Water Air Soil Pollut (2017) 228: 134

DOI 10.1007/s11270-017-3319-3

Fate of Fenhexamid in Water-Sediment Systems: Degradation

Under Aerobic/Anaerobic Conditions and Bioaccumulation
by Zebrafish (Danio rerio)
Zhenlan Xu & Xiuqing Hu & Min Wu & Tao Tang & Changpeng Zhang & Hongmei He &
Jianzhong Yu & Fangyuan Lou & Yuanyuan Wu & Yanhua Wang & Liezhong Chen &
Hua Zhao & Qiang Wang & Leiming Cai

Received: 19 December 2016 / Accepted: 24 February 2017 / Published online: 10 March 2017
# Springer International Publishing Switzerland 2017

Abstract Little is known about the environmental be-
havior of fenhexamid (FEN) in aquatic ecosystems such
as degradation and bioaccumulation, in spite of the fact
that it is critical for a comprehensive assessment of its
ecological risks. This study investigated for the first time
the degradation of FEN in water-sediment systems un-
der both aerobic and anaerobic conditions and also
bioaccumulation by zebrafish (Danio rerio). Water and
sediments from different natural waters including river
HR and lake HL were applied to build up water-
sediment microcosms in the laboratory. When FEN
was introduced into the aqueous phase, it would parti-
tion from water to sediment gradually and be
decomposed in sediment compartment. The dissipation
half-lives of FEN in water were 43.8, 75.9, 31.3, and
37.2 days for HR-aerobic, HR-anaerobic, HL-aerobic,
and HL-anaerobic microcosms, respectively. Moreover,
FEN degradation rate constants of whole systems varied
from 0.0045 to 0.0088 per day and the half-lives were
Electronic supplementary material The online version of this
article (doi:10.1007/s11270-017-3319-3) contains supplementary
material, which is available to authorized users.
Z. Xu (*) : X. Hu : M. Wu : T. Tang : C. Zhang : H. He :
J. Yu : F. Lou : Y. Wu : Y. Wang : L. Chen : H. Zhao :
Q. Wang : L. Cai (*)
State Key Laboratory Breeding Base for Zhejiang Sustainable Pest
and Disease Control, MOA Key Laboratory for Pesticide Residue
Detection, Institute of Quality and Standard for Agro-Products,
Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
Zhejiang, China
e-mail: xuzhenlan330@163.com
e-mail: caileiming2009@gmail.com
from 78.4 to 155 days. The aerobic circumstances were
demonstrated to be favor of FEN degradation. The
bioconcentration factor (BCF) was 2.6–3.1 obtained
from zebrafish exposure experiments at environmental-
ly relevant concentrations. Clearly, our results indicated
that FEN could be accumulated in the deeper layer of
sediment owing to the anaerobic condition against FEN
degradation, but FEN showed a low potential for bioac-
cumulation. These may aid in comprehensive under-
standing the fate and risk of FEN in aquatic
environment.
Keywords Fenhexamid . Degradation .
Bioaccumulation . Water-sediment system
1 Introduction
Fenhexamid (N-(2,3-dichloro-4-hydroxyphenyl)-1-
methylcyclohexanecarboxamide), named FEN for short
in this study, was introduced in 1999 as a
hydroxyanilide fungicide showing strong and preven-
tive activity against Botrytis cinerea and related patho-
gens, e.g., Monilinia and Sclerotinia in fruits, vegeta-
bles, and ornamental plants (Rosslenbroich and Stuebler
2000). This disease, namely grey mould, considerably
affects crops not only in the field but also during trans-
port and storage of ripe fruits, even at refrigerated tem-
peratures. FEN inhibits the biosynthesis of ergosterol
via depressing the 3-keto reductase of the enzymatic
complex of the sterol C-4 demethylation, which leads
134 Page 2 of 10
to the inhibition of germ tube elongation and mycelium
growth (Debieu et al. 2001). This new mode of action
makes FEN critically valuable for anti-resistance man-
agement because of no cross-resistance to any other
botryticides.
Like other pesticides, FEN is unavoidably released
into natural aquatic systems via spray drifting, drainage,
waste disposal, domestic or agricultural effluent, and
surface runoff (Narushima et al. 2014). The predicted
environmental concentration (PEC) of FEN in water
was estimated to be between 0.307 and 0.683 mg/L
when annual cumulative application rates of 921–
