ANALYSIS ARTICLE:

Long-term wastewater irrigation of vegetables in real agricultural

systems: Concentration of pharmaceuticals in soil, uptake and
bioaccumulation in tomato fruits and human health risk assessment

MASTER INTERNATIONAL WATER AND SUSTAINABLE

HORTICULTURE

BENABOUCHE NADA

2022/2023

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Abstract:

Wastewater (WW) reuse for vegetable crops irrigation is regularly applied worldwide. Such a
practice has been found to allow the uptake of pharmaceutical active compounds (PhACs) by
plants and their subsequent entrance to the food web, representing an important alternative
pathway for the exposure of humans to PhACs, with potential health implications. Herein we
report the impacts of the long-term (three consecutive years) WW irrigation of a tomato crop
with two differently treated effluents under real agricultural conditions, on (1) the soil
concentration of selected PhACs (i.e., diclofenac, DCF; sulfamethoxazole, SMX;
trimethoprim, TMP), (2) the bioaccumulation of these PhACs in tomato fruits, and (3)the
human risks associated with the consumption of WW-irrigated fruits. Results revealed that
the concentration of the studied PhACs in both the soil and tomato fruits varied depending on
the qualitative characteristics of the treated effluent applied and the duration of WW
irrigation. The PhAC with the highest soil concentration throughout the studied period was
SMX. DCF was not found in tomato fruits harvested from WW-irrigated plants during the first
year of the study. However, DCF displayed the highest fruit concentration throughout the
study (as a result of prolonged WW irrigation), followed by SMX and TMP. The calculated
fruit bioconcentration factors (BCFF) were extremely high for DCF in the 2nd and 3rd year of
the experimental period. The estimated threshold of toxicity concern (TTC) and hazard
quotients (HQ) values revealed that the consumption of fruits harvested from tomato plants
irrigated for long period with the WW applied for irrigation under field conditions in this study
represent a de minimis risk to human health.

Keywords: Bioconcentration- Human health risks- Pharmaceuticals- Tomatoes- Uptake -

Wastewater irrigation

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Introduction:

In the last years, developments in analytical instrumentation and techniques have enabled
researchers to detect and quantify organic micropollutants in environmental matrices. Thus,
pharmaceutical active compounds (PhACs) are now commonly detected in relevant
concentrations in both the aquatic and the terrestrial environments as a consequence of their
continual introduction through the disposal of treated municipal wastewater, biosolids and
manure. Despite the low concentration of PhACs usually found, their continuous introduction
into the environment from different pathways, as well as their bioactivity and known mode of
action, render them as environmental contaminants of emerging concern that may pose
potential ecotoxicological effects in aquatic and terrestrial organism, and public health risks.
The uptake and bioaccumulation of PhACs in the edible parts of food crops and fodders and
their subsequent entry into the human food chain have been gaining prominence over the
last decade. Moreover, numerous studies, mainly conducted under hydroponic or
greenhouse conditions, highlighted PhACs uptake and bioaccumulation in plants exposed to
known concentrations of individual or cocktails of PhACs. Such studies were proven to be
useful in elucidating the mechanism of PhACs uptake by plants, which was found to be
simply driven by the transpiration derived mass flow and largely depended on the chemical
properties of the compounds, especially their hydrophobicity and charge. Hydroponic and
greenhouse experiments though, even if conducted at environmentally relevant PhACs
concentrations, are unable to manifest the complexity of an actual agricultural environment. It
is widely accepted that the availability of PhACS in the soil is closely related to the properties
of the compound, as well as the soil properties, and the pH of the aqueous solution. The
absorption and retention, as well as desorption of PhACs in various clay minerals and soil
organic matter greatly determine both their potential transport in soil or bioavailability for
degradation and plant uptake. Interestingly, uptake of PhACs by plants and reported
bioaccumulation factors were found to be negatively correlated with soil clay and organic
matter content. In addition, the pH of the soil aqueous solution may greatly affect the uptake
of ionic rather than non-ionic PhACs. Moreover, soil properties (i.e., texture, pH) and soil
environment (i.e., water and air permeability) may affect the microbial degradation of
pharmaceuticals and therefore their bioavailability and potential for leaching to groundwater
concluded that soil constitutes a major sink which in turn determines the fate of PhACs in
agricultural environments, as they found that only 3.5% of the amount of PhACs accumulated
in soil was taken up by plants. Thus, only through field experiments, where actual farming
practices, or genuine soil, or ecological conditions typical for commercial agricultural farming
are incorporated and taken into account, can the potential uptake of PhACs by crops be
assessed and integrated into a database for risk assessment. Such studies are limited, and
primarily focused on biosolid application or to the irrigation with surface water that had been
mixed with treated wastewater. Recently, reviewed the human health risks of PhACs in plant
tissues due to biosolids and manure amendments, and WW irrigation, and concluded that
these practices represent a de minimis risk to human health, though when assuming
additivity, the mixture of PhACs could potentially represent a hazard. The aim of this study
was to evaluate the effects of the long-term (three years) irrigation of tomato crop under field
conditions with two distinctly tertiary treated WW, on the accumulation of selected PhACs
(diclofenac, DCF; sulfamethoxazole, SMX; trimethoprim, TMP) in soil and their uptake and
bioaccumulation in fruits, as well as the potential risks to human health associated with the
consumption of these fruits. Worth noting, the drip irrigation of vegetable crops with treated
WW is a common practice in all arid regions worldwide, as it complies with the majority of

