line Monitoring:
From Laboratory-Transposed Methods to Software Sensors . . . . . . . . . . 257
4.2 On-Line Monitoring Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
4.2.1 Physical and Aggregate Properties . . . . . . . . . . . . . . . . . . . . . . . . 258
4.2.2 Inorganic Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
4.3 Organic Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
4.3.1 Aggregate Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
4.3.2 Specific Organic Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . 263
4.4 Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
4.5 Non-Parametric Measurement for Detection of Accident or Disturbance . . . 266
5 Validation and Developments . . . . . . . . . . . . . . . . . . . . . . . . . . 266

5.1 Systems Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
5.2 Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
5.2.1 Optical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
5.2.2 Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
5.2.3 Software Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
246 O. Thomas · M.-F. Pouet
Abstract Sampling and laboratory analysis are not well adapted to wastewater quality mon-
itoring in a process control or hazards prevention context, for which on-line/on-site mea-
surement is preferable. Before considering the implementation and constraints of on-line
systems, the reasons for and ways of monitoring are discussed. The main existing and
up-and-coming solutions are then presented, showing that with respect to the number of
parameters and substances to be monitored, for regulation purposes for example, only a few
of them are measurable with on-line devices.
Keywords Wastewater quality · On-site measurement · On-line monitoring ·

Emerging pollutants
Abbreviations
COD Chemical oxygen demand
CSO Combined sewer overflow
ORP Oxido-reduction potential
PAH Polycyclic aromatic hydrocarbons
SAC Spectral absorption coefficient (UV 254)
TKN Total Kjeldhal nitrogen
TOC Total organic carbon
TSS Total suspended solids
UV Ultraviolet
UV/UV UV degradation + detection
VOC Volatile organic compounds
WW Wastewater(s)
1
Introduction
We all agree that the continuous on-line/in situ detection of pollutants in water
and wastewater should be the best practice for true quality monitoring. This is
particularly relevant for the monitoring of emerging pollutants if we consider
that for the other types of pollutants, there already exist some suitable systems.
The main topic of this chapter is to show that there are some available devices
for the on-line monitoring of emerging pollutants or, at least, some interesting
developments. But first, it is indispensable to outline several important points in
order to be sure that all potential users or developers of on-line/in situ measure-
ment systems do know the main limits and constraints of the exercise.
2
Measuring, Why and How?
First of all, let us explain that the term measurement used in this chapter must
be considered in its generally accepted meaning, including all efforts carried out
for giving end-users the quantitative or qualitative result that is more often use-
Wastewater Quality Monitoring: On-Line/On-Site Measurement 247
ful for a decision-making process. This obviously includes all analytical systems
but also all test kits, field-portable devices and on-/off-line instruments.
2.1
The Main Practical Objectives of Measuring
In the domain of wastewater quality monitoring, the main reason for measur-
ing is related to regulation compliance or contractual needs, in order to check
if concentrations of substances or parameter values are below the threshold
limits, for example at the outlet of a treatment plant, before discharge. More-
over, wastewater quality monitoring programmes are also planned for some
specific sewer parts, such as combined sewage overflow (CSO) or industrial
connections. For this application, the monitoring procedure is more often
based on the use of the automatic sampling–laboratory analysis scheme. Flow
measurement is generally coupled to wastewater quality measurement not only
for sampling assistance, but also for daily load calculations, complementary to
concentrations or parameter threshold values.
The second main reason for wastewater quality monitoring is related to
process control, particularly for treatment plants where analysers and sensors
are generally used with physico-chemical or biological reactors, including set-
tling tanks. This application is mainly encountered for important wastewater
treatment plants, either urban (majority domestic) or industrial, where the
storage capacities are rather small with regard to the flow to be treated. Obvi-
ously, on-line systems are preferable in this case, but the available instruments
often limit the choice.
Hazards prevention can also be a reason for wastewater quality monitoring,
in order to protect biological treatment plants from toxic shock loads, for ex-
ample, or to prevent potential toxic effects on the receiving medium. This ap-
plication is mainly found in industrial contexts where the presence of toxic pol-
lutants may occur. In this case, on-line systems are obviously preferable for
real-time warning.
The last reason is for improving the scientific knowledge of wastewater qual-
ity. This scientific need is of great interest with regard to the scope of this book.
A lot of research has been carried out in the domain of water considered as a
resource (drinking, surface and ground water) because of health considerations
and economic reasons, but somewhat less for wastewater quality itself, due to
poor interest. However, it is well known that wastewater constitutes a huge
problem, even in developed countries, for environmental protection from
chemical (including emerging pollutants) and other sanitary (pathogenic) risks
to human health. This is the reason why research on wastewater quality must
be encouraged with the development of suitable on-site measurement proce-
dures.
2.2
Sampling Versus On-Site Measurement
As previously presented, and depending on the objective of the measurement,

a procedure has to be chosen from two main ones (Fig. 1). The first is the clas-
sical procedure recommended and even required in official texts for regulation
monitoring, based on sampling and laboratory analysis, including several steps
between sampling and analysis: conditioning, storage, transportation and pre-
treatment. The other procedure, carried out on site, is based on the existence
of on-line measurement systems or on the use of field-portable devices or test
kits. Actually, the two approaches are often combined, taking into account
either the scientific relevance of some practices (e.g. on-site measurement of
dissolved gases and temperature), or the availability of systems for on-line
monitoring. The constraints and methodology related to this last procedure
will be explained in more in detail in the following sections.
With respect to the measurement objectives, the two procedures are not
equivalent, depending on the purpose (Table 1). If it is obvious that the classi-
cal procedure will still be preferred (at least because of the lack of suitable on-
line systems), the relevant control of the treatment process cannot be envisaged
without on-line monitoring. In fact, the two procedures might generally be con-
sidered as complementary for most applications.
Fig. 1 Measurement procedures for wastewater quality monitoring
Table 1 Choice of measurement procedure with respect to wastewater measurement needs
Sampling On-site
Regulation compliance ✓
Process control ✓
Hazards prevention ✓
Scientific knowledge ✓ ✓
2.3
Types of Results
Even if quantitative results are more often expected for wastewater quality
measurement, qualitative information is of great interest, as is the case for other
applications of the analytical sciences (in the health sector, the use of test kits
and biodiagnostic systems leads to quick and useful information, often far from
a classical analytical result). In fact, quantitative analysis gives the concentra-
tion not only of one substance, but also of a group of comparable substances
(surfactants, PAH, ...), and even the value of a specific (TOC, TKN, ...) or aggre-
gate (BOD, COD, toxicity, ...) parameter. In this context, total indices are often
proposed as parameters complementary to classical analytical results [1].
From semi-quantitative results to non-parametric measurement, there
exist several levels of qualitative information:
– The first one is a degraded response of quantitative analysis, given by quick
tests or kits designed for on-site use. The results obtained are then of a semi-
quantitative nature, often relying on a number of reagent drops or a colour
change. The sensitivity of the kits is very coarse, depending on the scale of
responses. This approach is very interesting for on-site studies from grab
sampling, as it can provide assistance for focusing on sites of interest (or
some period of time), in case of medium variability (pre-measurement).
– The most common type of qualitative analysis is related to a binary response,
with a “presence–absence”,“yes–no” or “lower than–greater than” answer to
a main assumption, as for example: is the concentration lower than a thresh-
old value? This method is interesting for the detection of unknown pollutants
and can be easily carried out with screening procedures coupling real qual-
itative analysis with semi-quantitative responses.
– A third mode of qualitative measurement is a non-parametric one [2]. This
concept is based on the direct exploitation of an analytical signal (absorbance,
intensity, potential, ...) without parameter calculation, leading to the simple
characterization of the studied sample (Fig. 2). For example, fingerprinting
or image analysis can be considered as non-parametric measurement.
With respect to wastewater quality monitoring, quantitative and/or qualitative
results can be chosen (Table 2).
Table 2 Adequacy between measurement needs and types of results
Quantitative Semi- Qualitative Non-