2049 g active ingredient (a.i.) per hectare for grapes,
ornamentals, and strawberry were considered (PMRA
2003). Owing to its very low vapor pressure (4 × 10−7
Pa, 20 °C), stability to hydrolysis, low solubility in
water (20 mg/L at pH 5–7) and partition coefficient
log Kow of 3.5 (pH 7, 20 °C) (Abbate et al. 2007),
FEN is prone to partition to sediment in aquatic systems.
Thus, it is of environmental relevance to carry out
extensive studies on the degradation of FEN in aquatic
systems, especially the biotic degradation in sediment.
However, little is known about the degradation of FEN
in sediment.
In aquatic systems, fishes are particular sensitive to
waterborne pesticides since they are able to uptake and
retain pesticides through any route, including inhalation,
ingestion, or direct contact (El-Amrani et al. 2012). FEN
is a potential endocrine disruptor and has been observed
to be an ERα agonist in vitro and act like ER agonist
with endogenous ERα in MCF-7 cells (Medjakovic
et al. 2014). The accumulated FEN may induce physio-
logical impairment of fish body, and/or have a poten-
tially adverse effect on human health when people con-
sume the contaminated fishes. In addition to the degra-
dation of FEN in sediment, whether and to what extent
FEN can be accumulated by fish are remained un-
known. Zebrafish (Danio rerio) is proposed as a test
organism by the OECD (1998), and considered as an
alternative species for evaluation of anthropogenic
chemicals. The investigation of FEN bioaccumulated
by Zebrafish (D. rerio) can be used to as an indication
of its bioaccumulation potential via BCF
(bioconcentration factor) value.
In the present work, attempts were made to study the
aerobic and anaerobic degradation of FEN by native
microbial communities in two aquatic microcosms, re-
spectively and the bioaccumulation of FEN by zebrafish
(D. rerio) at environmentally relevant concentrations.
Water Air Soil Pollut (2017) 228: 134
The results would provide a deep insight on the fate of
FEN in water-sediment systems and its risk to aquatic
ecosystems.
2 Materials and Methods
2.1 Materials and Reagents
The FEN standard (purity, 99.5%) was obtained from
Dr. Ehrenstorfer (Augsburg, Germany). The FEN tech-
nical material (purity, 98%) was donated by the manu-
facturer. Acetonitrile (chromatography grade) was sup-
plied by Burdick & Jackson (Ulsan, Korea). Other sol-
vents and reagents used in this study were analytical
grade and purchased from Shanghai Lingfeng Chemical
Regent Co., Ltd. (Shanghai, China). Sorbent PSA (par-
ticle size of 40–60 μm) and C18 (particle size of 40–
60 μm) were purchased from Agela Technologies (Tian-
jin, China).
Standard stock solution of FEN standard (99.5 mg/L)
was prepared in acetonitrile, and then diluted to obtain
eight working solutions for building up the calibration
curve. Stock solutions of 98% FEN technical material
dissolved in acetone were established at concentrations
of 1.0 × 103 and 1.08 × 104 mg/L, which were used in
the degradation and bioaccumulation experiments, re-
spectively. All solutions were stored in the dark at 4 °C.
2.2 Sediments
Two types of sediment (5–10 cm upper layer) and
associated water were collected from two sites located
at Beijing-Hangzhou Grand Canal (Hangzhou section,
120.14° E, 30.33°N) and West Lake (120.13° E,
30.24°N), Hangzhou, respectively. The two sampling
sites were denoted as HR and HL, respectively. After-
ward, the sediments were wet-sieved through a 2-mm
sieve. The water and sediment were stored at 4 °C until
the setup of experimental microcosms. The storage du-
ration was shorter than 3 weeks. The properties of water
and sediment are shown in Table 1.
2.3 Zebrafish
Adult zebrafish (D. rerio) of both sexes (fork length of
2.9–3.1 cm and wet body weight of 0.18–0.26 g) were
purchased from China Zebrafish Resource Center (Wu-
han, China) and allowed to acclimate to laboratory
Water Air Soil Pollut (2017) 228: 134 Page 3 of 10 134

Table 1 The physicochemical properties of water and sediment range. Three fish and 10 mL of water per tank were
Parameters HL HR collected on 0, 1, 2, 4, 6, and 8 days.