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existing guidelines for the safe reuse of WW for irrigation, with tomatoes being the most
commonly WW-irrigated vegetable crop.

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2. Material and methods:

2.1. Experimental design and treatments:

Tomato seedlings (Lycopersicon esculentum M. variety Tovi Roca F1) were grown in a field
dominated by sandy clay loam soil at the experimental station of the Agricultural Research
Institute in Nicosia, Cyprus, for three consecutive years (from 2011 to 2013). The growing
period lasted from mid-April to late August and was representative of that of open-field
tomato cultivation in the Mediterranean region. Overall, tomato plants were subjected to
three treatments, based on the water source applied for their irrigation. More precisely,
treatments concerned the wastewater (WW) irrigation of tomato plants with tertiary treated
effluent produced by two municipal wastewater treatment plants (MWTP), as well as the
control tubewell water irrigation (Control TW) with water abstracted from a nearby borehole
within the experimental station. Tertiary treatment and disinfection in MWTP I are
accomplished through Slow Sand Filtration (SSF) and chlorination process, whereas, in
MWTP II through Membrane Bioreactor (MBR) and UV treatment. The climate in the
experimental region during the growing season is characterized by high temperatures, and
low relative humidity, having as a result high evapotranspiration rate. A completely
randomized block design was applied, while each treatment was independently run in five
replicates; each replicate consisted of 20 individual tomato plants, planted in 1 m distance
from each other in the drip irrigation line. As a result,300 tomato plants per annum were used
in this experiment. The experimental plots (221 m long width) were isolated from each other
with the use of stainless-steel sheets in order to prevent cross contamination among
treatments. Tomato plants were surface drip irrigated with inline drippers based upon direct
measurements of soil moisture status by the use of tensiometers, while all the farming
practice undertaken (weed, and pest and diseases control) were typical for the tomato
cultivation. It is worth noting that no fertilization was practiced during the experimental period
and that the experimental field was cultivated with rainfed barley ever since, whereas biosolid
or manure was never applied. The tomato growing season lasted approximately 140 d in
each growing season and seven to eight harvests took place per annum. The examined
PhACs in this study include two bactereostatic antibiotics (SMX, TMP) and one nonsteroidal
anti-inflammatory drug (DCF), that are widely prescribed and found in treated wastewater
effluents worldwide. Moreover, the studied compounds are both anionic (DCF, SMX) and
zwitterionic (TMP) PhACs that have been reported to be taken up by crop plants. The
concentration of the selected PhACs, i.e., DCF, SMX and TMP, in the water sources applied
for irrigation, as well as in the soil and fruit samples, were quantified.