quantitative parametric
Regulation compliance ✓ – – –
Process control ✓ ? ✓ ✓
Hazards prevention ✓ ✓ ✓ ?
Scientific knowledge ✓ ✓ ✓ ✓
Fig. 2 Non-parametric measurement concept
2.4
Analytical Characteristics and Measurement Objectives
If we try to refine the adequacy between the measurement procedures and the
practical needs for wastewater quality monitoring, different metrological (ana-
lytical) characteristics have to be considered, such as detection limit, reliability
and robustness (Table 3). Even if it is very difficult to compare the analytical
methods carried out in the laboratory with on-site measurements (with on-line
or tests kits), this presentation points out the main features of the measurement
required for different needs. These characteristics define the quality of the
available information [3], which constitutes one of the major problems that
Table 3 Measurement needs versus analytical characteristics for wastewater monitoring
Regulation Process Hazards Scientific

compliance control prevention knowledge
Low detection limit, ✓ ✓

Sensitivity
Rapidity ✓ ✓
Reliability ✓
Simplicity ✓ ✓ ✓
Relevance ✓ ✓ ✓ ✓
Robustness
Availabilitya ✓ ✓ ✓
a
Ratio of number of exploitable results and number of produced results.
analysts have to face.A rapid response, even coarse, is sometimes preferable for
decision making.
In fact, there exists a great difference between the two types of needs in
terms of traceability. On the one hand, the regulation compliance need and
more often the scientific need require us to be as confident as possible about
the trueness of the results (or at least the closeness between the results and true
values) and to store the data for further exploitation. On the other hand, process
control and hazards prevention are based on the real-time exploitation of the
results.
2.5
Parameters and Substances
A lot of substances and components are present in wastewaters and can be mea-
sured, especially the emerging pollutants. However, in practice, the aggregate pa-
rameters (BOD, COD, TSS, ...) and the physico-chemical ones (temperature, pH,
dissolved oxygen, conductivity, turbidity, ...) are more often monitored. The only
specific compounds generally considered are the N and P forms, and in case
of industrial wastewaters, some specific pollutants such as organics (phenolics,
hydrocarbons, ...) or metallic compounds.
But if we take into account the emerging pollutants and compounds, the
choice of which is guided by environmental considerations (mainly risks for
health), then surfactants, endocrine disruptors, pesticides, other industrial
organics (PAH, aromatic amines, ...) or inorganics (sulphides, arsenic, ...) and
microbiological indicators (pathogens) must also be considered.
3
On-Site Measurement: From Needs to Solutions
The previous chapters have shown that the classical procedure based on sam-
pling and laboratory analysis is not suitable for the majority of cases, especially
in an industrial context, where it is obvious that the efficiency of (treatment)
processes must be always guaranteed or at least, most of the time. But there are
other reasons for considering on-site (on-line) measurement. Experimental
errors related to the numerous steps between sampling and analysis, medium
variability with space and time, and sample aging are some good reasons.
3.1
Minimizing the Measurement Error
The usual way to get information on wastewater quality is first sampling using
an autosampler and then transportation of samples to the laboratory for analy-
sis. Between sampling and analysis, several steps are needed: storage/condi-
tioning, transportation, preparation (filtration, pre-concentration, cleanup, ...).
Table 4 Comparison between technical steps used for the three main measurement proce-
dures
On-line/off-line In situ Classical

(grab sample)
Sampling –/✓ ✓ ✓
Storage ✓
Transportation ✓
Preparation ✓? ✓
Analysis ✓ ✓ ✓
Transcription/transmission ✓ ✓ ✓
These steps are sources of error, and thus some recommendations must be
carried out in order to minimize the final error in the analytical result [4]:
– Sampling, including position of sampler inlet or device in the flow or tank,
size of strainer, hose diameter and minimum flow rate for the sampling
line, ... [5, 6]
– Cooling or chemical preservation in adapted flasks, after sampling [7]
– Rapid transportation from sampling sites to laboratory
– Recovery tests or use of internal standards for pre-treatment and use of
reference materials for analytical calibration and traceability [5].
Depending on the type of measurement chosen, some of the previous steps can
obviously be avoided (Table 4). By minimizing the number of technical steps
between sampling (or even on-line sensing) and the analytical result, the global
error of measurement will be reduced.
3.2
Taking Account of Variability
Wastewater is usually a very complex medium as explained further, the com-

position of which (urban or industrial) is changing with time and space. If
time variability is well known for the load variation between night and day for
urban wastewater or between weekdays and weekends for industrial waste-
water, then space variability has been less studied.We easily understand that for
an urban wastewater network, the composition of the medium will change with
industrial discharge or with the presence of a hospital for example (these
establishments being responsible for emerging pollutant discharges depending
on the efficiency of the wastewater treatment, if any). But space variability can
easily be studied inside industrial sites [2]. Table 5 shows the evolution of the
qualitative variability of a refinery wastewater network, decreasing from up-
stream units (desalter) to the outlet of the treatment plant (biofilter). This study
was based on the use of UV spectrophotometry for the estimation of phenols
and sulphides [9] and on the study of hidden isosbestic points in UV spectra
Table 5 Variability study along a refinery wastewater network [2]
Effluents Comments Normalized UV spectra
Inlet of the desalter

pH 7.6, – Mixture of outlets of
Ammonium 49.8, different strippers.
Sulphide 12, – UV spectra very
COD 2000, structured.
Phenols 200, – lHIP=248 nm
TSS 100, – V=70.8%
Q 50 m3/h
Outlet of a storage basin