Water pH 7.53 7.76 2.5 Degradation Experiment

Oxidation reduction potential 141 160
(ORP, mv)
Sediment pH 7.22 7.99
Content of organic matter (%) 18.3 1.26
Cation exchange capacity 47.1 10.0
(CEC, meq/100 g)
Oxidation reduction potential −123 −159
(ORP, mv)
Water content (%) 230.8 63.53
Sand (>0.05 mm) 21.4 19.4
Silt + clay (<0.05 mm) 78.6 80.6
conditions for 2 weeks prior to FEN exposure. The fish
were maintained in charcoal-dechlorinated water at a
constant temperature of 23 ± 1 °C with a photoperiod
of 14 h/10 h (light/dark). They were fed with a commer-
cial fish food twice a day until 1 day before bioaccumu-
lation experiments. The aquarium was equipped with
aeration devices. The total hardness of water was 72 ±
1 mg/L as CaCO3. The pH was 7.5 ± 0.5 and the dis-
solved oxygen content was 80 ± 5% of saturated dis-
solved oxygen. All zebrafish were healthy, and during
acclimation, the zebrafish mortality rate was less than
2%.
2.4 Fish Exposure and Sample Collection
Bioaccumulation experiments were carried out follow-
ing the OECD Guideline 305 (OECD 2012). Fish were
randomly selected for exposure experiments and not fed
during the exposure. Exposure microcosm consisted of
a glass aquarium containing 50 adult zebrafish and 15 L
of water. FEN was spiked at concentration of 1 or
0.1 mg/L. Final concentration of solvent (acetone) in
the exposure media was <1 μL/L. Two replicates for
each concentration level were performed. One control
with the same amount of acetone but without FEN was
set. Exposure doses selected in this study were on the
basis of LC50 (96 h) value of 11.4 mg/L obtained in
previous acute toxicity test (unpublished data), and cov-
ered the PEC of FEN in water of 0.307–0.683 mg/L
(PMRA 2003). The experiments were conducted under
semi-static condition and lasted 8 days. The solutions in
the aquaria were renewed every 96 h to ensure a con-
sistent exposure concentration during the entire time
The degradation experiment was conducted according
to the OECD Guideline 308 (OECD 2002) in 500 mL
serum bottles. Four sets of degradation experiments
were performed: (1) aerobic degradation in HL water-
sediment microcosm, (2) aerobic degradation in HR
water-sediment microcosm, (3) anaerobic degradation
in HL water-sediment microcosm, and (4) anaerobic
degradation in HR water-sediment microcosm. For each
set, 30 bottles were prepared and each bottle was filled
with 170 g (wet weight, ww) of sediment and certain
amount of associated water (a water/sediment volume
ratio of 3:1). The bottles were ventilated with nitrogen
by gentle bubbling for anaerobic degradation experi-
ment, while they were ventilated with air for aerobic
degradation. They were kept with ventilation at 22 ±
2 °C in the dark for 16 days. Twenty-eight out of 30
bottles were spiked with FEN to obtain a concentration
of 2.0 mg/L in water (approximately 3–5 times of PEC
value in water) (PMRA 2003) and the water phase was
gently stirred for approximately 1 min. Two bottles for
each set were collected periodically, and water and
sediment samples were then processed separately for
analysis of FEN residue. The remaining two bottles
were without FEN and performed as controls. They
were collected at the end of the incubation for Biolog-
ECO plates (BIOLOG, Hayward, USA) test (section 1
in the Supplementary Information).
2.6 Sample Preparation and Instrumental Analysis
Water samples: 20 μL of formic acid was added into
10 mL of water sample. Then, the water sample was
extracted by dichloromethane twice with 20 mL for
each. The dichloromethane phase was collected and
combined. An aliquot of 10 mL extract was evaporated
and reconstructed with 5 mL of methanol. An aliquot of
2 mL final sample was filtered by a 0.22-μm nylon
syringe filter for analysis.
Sediment samples: 20 g of sediment sample was
weighed into a 100-mL centrifuge tube. 60 mL of ace-
tonitrile was added. The tube was capped and then
shaken for 60 min. Afterward, 10 g of NaCl was added,
and the tube was vortexed for 1 min. The mixture was
centrifuged at 4000 r/min for 10 min. 5.0 mL of the
134 Page 4 of 10
upper layer (acetonitrile) was transferred into a tube
containing 200 mg of C18, 20 mg of PSA and 500 mg
of anhydrous MgSO4. 50 μL of formic acid was added,
and then the sample was vortexed for 1 min. After being
centrifuged at 4000 r/min for 5 min, the supernatant was
then filtered with a 0.22-μm nylon syringe filter for
sample injection.
Fish samples: Fish sample collected was firstly rinsed
with distilled water for five times to remove FEN on fish
surface. Then, the fish sample together with 2 g of
anhydrous sodium sulfate were grounded manually
and extracted with 20 mL of acetonitrile. The superna-
tant was decanted after centrifugation at 10,000 r/min
for 5 min. Extraction was repeated again with another
20 mL of acetonitrile by votexing for 2 min and centri-
fugation. Then, the combined extracts were cleaned by
acetonitrile-hexane partitioning (1/1, v/v) twice. After
solvent evaporation by arotary evaporator, extract was
dissolved with 2 mL of methanol and passed through a
0.22-μm nylon syringe filter before injection.
FEN was measured using an ultra-fast liquid chro-
matography coupled with tandem mass spectrometry