2.2. Sample collection and preparation, and chromatographic analysis:

2.2.1. Sample collection:
Water samples (500 mL each; 8e10 samples per year) were regularly taken in pyrex storage
bottles from randomly selected drips in the drip irrigation pipeline and immediately transferred
to laboratory and stored at 18 C, until analysis. Soil samples (five replicates) representing
each treatment replication were collected at the end of the growing period of each year with
an auger sampler from the 0e20 cm topsoil layer as composite samples, consisting of 20
subsamples (each plant represented a subsample). Soil samples were subsequently air dried
laid in a glasshouse, ground, sieved, and stored in plastic bags at 18 C, until analysis. The
physico-chemical and microbial properties (i.e., E. Coli, Helminths eggs) of sampled irrigation

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water sources, as well as the electrical conductivity and pH values of soil samples were
determined based on the methodology described. All sampled fruits were hand harvested at
the fully ripe stage (fully red) at the end of the harvest period (last harvest) for the first two
years of the experimental period, while for the 3rd year tomatoes were sampled at the
beginning (1st harvest), middle (mid harvest; 4th harvest) and the end of the harvest period
(last harvest; 7th harvest). Tomato samples (five replicates per treatment per harvest event)
in each harvest event were constituted from fruits randomly harvested from all 20 plants in
individual replicates of each treatment. Tomatoes were then thoroughly washed with tap
water followed by rinsing with ultrapure deionized water to remove any soil particles, blotted
dried with tissue paper and stored in plastic bags at 18 C until analysis.

2.2.2. Sample preparation:

2.2.2.1. Preparation of water samples for analysis:
The frozen water samples were left to thaw at room temperature and after well shaking were
successively filtered glass filters.

2.2.2.2. Preparation of soil samples for analysis:

The soil samples before the analysis of DCF, SMX and TMP were further dried for 24 h at
room temperature before the extraction. Five grams of each sample were then weighted in a
50 mL polyethylene tube. The tube was vortexed for 1 min and sonicated in a sonication bath
for 20 min followed by 1 h shaking on a rotary shaker (230 rpm). The tube was then
centrifuged at 3900 rpm for 30 min. The supernatant was carefully collected and the residue
was re-suspended in 5 mL of 1:1 MeOH:6.25 mM NaOH solution. The extraction was
repeated and the combined extracts after filtering through 0.22 mm glass filter were diluted to
about 300 mL with ultra-pure water. Soil samples collected before the initiation of the
experimental treatments were considered as blanks and were spiked and analyzed along
with the soil samples.

2.2.2.3. Preparation of fruit samples for analysis:

The frozen tomato samples were left to defrost at room temperature. About 500 g of each
tomato sample were chopped and blended in a household blender at high speed to a thick
homogenous slurry. Two 10-g samples of the slurry (S1 and S2) were then accurately
weighted in two separate 150 mL conical flasks. For the extraction of TMP and DCF the pH
of S1 was adjusted to 6e7 with the addition of small amount of solid NaHCO3 while for the
extraction of SMX the pH of sample S2 was left as it is (pH 3e4). 10 mL of MeOH was then
added to each sample and the flasks were transferred for extraction in a sonication bath for 1
h. The mixture in each conical flask was then transferred in a centrifuge tube and centrifuged
at 3900 rpm for 10 min. The supernatant was carefully withdrawn and kept, while the residue
was re-suspended in 10 mL of 1:1 MeOH: H2O, sonicated for 30 min and centrifuged. The
combined extracts were then filtered through a 0.22 mm glass fiber filter and diluted with
H2O to 400 mL. The diluted extract was further handled as previously described. Blank
samples, starting from tomatoes produced in fields that have never been irrigated with
treated WW, and spiked blank samples with mixture of the three substances, were also
carried out along with each series of measurements.