ph 8.4, – UV spectra few
Ammonium 46.4, structured.
Sulphide 1.7, – lHIP=224 nm
COD 480, – V=44.4%
Phenols 9.1,
TSS 59,
Q 30 m3/h
Outlet of the biofilter

ph 7.1, – Few structured spectra.
Ammonium 37.8, – Presence of nitrate.
Sulphide 0.1, – lHIP=224 nm
COD 83, – V=7.7%
Phenols 0,
TSS 15,
Q 200 m3/h
HIP: hidden isosbestic point; Q: flow rate; V: variability calculated from the number of
spectra not crossing at the HIP divided by the total number of spectra.
sets [10]. Taking account of time and space variability, on-site measurement is
the only suitable procedure (unfortunately limited by the available systems).
3.3
Preventing Sample Aging
Another factor to be considered is that wastewater is rarely stable in its compo-

sition after sampling. Mainly due to physico-chemical or biological transfor-
mation, sample aging is very difficult to prevent even with low-temperature
conservation after sampling, which is proposed for biodegradation inhibition.
Among the chemical substances involved in physico-chemical aging, surfactants
play a major role in aggregation/adsorption phenomena (Fig. 3). This explains
why the surfactant concentration in the liquid phase can decrease by up to 30%,
and at the same time total suspended solids can increase by up to 30% [8].
Fig. 3 Aggregation/adsorption phenomena during aging processes [8]
A biologically treated wastewater is more stable than a raw one or a physico-

chemically treated effluent. Before biological treatment, raw urban wastewater
may show strong variation in its composition due to adsorption biodegradation
even at low temperature. This problem depends on the nature of the wastewater
(a raw urban one being generally less stable than an industrial wastewater, except
from the food industry), but seems to be sufficiently important for a lot of sub-
stances and parameters possibly to be transferred from dissolved to colloidal or
solid phases between sampling and analysis, with a risk of adsorption–com-
plexation–release for metallic compounds, and degradation into by-products
for organics. This reinforces the interest in on-site measurement.
3.4
On-Site Measurement Constraints and Solutions
On-site measurement thus seems to be the optimal solution for wastewater

quality monitoring. However, as seen previously, wastewater composition is
very complex and varyies with time and space. The implementation of on-site
measurement must take into account some constraints related to the risk of
sampling line clogging or sensor fouling (in the case of on-line measurement).
In order to prevent this risk, or at least to space maintenance procedures,
on-site measurement must be carried out carefully, depending on the type of
system used.
The principal factor of complexity is obviously related to the origin of the
wastewaters and to the presence of fouling and/or clogging materials (Table 6).
Even if raw suspended matter seems to be at first responsible for preventing
measurement, some other components such as soluble substances (grease, pe-
troleum by-products, ...) or living organisms (from bacteria to mussels) can be
the main problem.
Except for treated wastewater, almost all raw effluents contain solid com-
pounds or grease and hydrocarbons responsible for fouling/clogging. As seen
previously, on-site measurement is thus preferable, in order to place the instru-
Table 6 Wastewater types and risk of fouling/clogging
Wastewater (WW) type Suspended Fouling/clogging Factor

solids occurrence
Raw urban WW
Towns + + Grease, bacteria
Small communities Variable + id
Industrial WW
Refinery, chem. plants – ++ Hydrocarbons
Pulp and paper +++ +++ Fibres (cellulose)
Textile + + Fibres (textile)
Agrofood +++ +++ Biological solids
Metal transforming + – None
Agricultural WW + + Variable
Treated WW – – None
ment as close as possible to the medium to be characterized, and three main

possibilities exist (Fig. 4). The first is on-line measurement, with the sensor
placed inside the flow to be monitored, and in this case without sampling. The
second method is off-line measurement, for which the sensor is placed in a sam-
pling loop with a high flow rate. If an automatic analyser is used for monitoring,
a feeding line is connected to the “rapid” sampling loop. The third case is sim-
pler, as measurement is carried out with a field-portable system, more often af-
ter grab sampling.
On-line and off-line measurements are often grouped and considered as per-
manent or continuous measurements, when results are given with a more or less
regular time interval. Taking into account the complexity of some wastewaters
(mainly industrial), it is thus quite impossible to carry out perfect sampling for
a complete representation of the medium. But if we assume that emerging pol-
lutants are mainly present in the soluble fraction of wastewater, the only re-
Fig. 4 On-site measurement types

Table 7 On-line sensor/analysing equipment “properties” (from [11])
“Property” Main possibilities
Placement of on-line sensor On-line, off-line, in situ, ...

Principle of sampling External sampling,
no external sampling
Principle of sample pre-treatment No treatment, filtration,
sedimentation, ...
Principle of measuring Continuous, batch, ...
Principle of chemical/ Photometric, colorimetric,
physical method enzymatic, titrimetric,...
Number of determinants Single parameter,
multiple parameters
Need for supplies Consumables, no consumables
Service intervals Long, medium or short intervals
maining question is: does the presence of heterogeneous fractions in waste-

waters affect directly or indirectly the representativity of the measurement?
Directly means that, in the case of on-site measurement, the response may be
affected by clogging (off-line), fouling (on-line) or existing interferences.
On-line sensor/analysing equipment is an automatic measuring device
giving an output signal linked to the value of one determinant (or more) from
a medium, continuously or at regular time intervals. The choice and imple-
mentation of an on-line sensor must take into account several items or “prop-
erties” related to use constraints, which are presented in Table 7 (derived
from [11]).
To conclude this short discussion of on-site measurement implementation,
Table 8 presents the main problems concerning the representativity of the
Table 8 Main on-site measurement problems
Potential problems Recommendations
On-line Representativity Good positioning of sensor

Fouling Anti-fouling devices
Availability Adapted service
Double sensing preferable
Off-line Representativity Good positioning of strainer
Clogging Design of the strainer
Suitable flow rate and hole diameter
Availability Adapted service
In situ Representativity Repeated samples and measurement
measurement, the fouling/clogging risk for sensor or sampling line and the
availability of the results. In fact, this last criterion integrates the others, as it
is the judge of the final efficiency of the measurement chain. The availability
is thus the percentage of “good” response of the system (in terms of metrol-
ogy).
4
Parameters and Substances Monitored by On-Line Systems
Before considering some existing solutions for permanent measurement of

wastewater quality, let us describe briefly the different types of approaches.
4.1
On-Line Monitoring: From Laboratory-Transposed Methods to Software Sensors
The permanent measurement methods can be grouped into three classes,