(LC-MS/MS, LCMS 8050, Shimadzu, Japan). The LC
was equipped with a Waters Acquity UPLC BEH C18
column (2.1 mm × 100 mm, 1.7 μm particle size, Mil-
ford, MA, USA). The mobile phase consisted of 0.1%
formic acid in water (A) and acetonitrile (B) with a ratio
of 70/30 (v/v), and the flow rate was 0.20 mL/min. The
sample injection volume was 2 μL. The column and
sample manager were kept at 40 and 4 °C, respectively.
The mass system was equipped with an electrospray
ionization (ESI) source operating in the positive mode.
The nebulizer and drying gas were 99.95% nitrogen,
and their flow rates were 3.0 and 10.0 L/min, respec-
tively. The heating gas was 99.95% air with a flow rate
of 10.0 L/min. The collision gas was 99.99% argon with
a pressure of 270 kPa. Other parameters were as follows:
interface voltage 4.0 kV, interface temperature 300 °C,
DL temperature 250 °C, heat block temperature 400 °C,
and detector voltage 1.82 kV. Data was collected in the
multiple reaction monitoring (MRM) mode. MRM tran-
sitions of 302.0 > 97.2 and 302.0 > 55.1 were used as
quantifier and qualifier, respectively. The retention time
of FEN was approximately 2.0 min (Fig. 1).
2.7 Sediment Microbial Biomass
The sediment microbial biomass (SMBC) was deter-
mined using chloroform fumigation-extraction method
Water Air Soil Pollut (2017) 228: 134
(Vance et al. 1987). The soluble carbohydrate was ex-
tracted with 0.5 M K2SO4. The carbon content of both
fumigated and unfumigated samples was analyzed using
a total organic carbon analyzer (Shimadzu, TOC-V
CSH, Japan).
2.8 Calculations
The Origin 8.0 software (OriginLab, Northampton, MA,
USA) was used for data analysis. Values below the
LODs were set as zero in the calculation. FEN in the
whole system was expressed as the absolute amount of
FEN (μg), which was the sum of FEN in water and
sediment. A kinetic study of FEN in water and whole
system was performed and the degradation rate constant
k was calculated from Eq. (1), and the half-life (T1/2, d)
was estimated from Eq. (2) (Zhang et al. 2015):
C t ¼ C 0 e−kt ð1Þ
ln2
T 1=2 ¼ ð2Þ
k
The sediment microbial biomass carbon (SMBC)
was calculated using Eqs. (3) and (4).
SMBC ¼ 2:64 EC ð3Þ
EC ¼ C f ðextracted from fumigated sedimentÞ−C n
ðextracted from non fumigated sedimentÞ
ð4Þ
When the equilibrium state was achieved, the
bioconcentration factor (BCF) of FEN in zebrafish was
estimated as the ratio between the concentration in fish
(Cf) and that in the surrounding water (CW) at equilib-
rium. The t test was used to explore whether the SMBCs
were significantly different between various micro-
cosms with a significance level of 0.05.
3 Results and Discussion
3.1 Analytical Method Validation
Linear range, recoveries, accuracy, and sensitivity were
examined to evaluate the analytical method used in this
study. Spiked samples with 0.0095 and 3.98 mg/L of
Water Air Soil Pollut (2017) 228: 134
Fig. 1 Chromatography of FEN in fish sample collected on the eighth day (exposure level 1.0 mg/L)
FEN for water, 0.0995 and 4.98 mg/kg for sediment and
0.00498, 1.99 and 240 mg/kg for fish, were analyzed
according to the described methodology to check recov-
eries. An external standard calibration curve with the
linear range of 10 to 200 μg/L (R2 = 0.999) was used to
calculate the amount of FEN. The recoveries were in the
range of 77–106% for water, 81–107% for sediment and
86–105% for fish, respectively. The RSDs (n = 5) of the
method varied from 1.3 to 7.0% for three matrices. The
LOD, value corresponding to a signal-to-noise ratio of 3
(S/N = 3), was 1.4 μg/kg for water and sediment, and
1.6 μg/kg for fish. The LOQ was determined as a value
corresponding to an S/N of 10. It was 4.5 μg/kg for
sediment and water, and 5.4 μg/kg for fish. The method
for FEN analysis in crops was intensively developed on
the basis of enzyme-linked immunosorbent assay, GC,
GC-MS, and LC-MS/MS with LOQs of 5–120 μg/kg
(Čuš et al. 2010; Francesc et al. 2011; Hem et al. 2011;
Łozowicka et al. 2012). Whole fish was used for anal-
ysis of FEN in this study, and fats and lipids in fish
sample may induce matrix effects or decrease the sensi-
tivity of the method. The acetonitrile-hexane
partitioning method with low cost was applied into the
Page 5 of 10 134
clean-up of fish samples, resulting in satisfactory recov-
eries of 86–105%. Also, the LOQ was 5.4 μg/kg, which
is at the lower end of LOQ range documented for crops
(Čuš et al. 2010; Francesc et al. 2011; Hem et al. 2011;
Łozowicka et al. 2012).
3.2 Degradation of FEN in Different Microcosms
Chemical characterization of the freshly collected sedi-
ment and associated water samples revealed that they
did not contain FEN at concentrations above LOD
values. The degradation of FEN in two water-sediment
systems was investigated under both aerobic and anaer-
obic conditions. Figure 2 shows the dynamic curves of
FEN in water phase, sediment phase and the whole
system. The concentration of FEN in sediment was
initially increased and then descended gradually. It was
peaked approximately on the 40th day in the HL micro-
cosm, while it reached the maximum value during the
50th–60th day in the HR microcosm. In the initial stage,
the increase of FEN concentrations in sediment com-