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2.3. PhACs translocation and bioconcentration in tomato fruits:
The ability of tomato plants to accumulate the studied PhACs from the WW irrigated soil in
their fruits, during the three years of this study, was estimated using the bioconcentration
factor (BCFF), which was calculated as the ratio of each PhAC concentration in the tomato
fruit to the PhAC concentration in the soil (all in dry weight base):

2.4. Public health risk assessment:

The public health risk assessment, associated with the intake of the selected PhACs through
the consumption of tomatoes irrigated with treated WW, was conducted based both on the
threshold of toxicological concern (TTC) approach using the Toxtree software, and the
hazard quotient (HQ) for both adults and toddlers. The default average body weight of the
European adult (>18 years) and toddler (1e3 years) population, i.e., 70 and 12 kg,
respectively (EFSA Scientific Committee, 2012), was used in this study. Risk assessment
was performed for each of the three years of this study using the greatest concentration
quantified in tomato fruits in each year of the study. The TTC concept is used to provide an
abbreviated risk assessment for the thousands of low molecular weight contaminants and
other chemicals in food, provided that there is a sound intake estimate. The TTC values and
compounds classification were determined based on Gramer rules using the Toxtree
software. The TTC is defined by examining the distribution of threshold values for each
structural class. As a result, PhACs were classified in three structural classes (I, II, III). Class
I contains substances with simple chemical structures and for which efficient modes of
metabolism exist, suggesting a low order of oral toxicity. Compounds that are less innocuous
than those in class I but they do not contain structural features that are suggestive of toxicity
are categorized to Class II, whereas compounds with chemical structures that permit no
strong initial presumption of safety or may even suggest significant toxicity or have reactive
functional groups, are categorized to class III. All three studied PhACs fall into class III.
Consumption of a PhAC at a rate above TTC value poses potential risk of exposure and
demands specific toxicity analysis of the PhAC. The HQ was calculated as the ratio of the
estimated daily intake (EDI) of PhACs to the acceptable daily intake (ADI) which is the 28
amounts of PhAC that can be consumed daily over a person's lifespan without evocating an
adverse effect. ADI values for the studied PhACs were determined by dividing the lowest
daily therapeutic dose (mg day1) by a safety factor of 1000 and a body weight of 70 kg
(WHO, 2011) (DCF 1.4; SMX and TMP 5.7), whereas EDI was calculated for each of the
three years of the study using the greatest concentration quantified in tomato fruits in each
year, following the equation proposed by Prosser and Sibley (2015). The level of concern in
the risk assessment was set to HQ 0.1, not the more traditional value of 1, in order to add an
additional order of magnitude of conservatism/protection to the assessment.

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3. Results and discussion:

3.1. Irrigation water sources quality and concentration of PhACs in WW:

The average values of physico-chemical and microbial properties of the two treated effluents
and the control TW during the studied period met irrigation water quality standards according
to FAO's guidelines, with the sole exception of the high EC. Tubewell water's high salinity
may be attributed to the deterioration of groundwater quality, due to the prolonged
groundwater pumping for irrigation and the consequent saltwater upcoming. The averaged
BOD5 and COD levels of the two treated flows used imply that the MWTP I WW has higher
organic content compared to that of MWTPII WW. This may affect the bioavailability of
weakly acidic PhACs, as polar interactions between acidic PhACs and dissolved organic
matter present in the WW can lead to the formation of water-soluble complexes that are not
available for uptake and/or co-sorption to solid phases in the soil, which also reduces their
concentration in the soil solution. In addition, both Escherichia coli and Helminths eggs were
regularly not detected in all the water sources used for irrigation. Moreover, the qualitative
characteristics of the two treated effluents reused for irrigation were fully in line with both the
WHO and the USEPA guidelines for WW reuse in agriculture. Importantly, the pH values,
which greatly determine the dissociation of studied PhACs, were in the alkaline region in all
the three water sources applied for irrigation; pH values ranged from 8.04 to 8.45. Therefore,
it was expected that DCF (pKa 4.16) and SMX (pKa 1.85/5.60) to be almost entirely present
in their ionic form, whereas TMP (pKa 7.12) to be present mostly in its neutral form (almost
93% in MWTP I WW and 89% in MWTP II WW), rather than cationic form. All selected
PhACs were absent from the control TW, as the borehole used in this study was in an area
where biosolids or manure or WW irrigation were never applied. The concentration of all
three PhACs in WW varied significantly both between the years of the study, as well as
between the two WWs used, because the release of PhACs in WW is characterized by both
spatial as well as temporal (intra- and inter-day, seasonal and inter-annual) variability. As a
result, both the period of irrigation (1st, 2nd or 3rd year) and the origin of WW (MWTP I or
MWTP II WW) significantly affected the concentration of the selected PhACs in the irrigation
water. More precisely, DCF.