depending on the principle used in terms of closeness to an existing laboratory
method, generally considered as a standard (or reference) method:
– Transposed laboratory methods – This first group is historically the most im-
portant as the first developments were carried out in that way. All colori-
metric systems using automatic sampling feeding a fast reaction/detection
line (for example, with a flow-injection procedure) have been developed
from classical procedures, first to increase the analytical rate in laboratories
before being transposed for off-line measurement.
– Rapid equivalent methods – Generally based on a principle different from
that of the corresponding laboratory method, alternative or surrogate sys-
tems are used more and more often for on-line and off-line monitoring. For
example, the spectrophotometric methods or biosensors proposed for the
measurement of organic compounds or electrochemical techniques for
metals must be considered as alternative methods.
– Software sensors and related methods – This last group is considered be-
cause of the complexity of wastewater composition and of treatment
process control. As all relevant parameters are not directly measurable, as
will be seen hereafter, the use of more or less complex mathematical mod-
els for the calculation (estimation) of some of them is sometimes proposed.
Software sensing is thus based on methods that allow calculation of the
value of a parameter from the measurement of one or more other para-
meters, the measurement principle of which is completely different from an
existing standard/reference method, or has no direct relation. Statistical
correlative methods can also be considered in this group. Some examples
will be presented in the following section.
4.2
On-Line Monitoring Systems
In the past 5 years, some recommendations have been made for the develop-
ment of new on-site sensors/analysing equipment [12, 13]. Two recent reviews
present the state of the art of wastewater quality monitoring. The first [14] fo-
cuses on existing and innovative technologies for the main parameters used for
the measurement of organic load (BOD, COD, TOC) for regulation needs, and
the second [15] is a review of on-line monitoring equipment designed for
wastewater treatment processes. These studies do not refer to emerging pollu-
tants, and can be completed for the other parameters by the following consid-
erations.
4.2.1
Physical and Aggregate Properties
Relatively far from the present topic and well known, the on-line measurement
of the physical and aggregate properties of wastewater does not present any
problem. Conductivity, temperature, turbidity and oxido-reduction potential
(ORP) are easily measured by well-designed sensors, because these parameters
are also used for treatment process control. In practice, turbidity is more used
for the treatment of natural water, and ORP for the biological treatment of
wastewater. However, conductivity and temperature are often monitored at the
same time as the other parameters in this section.
The measurement of total suspended solids (TSS) in wastewater always con-
stitutes a challenge because of the difficulty of transposing the laboratory pro-
cedure (filtration, drying and weighing). Turbidity measurement is sometimes
used for TSS estimation by correlation, but generally without good agreement
(because of solids heterogeneity and the effect of colloids). However, a non-con-
tact device coupling both scattered light for TSS and fluorescence for organic
load (COD) estimation has been proposed [16], giving good results around
100–200 mg L–1 for crude wastewater. Another study shows the interest of con-
sidering the UV absorption response of the heterogeneous fraction for TSS
study and estimation [17].
4.2.2
Inorganic Constituents
There are a lot of interesting inorganic constituents to be measured in waste-

water, either metallic or non-metallic, but few of them can be measured with
on-line systems. Table 9 presents a selection of recent works dealing with on-
line/on-site monitoring systems for inorganic analytes.
For metallic constituents, some attempts have been made using electro-
chemical techniques but without real success, because of the existing inter-
ferences in wastewater and the high detection limit required with respect to
Table 9 Selected on-line/on-site methods for inorganic constituent monitoring
Analyte Principle Reference
Metals
Cu, Pb, Cd Electrochemistry [18]
Cd Biosensing [19]
Cr(VI) UV spectrophotometry [20]
Nitrogen compounds
Nitrate UV spectrophotometry [21, 22]
Nitrite Biosensing [24]
Ammonium Electrochemistry [23]
Nitrate + ammonium UV/UV [25, 26]
TKN [26]
Phosphorus compounds
Colorimetry [29]
UV spectrophotometry [27]
Biosensing [28]
Other
Sulphide UV spectrophotometry [32]
Multi-ions
Phosphate, iron, sulphate Continuous-flow analysis [30]
Chloride, sulphate, phosphate, ... Capillary electrophoresis [31]
regulation needs. A recent study [18] proposed the use of stripping voltam-
metry with thick-film graphite and screen-printed electrodes. The analysis is
performed in three steps: sample pre-treatment, accumulation of the analyte on
the electrode surface, and measurement. Cu, Pb and Cd can be determined with
a detection limit around 5 mg L–1. Unfortunately, this technique has not been
sufficiently checked for wastewater. Some commercial systems based on po-
larography can be envisaged, but they not really efficient for wastewater.
Another method is explored with gene expression-based biosensors [19] for
Cd measurement. A Cd-responsive promoter from E. coli is fused to a pro-
moterless lacZ gene and monitored with an electrochemical assay of b-galac-
tosidase activity. The expected detection limit is about 0.1 mg L–1 with a response
of a few minutes.As for stripping voltammetry, no real tests have been carried out
for wastewater. However, biosensors can be considered as a promising technique
for wastewater monitoring.
Hexavalent chromium is also a toxic compound (like lead, cadmium, mer-
cury) and can be easily detected with UV spectrophotometry [20]. This system
works for the quality control of electroplating treated wastewater with a de-
tection limit of 5 mg L–1.
For non-metallic constituents, several systems exist especially for nutrients
monitoring, considering their importance in the eutrophication phenomenon.
The on-line measurement of some nitrogen compounds (nitrate and ammo-
nium) has been possible for at least 20 years with the use of selective electrodes
at the beginning and UV detection more recently. For nitrate measurement, UV
spectrophotometry is the best method because the UV-specific signal of nitrate
can be simply extracted from the raw spectrum of a wastewater [21, 22]. An in-
tercomparison study was carried out some years ago for NH4+ on-line analysers
with several instruments using selective electrodes or UV spectrophotometry
[23], showing that the performances were acceptable but installation and main-
tenance are crucial.
For other forms of nitrogen, nitrite and organic constituents, few techniques
have been proposed due to the lesser importance of nitrite in wastewater man-
agement (low concentration and non-stable), and to the difficulty in being
selective for the organic forms.
A biosensor for nitrite [24] was recently proposed for monitoring nitrite con-
centration in activated sludge exposed to oxic/anoxic cycles. The biosensor con-
tains bacteria reducing only NO2– into N2O, which is subsequently monitored by
a built-in electrochemical sensor. Up to 90% of the response is obtained in about
1 min, and the detection limit is around 5 mg L–1, a concentration sufficient for
treatment process monitoring. Unfortunately, the maximum operational lifetime
of the NO2– biosensor is 6 weeks and some problems may occur with time.
A new in situ probe [25] was presented for the continuous measurement of
ammonium and nitrate in a biological wastewater treatment plant. Based on the
use of electrochemical measurement, the sensor can be immersed and requires
minimum maintenance. The tests carried out to compare its performance with
those of other procedures (including UV for nitrate) showed that the results
were rather close, with a detection limit of 0.1 mg L–1 for both analytes.
Another principle, based on the use of UV photo-oxidation of reduced forms
of nitrogen (ammonium and organic) into nitrate (measured by UV spec-
trophotometry), allows the selective determination of nitrate, ammonium and
organic nitrogen, and thus of TKN [26], with a detection limit of 1 mg L–1. This
method is commercially available.
On-line phosphate measurement is more often limited to orthophosphates,
the total phosphorus measurement needing a mineralization step that is diffi-
cult to carry out on site. However, some recent works have been published [27,
28] based on the use of a biosensor or of UV spectrophotometry (after reagent
addition). The limit of detection is rather high for this analyte (0.5 mg L–1 or
higher).
Some coupled systems allow measurement of the main N and P forms
(nitrate, ammonia and orthophosphates) [22, 27, 29], among which is a system
based on membrane technology in combination with semi-micro continuous-
flow analysis (mCFA) with classical colorimetry. With the same principle (clas-
sical colorimetry), another system [30] proposes the measurement of phosphate,
iron and sulphate by flow-injection analysis (FIA). These systems are derived
from laboratory procedures, as in a recent work [31] where capillary electro-
phoresis (CE) was used for the separation of inorganic and organic ions from
waters in a pulp and paper process. Chloride, thiosulphate, sulphate, oxalate,
sulphite, hydrogen sulphide, formiate, carbonate, phosphate and acetate are sep-
arated in 5 min after filtration.
Except for the development of on-line systems for nutrients monitoring, the
measurement of other inorganic non-metallic constituents is rather rare. Some
commercial systems based on electrochemical sensing are proposed for the
measurement of cyanide. A simple and rapid procedure for sulphide measure-
ment in crude oil refinery wastewater has been developed [32]. Based on the de-
convolution of the UV spectrum of a sample, this method has a detection limit
of 0.5 mg L–1 and has been validated for crude oil refinery wastewater.
Even if few systems are proposed for inorganic compounds (with regard to
the number of potential pollutants), instruments or sensors for parameters used
for treatment process control are available: UV systems for residual chlorine in
deodorization, electrochemical sensors for dissolved oxygen (with nowadays a
luminescent dissolved-oxygen probe utilizing a sensor coated with a lumines-
cent material) and a colorimetric technique for residual ozone.
In conclusion it must be noted that a lot of developments are still needed in
order to increase the possibility of on-line/on-site monitoring of mineral con-
stituents, including the speciation of metallic compounds with regard to health
risks.
4.3
Organic Constituents
The monitoring of organic constituents in wastewater concerns mainly the mea-