bined with a small decrease in its total amount in whole
system indicated the dominance of the partition of FEN
134 Page 6 of 10
from water to sediment. Though the log Kow value was
high for FEN (Abbate et al. 2007), the quick adsorption
of FEN to sediment was not observed. This is mainly
due to the lack of mixing and turbulence in the system in
order to mimic the natural aquatic condition, which
hindered the mass transfer between water and
sediment. Similar results were observed by Wang et al.
(2016) and Liu et al. (2015). The maximum concentra-
tions in sediment of HL microcosms were found to be
higher than those in HR microcosms, which was prob-
ably related with the content of the organic matter in
sediment (Huang et al. 2014). The content of organic
matter of 18.3% in HL sediment was higher than that of
1.26% in HR sediment. Pinna et al. (2008) have reported
that the organic fraction in solid lees was the reason for
the enhancement of FEN adsorption on soil amended
with solid lees.
A continuous decrease of FEN in water and sediment
in the next 150–170 days was observed in all micro-
cosms leading to the descending of its total amount in
whole systems. Since FEN was recalcitrant to the hy-
drolysis (Abbate et al. 2007), the degradation of FEN by
sediment microbes was attributed to the decrease of
FEN in whole system. The significance of sediment
microbes in degrading of FEN in aquatic environment
has been demonstrated by Wang et al. (2016). However,
FEN can be detected with concentrations above 0.1 mg/
L at 127 days, indicating the long-term existence of FEN
in water compartment. This was mainly because of the
relative static state of the water and sediment phase. The
phenomenon is in agreement with observations of Wang
et al. (2016) and Liu et al. (2015). However, it contra-
dicts the result of Volpe et al. (2017), who observed that
3-(4-methylbenzylidene) camphor was completely
adsorbed by sediment soon after spiking and it was not
detected in aqueous phase when a magnetic stir bar was
used to mix the sediment and water.
The dissipation kinetics of FEN in aqueous phase
was investigated. As listed in Table 2, FEN dissipation
rate constants are 0.016, 0.0091, 0.022, and 0.019 per
day for HR-aerobic, HR-anaerobic, HL-aerobic, and
HL-anaerobic microcosms, respectively. The corre-
sponding half-lives were 43.8, 75.9, 31.3, and 37.2 days.
Moreover, the degradation kinetic of FEN in whole
system was also explored. FEN degradation rate con-
stants varied from 0.0045 to 0.0088 per day and the half-
lives were from 78.4 to 155 days. The half-lives are two
magnitudes longer than those of <1 day in soil
(Brumhard and Bornatsch 1996), and also substantially
Water Air Soil Pollut (2017) 228: 134
Fig. 2 The degradation curve of FEN in water-sediment systems.„
a HL-aerobic microcosm. b HR-aerobic microcosm. c HL-
anaerobic microcosm. d HR-anaerobic microcosm
longer than values of 2–15 days in sediment observed
by Brumhard and Bornatsch (1997). In this study, the
degradation half-lives were in the order of HL-aerobic <
HR-aerobic < HL-anaerobic < HR-anaerobic. After
215 days’ incubation, a residual FEN rate of less than
10% was found in HL-aerobic, HR-aerobic, and HL-
anaerobic microcosms while 27% in HR-anaerobic mi-
crocosm. The high potential of FEN degradation was
observed under aerobic condition compared to that un-
der anaerobic condition, indicating that aerobic circum-
stance favored the degradation of FEN in sediment. The
microbial population is probably attributed to the supe-
rior degradation of FEN in aerobic circumstance (Chang
et al. 2012a; Peng et al. 2015; Zhang et al. 2015). The
SMBCs of HL-aerobic, HR-aerobic, HL-anaerobic, and
HR-anaerobic were 363.1 ± 22.6 (n = 3), 263.1 ± 18.0
(n = 3), 392.8 ± 74.8 (n = 3), and 211.7 ± 2.35 (n = 3)
mg/kg, respectively. The SMBC in HR-aerobic is sig-
nificantly larger than that in HR-anaerobic (t test,
p < 0.05). However, there is no significant difference
in SMBC between HL-aerobic and HL-anaerobic (t
test, p > 0.05), implying that factors other than micro-
bial population play a role in FEN degradation. As
reported, microbial community is a key factor in the
chemical decompositions in sediment; sulfate-
reducing bacteria and methanogens constituted a ma-
jor microbial component in sediment during
tetrabromobisphenol-A (TBBPA) anaerobic degrada-
tion (Chang et al. 2012b), whereas Bacillus pumilus
was the dominant bacteria in TBBPA aerobic degra-
dation in sediment (Chang et al. 2012a). Therefore,
communities in sediment under anaerobic and aerobic
conditions are probably responsible for the distinct
FEN degradation in this study. However, further study
is needed. Additionally, the sediment from HL had a
higher capacity of FEN degradation than HR sedi-
ment. This may be caused by the different microbial
biomass and diversity in sediment exposed to aerobic
and anaerobic conditions. Under both conditions,
SMBC in HL microcosms is significantly higher than
that in HR microcosms (t test, p < 0.05).
3.3 Bioaccumulation of FEN by Zebrafish
The concentration of FEN in the exposure media
and zebrafish were investigated (Fig. 3). No FEN
Water Air Soil Pollut (2017) 228: 134 Page 7 of 10 134
134 Page 8 of 10 Water Air Soil Pollut (2017) 228: 134

