Introduction
Fisheries depletion has become a global problem and aroused attention
for decades (Hutchings et al. 2000; Worm et al. 2016). Of 4,714
fisheries assessed in 2012 around the world, 68% have slipped below the
biomass target that supports maximum sustainable yield (Worm et al.
2016), and some rivers and lakes even have experienced many local
fishery collapses (Mcintyre et al. 2016). While invasive species and
overfishing are usually proposed to be contributors, early life stage
mortality of fish is also a factor, due to its effect on population
maintenance. For example, mass hatchery stocking failed to re-establish
self-sustaining lake trout populations in the Great Lakes, USA, partly
due to the fact that 50% of offspring died at an early life-stage
(Smith et al. 1994; Palace et al. 1998).
Several man-made chemicals, mainly organochlorines, have been associated
with larval and adult mortality in wild fish (Smith et al. 1994; Palace
et al. 1998; Hamilton et al. 2016; Sepúlveda et al. 2004). Notably, fish
undergo a series of rapid cell divisions and differentiations during
embryonic development, including large-scale de novotranscription, and therefore fish embryos are much more sensitive to
environmental pollutants than larval and adult fish (Mohammed et al.
2013; Cherr et al. 2017). Thus, organochlorines could cause embryonic
mortality in wild fish and, over time, compromise natural population
recruitment and ultimately induce fishery collapse. However, there is
sparse evidence that widespread chemicals are lethal to embryos of wild
fish since collecting fish embryo and tracking subsequent development
are big challenges in aquatic ecosystems. Crucian carp (Carassius
carassius ) is ubiquitous in the lakes, rivers, and reservoirs, and
therefore has been usually used as a bio-indicator of fish resources
(Zheng et al. 2015). During the reproductive season, crucian carp spawns
adhesive eggs over substrates such as shallow-shore aquatic macrophyte,
where eggs remain until hatching (Shin-ichiro et al. 2011), facilitating
large-sample-size egg collection and observation. Such reproduction
feature of crucian carp give us a chance to observe whether a pollutant
could cause embryo mortality.
Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), a
chloro-organophosphorus flame retardant, is produced industrially in
high volumes. TDCIPP has been reported to cause embryo mortality in a
zebrafish embryo exposure ex vivo (Volz et al. 2016; Kupsco et
al. 2017). Therefore, in the present study, we utilized reproductive
characterization of crucian carp in natural population to address
following questions. (i) Does mass mortality of crucian carp embryo
occur in aqueous ecosystem? (ii) Is the embryonic mortality observed in
field associated with embryonic TDCIPP concentration; (iii) Can TDCIPP
at environmental levels cause the mass mortality of crucian carp embryos
under controlled laboratory conditions? We performed mortality
investigation and causal analyses by clarifying the temporal and spatial
distributions of embryonic mortality and TDCIPP concentrations in
crucian carp over three years in a lake ecosystem, and laboratory
exposure experiments were also conducted to determine whether
environmental concentrations of TDCIPP could cause embryonic mortality
in crucian carp from natural population.
Materials and Methods
Embryo Collection in Taihu Lake. In 2016 and 2017,
embryos of crucian carp were
collected from water grass in SZ, GH, MlL and ZS (see Table S1 for
location information) during the crucian carp reproduction season (April
to May), with the permission of the fishery administration. Fingerling
identification was based on the external characteristics and DNA barcode
analysis, as described in Supporting Information (SI). After collection,
embryos were transported to laboratory immediately with incubators at 18
± 2℃, at which temperature crucian carp embryos developed slowly
(Laurila et al. 1987), Then, the embryos were cultured until hatching in
active carbon-treated drinking water (1 L in 1.5 L-glass dishes; each
dish contained approximately 500 embryos) at 26 ± 1°C and under a 16 h:
8 h (light: dark) light cycle. The transport and culture conditions were
identical for all sampling sites. In 2018, embryos were collected from
GH and ZS by constructing artificial fish nests for locally tracking the
developmental progress of crucian carp embryo. Artificial nest was made
by fishing net by cutting into pieces and tied together to increase the
area for crucian carp to spawn. Artificial nests were put into shallow
water nearshore in GH and ZS according to a time schedule (Fig. 2 A and
B), and five nests were used in each site.