surement of aggregate properties like oxygen demand parameters (BOD and
COD) and also the detection of specific compounds, generally expressed as the
total sum of the concentrations of their congeners. Table 10 displays a selection
of on-line/on-site methods for the monitoring of organic constituents and
related parameters.
4.3.1
Aggregate Properties
Among organic constituent measurements, that of aggregate properties (BOD

and COD) and specific parameters (TOC for example) has been well developed
for more than 20 years. Concerning BOD, a recent review on biosensors [33] has
been published. BOD biofilm-based sensors as well as respirometric systems,
other measuring principles, and the commercial BOD instruments are dis-
cussed and compared regarding their performance characteristics like linear-
ity, response time, precision, agreement between BOD values obtained from the
biosensors and the conventional 5-day test, as well as toxic resistance to vari-
ous compounds and operational stability.
Some new developments are also proposed such as a system based on the
use of electrochemically active bacteria in combination with a microbial fuel
cell [34], giving good responses over 60 days, or a biosensor developed for fast
Table 10 Organic constituents: on-line/on-site systems
Parameter Principle Reference
BOD Sensor array [37]

Respirometer-type BOD sensors [33]
Biosensor [34]
Short-term BOD [35]
COD, TOC, ... UV spectrophotometry [38, 39]
FTIR [40]
Hydrocarbons IR evanescent wave [41]
Organohalogenated ATR-FTIR [42]
compounds
Phenols Amperometric biosensor [43]
UV spectrophotometry [9]
Aromatic amines UV spectrophotometry [44, 45]
SPME–HPLC [50]
SPME–GC [51]
Surfactants UV spectrophotometry [46]
On-line titration [47]
Explosives SPME–biosensor [49]
Screening SPE (biosorbents)–HPLC–MS [50]
SPME–GC [51]
estimation of short-term biochemical oxygen demand (BODst) [35], leading

to generally good agreement with the reference method for process control
applications.
For on-site measurement from grab sampling, a compact optical device with
disposable strips for BOD determination has been developed [36]. The system
includes three pairs of light-emitting diodes and photodiodes, and the dispos-
able strips are made of inexpensive, transparent polycarbonate plates, where
Pseudomonas fluorescens is immobilized. Using the 2,6-dichlorophenol-indo-
phenol sodium salt as chromophore, a linear relationship was observed be-
tween the bioluminescence of the exposed strip and the BOD value.
Another way for BOD estimation is the use of sensor arrays [37]. An elec-
tronic nose incorporating a non-specific sensor array of 12 conducting poly-
mers was evaluated for its ability to monitor wastewater samples. A statistical
approach (canonical correlation analysis) showed a linear relationship between
the sensor responses and BOD over 5 months for some subsets of samples,
leading to the prediction of BOD values from electronic nose analysis using
neural network analysis.
In the same way (use of a principle very different from the reference method),
UV spectrophotometry is often proposed for BOD and even COD estimation.
Among numerous works two main approaches are used for the exploitation of
UV spectra. The first one is based on the absorbance measurement at 254 nm

(the emitting ray of a low-pressure mercury lamp), often corrected by another
absorbance measurement (for example at 280 or 350 nm) for compensation of
interferences. This method can be advantageously replaced by a semi-deter-
ministic deconvolution of UV spectra, taking account of the interferences for
their own estimation (TSS for example). Some industrial applications for the
pulp and paper industry or petrochemical plants [38, 39] have shown good
estimations of aggregate and specific parameters, with a good availability of
results with regard to other instruments (TOC-meter for example).
Before considering the on-line measurement of some specific organic
constituents, a final work dealing with the use of a Fourier-transform infrared
(FT-IR) spectrometer as an on-line sensor can be cited [40]. Although limited
to high concentrations, this method based on mid-IR analysis and calibration
gives a rapid estimation of chemical oxygen demand (COD), total organic
carbon (TOC), volatile fatty acids (VFA) and partial and total alkalinity (PA and
TA) in anaerobic digestion processes for the treatment of industrial waste-
waters.
4.3.2
Specific Organic Constituents
The first group of interesting organic constituents is hydrocarbons. Classically