Table 2 Degradation and dissipation kinetics of FEN in different microcosms

Microcosm Condition Compartment Regression equation Correlation coefficient (R2) Half-life

(T1/2, d)

HR microcosm Aerobic Water C = 1.970e−0.016t 0.982 43.8

Whole system C = 0.740e−0.0080t 0.893 86.1
−0.0091t
Anaerobic Water C = 2.048e 0.942 75.9
Whole system C = 0.737e−0.0045t 0.906 155
HL microcosm Aerobic Water C = 1.646e−0.022t 0.980 31.3
Whole system C = 0.768e−0.0088t 0.932 78.4
Anaerobic Water C = 1.679e−0.019t 0.990 37.2
−0.0066t
Whole system C = 0.726e 0.782 106

was detected in water and zebrafish of control on
any sampling day. The FEN concentration in water
phase was from 0.081 to 0.093 mg/L and from 0.83
to 0.90 mg/L for two exposure doses, respectively. It
is proposed that the FEN concentration in the expo-
sure media remained constant since deviations from
their nominal concentrations were always below
20%. The FEN concentration in zebrafish increased
from <LOD to 0.268 mg/kg for the low exposure
level, and from <LOD to 2.22 mg/kg for the high
exposure level. It was observed that FEN concentra-
tion in zebrafish was tended to be constant from the
fourth day, implying the equilibrium of FEN accu-
mulation by zebrafish. Therefore, the BCF was esti-
mated to be 3.1 and 2.6, respectively, which was
indicative of a low potential of FEN