Embryos from four sites in Taihu Lake were collected for TDCIPP
analyzing prior to culture (approximately 50 mg of embryos per sample; n
= 6). The dead embryos and contemporaneous live embryos during culture
were also sampled to determine TDCIPP concentrations [approximately 50
mg of embryos per sample; n = 6 in 2017; n = 3 in 2018 (due to limited
numbers of dead embryos)].
Exposure Experiment. One- to three-year old wild adult crucian
carp including females and males, aged by measurement of dorsal spines
and pectoral fin rays according to a previous study (Vilizzi et al.
2018), were captured from a natural population in October 2017, and then
were exposed in a laboratory setting to TDCIPP for six months (October
2017–April 2018) at nominal concentrations of 8, 40, and 200 μg/L.
Exposure was conducted in 420-L glass tanks (30 fishes per tank, with
each group containing three replicates) under a natural temperature and
light cycle (to imitate the natural maturation of ovaries). The fish
were fed on fish premix twice daily until the end of exposure. A
flow-through system (Fig. S3) was used to renew the water daily, and
water samples were collected monthly (n = 6) for TDCIPP analysis during
the exposure period. After six months of exposure, embryos were obtained
through artificial fertilization; a portion of these were sampled
(approximately 50 mg each sample, n = 6) for TDCIPP analysis and a
portion were prepared for culture to enable study of embryonic
development. Culture conditions of embryo and method for cell height
quantification were according to a paper reported previously (Kupsco et
al. 2017), and more details were provided in SI.
In April 2018, embryos were obtained from the adult crucian carp
cultured in the control group in the exposure experiment as described
above, and the larvae were exposed to TDCIPP from half-month-old
(beginning eating) to sex maturation (June 2019) at nominal
concentrations of 50, 500, 5000, and 50000 ng/L. Exposure was conducted
in 420-L glass tanks (50 fishes per tank, each group containing three
replicates) equipped with a flow-through system. During the exposure,
water samples were collected quarterly (n = 5) for TDCIPP analysis. Fish
were fed on live brine shrimp (Artemia nauplii ) twice daily until
fish were one-month-old, and then fed on fish premix twice daily until
the end of exposure. After a one-year exposure, embryos were obtained
through artificial fertilization, and samples were collected and
developmental processes were observed according to the same method
described above. After sample preparation, the TDCIPP concentrations in
water and embryos were determined by liquid chromatography-tandem mass
spectrometry (LC-MS/MS) analysis.
Chemical Analysis. All of the samples were collected in glass
vials, with all vials having been pretreated in a muffle furnace at
450°C for 6 h to prevent contamination. Samples were frozen immediately
at -20°C after collection, and stored at this temperature until
analysis.
Methods for sample preparation and TDCIPP quantification in water and
embryo samples were according to our previous study with some
modification (Zhao et al. 2018), and the details were provided in SI.
TDCIPP-d15 was used as the isotopic internal standard to correct the
loss of TDCIPP during the sample preparation and to compensate for
variations in LC-MS/MS instrumental response from injection to
injection. Recoveries of TDCIPP in the water and embryo samples were
73.4 ± 1.4% and 94.1 ± 1.7%, respectively. TDCIPP was detected in the
procedural blanks at concentrations of 0.1 ± 0.03 ng/L in water samples
and 0.9 ± 0.14 ng/g lw in embryo samples. The limits of quantitation
(LOQs) were defined as 10 times the standard deviation of the procedural
blanks, and thus the LOQ was 0.3 ng/L for water samples and 1.4 ng/g lw
for embryo samples. The final concentrations reported in this study were
blank-subtracted, and all of the chemical analysis experiments were
performed in triplicate.
Statistical Analyses. Differences in mortality and
developmentally delayed rates of embryos from different sites in Taihu
Lake were analyzed by the χ2 test. Concentrations of TDCIPP, mortality
of embryos from the exposure experiment, and relative embryo height were
analyzed by one-way analysis of variance (ANOVA) with Dunnett’s test
(95% confidence interval). When the concentrations were below the LOQ,
the values of the LOQs divided by 2 were used for calculation and
statistical analysis. Differences were analyzed with SPSS (v22.0, IBM,
Armonk, NY) and considered statistically significant at p< 0.05.