measured by mid-IR spectrometry after solvent extraction, they can also be
measured with near-IR devices such as a polymer-coated quartz glass optical
fibre and direct spectrophotometric measurement of the extracted species in
the polymer through the evanescent wave [41]. The proposed system can be
used for the quantitative in situ analysis of organic pollutants like chlorinated
hydrocarbons, aromatic hydrocarbons, or fuels in a broad concentration range
from around 200 mg L–1 up to a few hundred milligrams per litre.As for the ref-
erence method, this instrument provides a signal corresponding to the sum of
concentrations of the extracted organic compounds by measuring the integral
absorption at the C–H overtone bands in the near-IR spectral range.
Always based on the use of IR spectrophotometry, a novel attenuated total
reflection–Fourier-transform infrared (ATR–FTIR) sensor [42] was proposed
for the on-line monitoring of a dechlorination process. Organohalogenated
compounds such as trichloroethylene (TCE), tetrachloroethylene (PCE) and car-
bon tetrachloride (CT) were detected with a limit of a few milligrams per litre,
after extraction on the ATR internal-reflection element coated with a hydro-
phobic polymer. As for all IR techniques, partial least squares (PLS) calibration
models are needed. As previously, this system is promising for bioprocess con-
trol and optimization.
For phenolic compounds, amperometric biosensors have recently been de-
signed using bacterial cells [43]. For this purpose, Pseudomonas putida immo-
bilized on the surface of thick-film working electrodes made of gold, by using
a gelatin membrane cross-linked with glutaraldehyde, was used and the respi-
ration corresponding to phenol degradation was followed with a commercial

oxygen electrode. Phenol detection was performed in synthetic wastewater
samples. For refinery wastewaters, a UV spectrophotometry system, based on
spectrum deconvolution, has been proposed for the simultaneous determina-
tion of sulphides and phenols with a detection limit of 5 mg L–1 [9].
Aromatic amines from the (bio)degradation of azo dyes or nitroaromatic
explosives must also be monitored, mainly through the sum of their concen-
trations. However, taking account of the standard solution used for the calibra-
tion of the colorimetric reference method (4-nitroaniline), some attempts are
proposed for the on-line specific determination of the most important single
compounds [44, 45].
Both urban and industrial wastewater often contains high concentrations of
surfactants. Cationic (like alkylbenzene sulphonates) and non-ionic surfactants
(like alcohol ethoxylates) are among the most-used surfactants and are dis-
charged into sewers in widely varying concentrations. Two on-line methods have
been designed for the monitoring of cationic surfactants with UV spectropho-
tometry [46] and non-ionic surfactants by on-line titration [47]. The detection
limits are around 10 mg L–1.
Endocrine disruptors are nowadays considered among the most important
emerging pollutants in wastewater, but they are not actually monitored on-line.
A recent study [48] described the implementation of a broad-spectrum ana-
lytical scheme for the screening of more than 200 compounds (endocrine dis-
ruptors, pharmaceutical compounds, ...) in urban wastewater. For other specific
organic compounds, a study concerning the improvement of immunoassays
with a solid-phase extraction (SPE) membrane was reported for the on-site
detection in soils and water of energetic materials (i.e. explosives) [49], but
unfortunately it was not really tested for wastewater.
Otherwise, there are some on-line procedures for the screening and detec-
tion of several specific organic constituents. The first [50], concerning polar
pollutants, consists of a selective SPE based on antigen/antibody interactions,
followed by liquid chromatography and diode-array or mass spectrometric
detection. Class-selective immunosorbents have been developed for poly-
aromatic hydrocarbons, benzidine and congeners, nitroaniline and aromatic
amines. Another procedure [51], using an on-site manual step of solid-phase
microextraction (SPME) before gas chromatographic analysis, was designed for
the detection of organic compounds in industrial wastewater.
4.4
Toxicity
On-line or on-site toxicity evaluation is a great challenge due to the complexity

of measuring the different impacts (from trouble from to death of organisms)
of several substances, the effect of which is often increased by synergy. Fur-
thermore for wastewater, toxicity monitoring must be implemented in several
locations: raw sewer, treatment plant or discharge point. Toxicity can be eval-
uated through some effects on organisms such as respirometric modification,

bioluminescence or electrochemistry biosensing and even death (rarely used
for on-line systems).
Historically, respirometers have been used for wastewater biodegradability
evaluation. More recently [52], a mobile on-line respirometer was proposed and
tested for monitoring the activated sludge inhibition due to industrial discharges
in a sewer network. A derived portable device called a Baroxymeter [53], based
on monitoring the respiration of a bacterial culture by pressure measurements
and using respiration inhibition as a toxicity alert, was proposed for the rapid
detection of the toxicity effect of some toxic substances.
But the most-used toxicity tests are based on bioluminescence inhibition, the
responses of which are sometimes difficult to interpret particularly for waste-
water quality monitoring. A comparison between a bioluminescence test kit
(Microtox) and a respirometry approach for the toxicity study of seven organic
and five inorganic toxic compounds was performed [54]. The bioluminescent
response proved to have a higher sensitivity to toxicants but was less repre-
sentative of the effects on activated sludge compared to respirometry, due to the
nature of the microorganisms involved in each procedure.
Recent studies, including the use of Microtox and ToxAlert test kits [55, 56],
were carried out for the determination of the toxicity of some non-ionic sur-
factants and other compounds (aromatic hydrocarbons, endocrine disruptors)
before implementation on raw and treated wastewater, followed by the identi-
fication and quantification of polar organic cytotoxic substances for samples
with more than 20% inhibition. Furthermore, the study of their contribution
to the total toxicity was obtained using sequential solid-phase extraction
(SSPE) before liquid chromatography–mass spectrometry (LC–MS) detection.
This combined procedure allows one to focus only on samples containing toxic
substances.
A derived combined approach uses an amperometric biosensor [57] with a
whole-cell (E. coli) sensing part, for industrial application (textile and tannery
wastewaters) and detection of phenolic compounds, non-ionic surfactants and
benzenesulphonate compounds. As in the previous studies, chemical analysis
(SSPE followed by LC–MS) revealed the pollutants responsible for the observed
toxicity.
A portable microbial sensing system [58] was developed for detecting the
toxicity of pre-treated wastewater. The signal of the modified electrode con-
taining a bacterial culture renewed every 9 h, within 8 min of contact with toxic
solutions or samples, is roughly correlated with toxicity.
A novel slow-release biosensor delivery for on-line monitoring instrumenta-
tion [59] allows both simple toxicity testing and more complex toxicity finger-
printing of industrial effluents, with the exploitation of kinetic (dose–response)
and dynamic (response with time) signals. Furthermore, the slow release of bio-
sensors immobilized in a polyvinyl alcohol (PVA) matrix greatly improved
biosensor delivery, did not affect the sensitivity of toxicity testing, and demon-
strated great potential for inclusion in on-line monitoring instrumentation.
In conclusion, a review of the application of whole-cell biosensors for early