3.2. PhACs' concentration in soil:

The occurrence of PhACs in wastewater irrigated- and biosolid and manure-amended soils
has been well documented during the last years in several sites. Therefore, experimental
setups employing soil, especially under real agricultural systems, are imperative for
evaluating PhACs fate in the terrestrial environment, since soil is a living, dynamic system
that greatly influences the fate and bioaccessibility of organic micropollutants. Once
introduced to the soil via WW irrigation, PhACs may undergo changes through several
chemical, physical and biological processes. Thus, the fate and concentration of PhACs in
soil is closely related to their physicochemical properties and the characteristics of the soil.
PhACs may be retained in the top soil layer or they may be leached to deeper layers or even
reach the groundwater table, runoff into surface waters, interact with soil microbial
community and biodegraded, or up taken by plants. PhACs were not detected in the control
TW irrigated soil, because the TW water was free from PhACs and the presence of stainless-
steel sheets between the experimental treatments provided a proper isolation of the
treatments. This is of particular importance, found PhACs on average of 0.2 mg kg1 in soils
under rainfed agriculture, as a result of the deposition of wastewater aerosol or wastewater

8
irrigated soil due to wind erosion, or transport of soil material between field with farm
machinery. The concentration of PhACs in the topsoil was significantly affected by the
duration of irrigation and the origin of the WW applied for irrigation. None of the targeted
PhACs displayed an obvious cumulative or increasing pattern with increasing time of WW
irrigation. Noteworthy, the concentrations of targeted PhACs in the soil were higher and
followed different pattern compared to the concentrations found in the WW used for irrigation,
as higher PhACs in the WW used for irrigation does not necessary implies higher
concentration in the soil, as well. Indicative is the fact that despite the higher SMX and TMP
concentration in the MWTP I WW compared with MWTP II WW during the 3rd year of the
study, their respective values in the soil sampled from MWTP I WW irrigated soil were lower
compared to the ones from MWTP II WW irrigated soil. Moreover, SMX and TMP tend to
accumulate in the soil at higher levels compared with DCF. Similar concentrations were
reported as a result of short- (single growing period) and long-term (20e100 years) WW
irrigation. The variation of the concentration of studied PhACs throughout the growing
season in the 3rd year of study is in accordance with the findings, who monitored the
presence and distribution of TMP and SMX (among other PhACs) in WW irrigated soils
throughout a whole growing season. Given the alkaline nature of both the irrigation water
sources used (pH ranged from 8.04 to 8.45) and the soil (pH mean value 8.74), as well as
the acid dissociation constants of examined PhACs, both DCF and SMX were expected to be
exclusively found in their ionic form, whereas TMP in its neutral form. The negative charge of
DCF and SMX may counteract their absorption to the negatively charged clay and soil
colloids and moieties of soil organic matter, simultaneously facilitating their bioaccessibility
for degradation and plant uptake. On the other hand, TMP may be prone to leaching to
deeper soil layers, as well to microbial degradation and plant uptake. The lack of DCF
accumulation in soil during the three years of WW irrigation in this study is in line with
previous results reporting that the concentrations of DCF and other acidic PhACs in
agricultural soil in Mexico did not increase with increasing time of WW irrigation (90e100
years). The poor retardation of DCF in soils may be attributed to its poor absorption and
leaching potential, as well as to its high microbial degradation. Previous fate studies showed
that DCF is not persistent and that is readily biodegradable in soil; its degradation has been
found to follow the first-order exponential decay model, while its half-life (t1/2) ranged from
0.4 to less than 5 days. SMX and TMP displayed significantly higher soil concentrations
compared to DCF. SMX displayed the highest soil concentration among the three studied
PhACs at the end of the 3rd year of the study. The dissipation rate of SMX and TMP
depends on the physico-chemical properties of the soil and the extend of its aeration. The
reported half-life values of SMX are contradictive. reported SMX half-life value of 2 days in
clay loam under aerobic conditions, whereas half-life values of 9 and 20 days in medium
loam and clay loam were reported, respectively. On the other hand, reported that SMX is not
bioavailable and therefore persistent to microbial degradation. found that SMX
concentrations in soil rapidly increased with increasing duration of WW irrigation until no
further increase of concentration could be observed in soils irrigated for approximately 25
years. The reported half-life for TMP ranged from 4 days in aerated clay loam to more than
84 days in medium loam, highlighting its persistence in soils. In addition, TMP displayed
declining absorption coefficient with increasing pH values. Overall, both SMX and TMP
concentrations in soil varied during the experimental period in the present study, until a
steady state concentration between input and dissipation could be achieved.