Fig. 3 FEN concentrations in

water and zebrafish. a Exposure
level of 0.1 mg/L. b Exposure
level of 1.0 mg/L
Water Air Soil Pollut (2017) 228: 134
bioaccumulation by aquatic organisms. This is the
first study to determine the FEN bioaccumulation by
zebrafish; therefore, no comparison can be per-
formed with other studies. When compared the
BCF values of FEN to other pesticides, it is dramat-
ically lower than hexaconazole (BCF 82–144)
( Wa n g e t a l . 2 0 1 5 ) , a n d c o m p a r a b l e t o
fluoroglycofen-ethyl (BCF 1.2–2.4) (Zou et al.
2006) and omethoate (BCF 1.5–5.8) (Song and
Zhang 2016).
3.4 Environmental Implications
Normally, the conditions in natural aquatic environment
are aerobic in the upper water phase, either aerobic or
anaerobic in the surface layer of sediment, and anaero-
bic in the deeper sediment. Our study encompassed all
of these possibilities by conducting the degradation test
under both aerobic and anaerobic conditions. The results
implied that the degradation of FEN in natural aquatic
sediment systems was slow, and FEN would probably
be accumulated in the deeper layer of sediment. In spite
of the long-term existence of FEN in water, the potential
of its bioaccumulation by waterborne organisms may be
low when continuously exposed to environmentally
relevant concentrations. Additionally, the sediment mi-
crobial functional diversity was evaluated using Biolog-
ECO plates at the end of the incubation. The result
showed that there was no significant difference between
FEN-spiking treatment and non FEN-spiking control
(Table S1 in the Supplementary Information), indicating
that the long exposure to FEN would not lead to the
change in the sediment microbial community function.
4 Conclusions
Degradation of FEN in HL and HR microcosms was
explored. The degradation half-lives of FEN in whole
systems were in the order of HL-aerobic < HR-aerobic
< HL-anaerobic < HR-anaerobic, with values of 78.4–
155 days. Generally, the aerobic condition was favor of
FEN degradation. The BCF was 2.6–3.1 derived from
the zebrafish exposure experiments at environmentally
relevant concentrations. These results depict the main
environmental behaviors of FEN in aquatic environment
and can be a reference for the evaluation of its risks to
ecosystems. Future studies should be paid attention to
Page 9 of 10 134
the environmental behavior and toxicity of its
metabolites.
Acknowledgments This work was supported by the National
Natural Science Foundation of China [grant number 31401772],
Zhejiang Province of Natural Science Foundation [grant numbers
LQ16B070003, 2015C32039], the National Natural Science
Foundation of China [grant numbers 31501685, 31501668] and
Young Scientists Training Program of Zhejiang Academy of Ag-
ricultural Sciences. The authors are very grateful to anonymous
reviewers for helpful comments and suggestions.
References
Abbate, C., Borzì, D., Caboni, P., Baglieri, A., & Gennari, M.
(2007). Behavior of fenhexamid in soil and water. Journal of
Environmental Science and Health. Part. B, 42, 843–849.
Brumhard, B., Bornatsch, W. (1996). Aerobic degradation and
metabolism of KBR 2738 in soil. Interner Bericht, Bayer AG:
Leverkusen.
Brumhard, B., Bornatsch, W. (1997). Degradation and metabo-
lism of KBR 2738 in the system water/sediment. Interner
Bericht, Bayer AG: Leverkusen.
Chang, B. V., Yuan, S. Y., & Ren, Y. L. (2012a). Aerobic degra-
dation of tetrabromobisphenol-A by microbes in river sedi-
ment. Chemosphere, 87, 535–541.
Chang, B. V., Yuan, S. Y., & Ren, Y. L. (2012b). Anaerobic
degradation of tetrabromobisphenol-A in river sediment.
Ecological Engineering, 49, 73–76.
Čuš, F., Česnik, H. B., Bolta, Š. V., & Gregorčič, A. (2010).
Pesticide residues in grapes and during vinification process.
Food Control, 21, 1512–1518.
Debieu, D., Bach, J., Hugon, M., Malosse, C., & Leroux, P.
(2001). The hydroxyanilide fenhexamid, a new sterol bio-
synthesis inhibitor fungicide efficient against the plant path-
ogenic fungus Botryotinia fuckelinan (Botrytis cinerea). Pest
Management Science, 57, 1060–1067.
El-Amrani, S., Pena-Abaurrea, M., Sanz-Landaluze, J., Ramos, L.,
Guinea, J., & Camara, C. (2012). Bioconcentration of pesti-