warning systems [60] showed that electrochemical biosensors are well suited
for on-site use in the monitoring of general toxicity as well as hydrocarbons
and heavy metals.
4.5
Non-Parametric Measurement for Detection of Accident or Disturbance
A final up-and-coming application, based on non-parametric measurement, is

used more and more for process control and hazards prevention, for example
for shock load prevention or toxic events. This qualitative approach uses inte-
grated information coming from multiple physical signals:
– Several existing physico-chemical sensors, with data reduction algorithms
and filtering methods [61] (see “software sensing” in last part)
– A chemical sensor array (consisting of eight conducting polymer sensors)
derived from an electronic nose [62], for the characterization of headspace
gas from a sparged liquid sample
– Only one instrument giving several responses, such as the absorption spec-
trum of a UV detector [63].
5
Validation and Developments
5.1
Systems Validation
Generally speaking, alternative methods (including on-line, off-line or in situ

methods) may be used provided it can be demonstrated that equivalent results
with those of reference procedures can be obtained. The experiments are gen-
erally carried out with standard solutions and reference materials for the de-
termination of the method characteristics. The equivalence between methods
must be statistically verified by plotting the results (Fig. 5) and checking the
coordinates of the experimental regression line (comparison of the slope and
intercept values, which must be not statistically different from respectively 1
and 0 values of the theoretical line).
Once the equivalence between methods is confirmed, the validation proce-
dure results given for on-/off-line instruments (permanent measurement) must
be completed, taking into account that the sampling procedure is different for
a lab method compared to a permanent one. For example, considering that reg-
ulatory constraints require 24 hours of composite sampling before lab analy-
sis, the challenge is to obtain equivalent results with this procedure and with
permanent measurement. In this case, the results to be compared are the mean
of values for each measurement during the permanent acquisition, and the ref-
Fig. 5 Comparison of reference and alternative methods
erence value of the corresponding composite sample. Then, the final choice of
the measurement system must consider the specifications and performance
tests of the selected system [11] and the future availability of the measurement
estimated from other end users’ experiences [39].
For on-site measurement, such as colorimetric field kits giving semi-quan-
titative responses, a simple validation can be carried out [64] taking account of
the non-continuous distribution of measurement values and that the measur-
ing steps are often non-uniformly distributed over the measuring range. A
recent study dealt with new elements related to metrological analysis in the field
of (electrical safety) testing, such as measurement uncertainty and traceability
[65]. It is important that the measurement result and its uncertainty are correctly
evaluated so that the right conclusion of conformity or nonconformity with
specifications is made, as is the case for wastewater quality regulation needs.
In conclusion, one must insist on the importance of the main metrological
characteristic, the traceabilility (generally of a result), ensuring a clear (un-
broken) relationship between the final result and the complete measurement
scheme by using appropriate procedures, standards and calibrated equipment.
However, for chemical metrology and particularly for on-site measurement,
some adaptations are needed for a wider meaning of traceability [66].
5.2
Developments
At the end of this chapter, the main methods of on-site measurement system
development are briefly presented.
5.2.1
Optical Techniques
Historically used but still in progress are the optical methods, the applications
of which in wastewater quality monitoring have recently been reviewed [67, 68].
Before seeing a (future?) whole integrated system, mixing UV–visible–infrared

and fluorimetric methods, the first route is the development of UV-based
microsystems, including some relevant spectral exploitation techniques such as
the semi-deterministic one [69, 70].
Fluorescence data could be used to quantify oxygen demand values (chem-
ical and biochemical) and total organic carbon values. Furthermore, the fluo-
rescence spectral response can be apportioned to biodegradable (BOD) and
non-biodegradable (COD-BOD) dissolved organics [71]. Other studies outline
the advantages and drawbacks of the use of fluorescence techniques for waste-
water quality monitoring [72, 73].
Less suitable for the purpose are infrared techniques, which are limited
by the strong absorption of water. However, they can be envisaged for the mon-
itoring of highly concentrated organic pollutants [74], particularly with the
development of mid-infrared transparent optical fibres and waveguides, the
surface of which can be chemically modified to enhanced analyte recognition
based on tunable properties of enrichment or (bio)chemical recognition layers.
The use of attenuated total reflection (ATR) devices with Fourier-transform IR
spectroscopy is also proposed for organic compound monitoring [75].
The application of near-IR spectroscopy for real-time monitoring of glucose,
lactic acid, acetic acid and biomass in liquid cultures of microorganisms of the
genera Lactobacillus and Staphylococcus has been recently published [76]. The
NIR spectrum acquired by the optical-fibre probe immersed in the culture is
exploited using a partial least squares (PLS) calibration step, a classical method
for IR techniques.
A final optical application deals with the measurement of intracellular
nicotinamide adenine dinucleotide (NADH) by fluorescence [77], giving in-
formation about the physiological status of wastewater treatment plant bio-
mass. This indirect method could be envisaged for toxicity estimation.
5.2.2
Biosensors
Biosensors are based on the direct spatial coupling of immobilized biologically

active compounds with a signal transducer and an electronic amplifier. Due
to the reaction between the biorecognition molecule (receptor) and a target an-
alyte, a transducer signal is produced [27]. Biosensors are promising techniques
for environmental monitoring applications such as toxicity, bioavailability and
multi-pollutant screening, even if some limitations still exist [78]. With regard
to the diversity of compounds and the complexity of matrices (particularly
wastewaters), further developments have to be made before considering
biosensing as an operational solution for on-line monitoring.
Currently, a large spectrum of microbial biosensors have been developed
that enable the monitoring of pollutants by measuring light, fluorescence,
colour or electric current and electrochemical signal [60]. A recent study [19]
shows that whole-cell biosensors based on the detection of changes in gene
expression can be applied to environmental alterations of the cellular response