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3.3. PhACs' concentration in tomato fruits and bioconcentration factor:
The concentration of selected PhACs in tomato fruits varied depending on the duration of
WW irrigation and the irrigation water source applied (MWTP I and II WW). DCF was found
below the detection limit in fruits harvested during the 1st year of the experiment from MWTP
I WW irrigated plants, while it was also absent in fruits harvested during the first two years
and the 1st harvest of the 3rd year from MWTP II WW irrigated plants. Moreover, DCF
concentration in fruits displayed an increasing pattern from the 1st to the last harvest in the
3rd year of the experiment, while also displayed the highest fruit concentration among all
tested PhACs. SMX and TMP were detected in tomato fruits in all harvest events throughout
the study period. The highest fruit concentration for both the SMX and TMP was recorder in
fruits harvested during the 3rd year of the study. Intriguingly, the concentration of both SMX
and TMP in fruits displayed an increasing pattern during the three harvests taken place at the
3rd year in MWTP II WW irrigated plants, whereas no obvious pattern was recorded in
MWTP I WW irrigated plants . The absence of DCF in tomato fruits harvested during the 1 st
year of the experiment from MWTP I WW irrigated plants, and from the first two years and
the 1st harvest of the 3rd year from MWTP II WW irrigated plants may be attributed to the
rapid dissipation of DCF in soil, to its low lipophilicity, as well as to its anionic form found in
soil and soil solution, which restricts its uptake by roots and transport to the aboveground
part (fruits) of tomato plant. However, the results of this study revealed that the long-term
(more than a single year) WW irrigation may result in the significant uptake and
bioaccumulation of DCF in tomato fruits. The concentration equilibrium reached between
DCF input and dissipation during a prolonged WW irrigation period may result in the
existence of a readily available DCF concentration in soil for plant uptake. DCF in its ionic
form in soil may penetrate the root by water mass flow but to a lesser extent than neutral
compounds, since it is repelled by the negatively charged cell walls. However, the pKa
values of DCF suggest its partial existence in non-ionic form in the rhizoplane, which allows
its dissolution into the membranes and release to the cytosol. Though, once in the cytosol,
DCF retains its ionic form, which may result to its ion trapping in the cytosol. As a result,
DCF, as well as other weakly acidic PhACs, such as SMX, are expected to preferentially be
translocated in the phloem rather than in the xylem, having as a result their accumulation in
the fruits rather than in the leaves. SMX is more hydrophilic with slightly higher pKa value
than DCF; thus, it may exhibit similar uptake pattern with DCF. The continuous detection of
SMX in all harvest events throughout the three years of the experimental period, in contrast
to DCF, may be attributed to the fact that SMX was found in higher concentration in soil
compared to DCF, due to its higher persistence. Interestingly, found SMX in the leaves but
not in the fruits of tomato plants irrigated with WW for a single growing period, while reported
the absence of SMX from the fruits of pepper, cucumber and tomato plants grown in field
during a single growing period and irrigated with WW. TMP exhibits several charged species
within the soil and plant pH range. Its neutral form in the soil facilitates its uptake from the
roots, while it may be also translocated through the transpiration mass flow as it partially
retains its neutral form (~30%) in the cytosol (pH 7.5) (it may be also trapped in the cytosol in
its cationic form). These can potentially explain why TMP was regularly found in tomato fruits.
The ability of the studied PhACs to translocate within the tomato plant vascular system and
accumulate in the fruits was estimated based on the BCFF. Results revealed that DCF
displayed the highest ability for bioconcentration in tomato fruits after prolonged wastewater
irrigation in field. DCF was not accumulated in tomato fruits irrigated with both treated
effluents used during the 1st year of the study, neither in the 2 nd year in MWTP II WW
irrigated plants. However, it displayed high BCFF values during the 3rd year of the study, as
it was found in high concentration in fruits compared to the other PhACs studied. They found
higher SMX concentrations in the leaves of cucumber and tomato plants grown in sand