cides in zebrafish eleutheroembryos (Danio rerio). Science of
the Total Environment, 425, 184–190.
Francesc, A. E.-T., Antonio, A.-F., & Josep, V. M. (2011).
Determination of fenhexamid residues in grape must, kiwi-
fruit, and strawberry samples by enzyme-linked immunosor-
bent assay. Food Chemistry, 124, 1727–1733.
Hem, L., Choi, J.-H., Park, J.-H., Mamun, M. I. R., Cho, S.-K., El-
Aty, A. M. A., & Shim, J.-H. (2011). Residual pattern of
fenhexamid on pepper fruits grown under greenhouse condi-
tions using HPLC and confirmation via tandem mass spec-
trometry. Food Chemistry, 126, 1533–1538.
Huang, X. L., Wu, C. X., Xiong, X., Zhang, K., & Liu, J. T.
(2014). Partitioning and degradation of triclosan and forma-
tion of methyl-triclosan in water-sediment systems. Water,
Air, & Soil Pollution, 225, 2099.
Liu, M., Liu, D., Xu, Y., Jing, X., Zhou, Z., & Wang, P. (2015).
Fate and stereoselective behavior of benalaxyl in a water-
sediment microcosm. Journal of Agricultural and Food
Chemistry, 63, 5205–5211.
134 Page 10 of 10
Łozowicka, B., Jankowska, M., & Kaczyński, P. (2012). Pesticide
residues in Brassica vegetables and exposure assessment of
consumers. Food Control, 25, 561–575.
Medjakovic, S., Zoechling, A., Gerster, P., Ivanova, M. M., Teng,
Y., Klinge, C. M., Schildberger, B., Gartner, M., &
Jungbauer, A. (2014). Effect of nonpersistent pesticides on
estrogen receptor, androgen receptor, and aryl hydrocarbon
receptor. Environmental Toxicology, 29, 1201–1216.
Narushima, T., Sato, T., Goto, Y., & Takahashi, Y. (2014).
Pesticides in river and tap water in a rice production area of
Niigata, Japan. Water, Air, & Soil Pollution, 225, 2229.
Organization for Economic Cooperation and Development
(OECD). (1998). Guide-lines for testing chemicals. No.
212. Paris.
Organization for Economic Cooperation and Development
(OECD). (2002). Guide-lines for testing chemicals. No.
308. Paris.
Organization for Economic Cooperation and Development
(OECD). (2012). Guide-lines for testing chemicals. No.
305. Paris.
Peng, Y. H., Chen, Y. J., Chang, Y. J., & Shinh, Y. H. (2015).
Biodegradation of bisphenol A with diverse microorganisms
from river sediment. Journal of Hazardous Materials, 286,
285–290.
Pinna, M. V., Budroni, M., Farris, G. A., & Pusino, A. (2008).
Fenhexamid adsorption behavior on soil amended with wine
lees. Journal of Agricultural and Food Chemistry, 56,
10824–10828.
PMRA-Pest Management Regulatory Agency of Health Canada.
(2003). Fenhexamid proposed regulatory decision document.
Water Air Soil Pollut (2017) 228: 134
http://publications.gc.ca/collections/Collection/H113-9-
2003-4E.pdf. Accessed on 19 Dec 2016.
Rosslenbroich, H.-J., & Stuebler, D. (2000). Botrytis cinerea-
history of chemical control and novel fungicides for its
management. Crop Protection, 19, 557–561.
Song, Z. H., & Zhang, Y. Y. (2016). Toxicity and bioaccumulation
of omethoate in Danio rerio. Journal of Hydroecology, 37,
89–94 (in Chinese).
Vance, E. D., Brookes, P. C., & Jenkinson, D. S. (1987). An
extraction method for measuring soil microbial biomass C.
Soil Biology and Biochemistry, 19, 703–707.
Volpe, A., Pagano, M., Mascolo, G., Grenni, P., & Rossetti, S.
(2017). Biodegradation of UV-filters in marine sediments.
Science of the Total Environment, 575, 448–457.
Wang, Y., Xu, L., Li, D., Teng, M., Zhang, R., Zhou, Z., & Zhu, W.
(2015). Enantioselective bioaccumulation of hexaconazole
and its toxic effects in adult zebrafish (Danio rerio).
Chemosphere, 138, 798–805.
Wang, S., Seiwert, B., Kästner, M., Miltner, A., Schäffer, A.,
Reemtsma, T., Yang, Q., & Nowak, K. M. (2016).
(Bio)degradation of glyphosate in water-sediment micro-
cosms-A stable isotope co-labeling approach. Water
Research, 99, 91–100.
Zhang, Q., Zhou, L. L., Yang, Y., Hua, X. D., Shi, H. Y., & Wang,
M. H. (2015). Study on the stereoselective degradation of
three triazole fungicides in sediment. Ecotoxicology and
Environmental Safety, 117, 1–6.
Zou, J. X., He, X. K., Tao, C. J., Piao, X. Y., & Jiang, H. (2006).
Acute toxicity and bio-concentration of fluoroglycofen-ethyl
to Brachydonio rerio. Chinese Journal of Pesticide Science,
8, 375–378 (in Chinese).