(promoters responding to some physical parameters such as metabolites or
environmental stress agents). A biosensor for measurement of inhibitors of
nitrification in environmental samples has also been developed. The biosensor
consists of a Clark oxygen probe as a transducer and an immobilized mixed
nitrifying culture as the microbial component. The measuring principle is
based on the direct determination of bacterial metabolic activity by measuring
the oxygen consumption rate of the microbial immobilizate [79].
In conclusion, more than 40 years after the first electrode with an immo-
bilized-enzyme membrane was produced, future developments in biosensor
design will inevitably focus upon the technology of new materials, especially
the new copolymers that promise to solve the biocompatibility problem and
offer the prospect of more widespread use of biosensors in clinical (and envi-
ronmental) monitoring [80].
5.2.3
Software Sensors
As seen previously for some specific applications such as wastewater treatment

plants, software sensors can be envisaged to provide on-line estimation of non-
measurable variables, model parameters or to overcome measurement delays
[81–83]. Software sensors have been developed mainly for monitoring bio-
processes because the control system design of bioreactors is not straightfor-
ward due to [84] significant model uncertainty, lack of reliable on-line sensors,
the non-linear and time-varying nature of the system or slow response of the
process.
A software sensor combines the theoretical knowledge of a system through
a mathematical model, and the practical knowledge of its actual functioning
through measurements. If the inputs acting on the system are known (and pro-
vided that theoretical conditions are fulfilled) and if, moreover, the model is a
sufficient approximation of the real system, then the software sensor estimates
the whole state of the system [81]. There are two types of approaches in devel-
oping software sensors [85], the first estimating the required parameters on the
basis of a deterministic model and the second being a black-box approach de-
pending only on the observed values. In practice, for wastewater treatment ap-
plications, the main techniques available are [82]:
– The representation of the biological conservation of substrate to cell mass

by an overall chemical reaction. The stoichiometric relationships are then
used to calculate various rates such as cell mass concentration [83].
– The previous method supposes complete knowledge of the system and de-
pends on the measurement quality of instruments (errors, availability), lead-
ing to severe effects on the accuracy of the on-line estimates. Therefore, a good
noise filtration algorithm (like the Kalman filter or derivative) should be
employed to improve the reliability of the estimated values before their use.
Table 11 Software sensor main types for wastewater treatment applications
Types Objectives References
Estimation through elemental Anaerobic bioreactor parameters [81]

balances estimation
Observer and filtering techniques Anaerobic bioreactor control [86]
Nitrification bioreactor control [88]
Artificial neural networks TKN estimation [85]
or hybrid ANN Fluorescence spectra exploitation [89]
for organic constituents estimation
Estimation of wastewater [90]
parameters (COD, NH4+, ...)
– Another procedure uses artificial neural networks (ANN) derived from ar-
tificial intelligence techniques.Among several ANN algorithms, the feed-for-
ward one, made up of interconnected neurone-like elements, can model
complex non-linear systems easily, depending on the status of the training
data. If there are noise and uncertainty in the training data, a problem of
overfitting often arises but can be solved by data pre-processing, using a
principal component analysis (PCA) for example [85].
Table 11 gives some examples of different software applied to the monitoring
of bioreactors and the estimation of wastewater parameters.
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The Handbook of Environmental Chemistry Vol.

Estrogens and Progestogens in Wastewater, Sludge,

Keywords Estrogens · Progestogens · Environmental analysis · Occurrence · Fate

Chemicals used in a wide range of applications in our modern society are

alkyl phenol ethoxylates, phthalates, bi-phenolic compounds, fitoestrogens and

Large quantities of pharmacologically active substances are used annually in

tered as food additives for preventing illness, as growth promoters or parasiti-

In terms of binding to the human estrogen receptor, estradiol is the principal

Estrogens and progestogens are mainly used as growth promoters in animal

and testosterone, and synthetic chemicals such as zeranol (estrogenic), melen-

Figure 1 shows the principal routes of environmental exposure to estrogens and

Fig. 1 Routes of environmental exposure to estrogens and progestogens

liver with some undergoing enterohepatic recycling. Excreted hormones and

or estrone and that synthetic estrogens in general exhibit greater recalcitrance

The introduction of estrogens and progestogens into the environment is a func-

environment. Table 1 lists the main physicochemical characteristics of the most

Estradiol 000050-28-2 272.39 3.6 (27 °C) 4.01 1.26E-008 3.64E-011

Table 2 Environmental occurrence of estrogens and progestogens

Matrix (location) Compounds Concentration Ref.

As previously mentioned, discharged domestic effluents represent the most

Fig. 3 General scheme of TIE procedure

extracts into fractions where the identification of the compounds responsible

Table 3 Relative estrogenic potencies as determined by different bioassays (expressed as

Compound/bioassay YES MCF-7 ER-CALUX

The analysis of steroid sexual hormones and related synthetic compounds

Sample Compound Sample preparation Analytical method LOD Ref.

Sample Compound Sample preparation Analytical method LOD Ref.

The choice of the sampling procedure is essential to obtain significant, repre-

An essential step in the analysis of trace pollutants in environmental matrices

The analysis of WW samples has been dominated by the use of immunoassays

Fig. 4 Reconstructed SRM chromatograms obtained from the LC–ESI-MS/MS analysis of a

1. Stone R (1994) Science 265:308

Organic Compounds in Paper Mill Wastewaters

1 Pulp and Paper Mill Wastewaters . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2 Legislation Related to Pulp and Paper Mill Industries . . . . . . . . . . . . . . 30

3 Chemical Characterization of Pulp and Paper Mill Waters . . . . . . . . . . . . 32

4 Toxicity of the Effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

7 Conclusions and Future Recommendations . . . . . . . . . . . . . . . . . . . . 48

Keywords Paper mill · Whitewaters · Effluents · Organic compounds · Analytical methods ·

Table 1 List of acronyms ordered by compounds and techniques

AOX Absorbable organic halides

APCI Atmospheric pressure chemical ionization

The accumulation of organic matter in whitewaters is due to the paper-mak-

of paper [5]. Wastewater is either released untreated, treated or recycled. Pulp

Type of pollutant Typical example Levels

Air emissions Malodorous gases of reduced 0.3–3 kg/t

As highlighted by the EU, in order to improve the management and control

Country Type of pulp COD BOD5 TSS AOX

New: 25 kg/t New: 30 mg/L (part of COD)

levels of various priority pollutants in surface waters, sediments and biota

The analysis of organic pollutants in whitewaters and effluents requires

Fig. 2 Chemical structure of organic compounds identified in paper mill effluents

Depending on the type of paper produced, it is common to dose biocides for

Compounds Matrix Method LOD Recoveries Ref.

FA esters Primary effluent SPE GC MS n.r. n.r. 25

Compounds Matrix Method LOD Recoveries Ref.

NP1EC, OP1EC Paper-recycling SPE LC ESI-MS 10–80 mg/L <90 17

Hemicelluloses Pulp fibres 1% Acid methanolysis GC FID n.r. n.r. 38

Compounds Matrix Method LOD Recoveries Ref.

OC, PCB, Nesting along GPC GC EDC n.r. n.r. 55

Chlorophenols, Contaminated sediment Dean–Stark Soxhlet GC MS n.r. n.r. 52

Drinking water HS-SPME GC MS 1.2–3.3 ng/L n.r. 73

pend on the paper-making procedure. Concentrations of 20 to 400 mg/L

effluent discharge, the concentration of resin acids found in river sediment is