10
rather than in aeollian and alluvial soil as a result of irrigation with WW or spiked WW, while
no SMX was found in the fruits of these plants. Overall, BCF values in the current study
suggest that both SMX and TMP may be translocated within the tomato plant vascular
system at moderate rates, whereas DCF can be translocated in higher rates, only if plants
are exposed to this PhAC for a prolonged period.

3.4. Public health implications/risk assessment:

Health risks associated with the consumption of wastewater irrigated tomato fruits throughout
the three years of the experimental period were estimated by using the TTC value, as well as
the HQ approach, for all of the three studied PhACs. The estimated TTC values demonstrate
that the daily consumption of tomatoes irrigated for a long term with the tertiary treated
wastewater used in this study does not pose a health threat, as the weight of tomato
consumption for an adult or a toddler in order to reach the TTC for any of the studied PhACs
is 9.04 and 1.55 kg day1 , respectively. The absence of health implications due to the
consumption of wastewater irrigated tomatoes is further corroborated by the estimated low
values . Although the present results suggest that the consumption of one kind of vegetable
species (i.e. tomatoes) grown in field and irrigated for consecutive years with the studied
treated effluents with the particular concentration of PhACs should be considered as safe,
more studies are needed in order to reach a definite conclusion for the classification of
wastewater reuse as a safe practice regarding human health. Such studies should take into
account the consumption, at the same time, of various other vegetables that may
bioaccumulate higher PhACs concentrations, the potential additivity of the mixture of few
dozen PhACs that may present in WW, the metabolites of PhACs that may present in plant
tissues in concentrations that may reach or exceed the ones of the parent compound while
also sometimes being more toxic than the parent compound, the potential sensitivity of
subgroups of the population (i.e. pregnant, infants, elderly people, and chronic sufferers), the
dietary habits of the population studied, as well as the fact that vegetables consumed in a
region may be both wastewater- or tube well and surface water-irrigated.

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4. Conclusion:

The reuse of treated WW for irrigation and the application of biosolids and manure as soil
amendments have been found to facilitate the uptake of PhACs by plants and their
subsequent entrance to the food web, representing important alternative pathways for the
exposure of humans to PhACs, with potential health implications. The present study sheds
light to the fate of the studied PhACs in real agricultural systems and their uptake and fruit
bioaccumulation by a widely consumed vegetable crop, such as tomato, by incorporating two
distinguished treated flows for irrigation under field conditions for a prolonged period (three
years), as well as by analyzing the concentration of PhACs in all involved matrices (WW, soil,
tomato fruits). Results revealed that the concentration of the studied PhACs in soil and fruits
varied, depending on the duration of irrigation and the origin of the WW applied, as well as
the physicochemical properties of PhACs. Noteworthy, the long-term (more than a single
growing period) WW irrigation may facilitate the uptake of acidic PhACs (i.e., DCF, SMX) by
plants and their subsequent bioconcentration in fruits, in contrast to their absence from fruits
harvested from plants that have been wastewater-irrigated for a single growing period. The
concentration of the studied PhACs in tomato fruits varied both inter-annually and
seasonally, and increased with increased duration of WW irrigation, reaching maximum
concentration during the last year of study, highlighting the need for long-term studies
utilizing WW irrigation under field conditions.
Although the estimated TTC and HQ values revealed that the consumption of fruits harvested
from tomato plants irrigated for long period with the WW applied for irrigation under field
conditions in this study represent a de minimis risk to human health with regard to the studied
PhACs.

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