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Insights about the toxicity of tannery effluent on chicken (Gallus gallus domesticus) embryos
Journal Pre-proof INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS
Alex Rodrigues Gomes, Julya Emmanuela de Andrade Vieira, Amanda Pereira da Costa Araújo, Guilherme Malafaia PII:
S0045-6535(19)32643-8
DOI:
https://doi.org/10.1016/j.chemosphere.2019.125403
Reference:
CHEM 125403
To appear in:
Chemosphere
Received Date:
03 November 2019
Accepted Date:
17 November 2019
Please cite this article as: Alex Rodrigues Gomes, Julya Emmanuela de Andrade Vieira, Amanda Pereira da Costa Araújo, Guilherme Malafaia, INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS, Chemosphere (2019), https://doi. org/10.1016/j.chemosphere.2019.125403
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Journal Pre-proof INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS Alex Rodrigues Gomes1, Julya Emmanuela de Andrade Vieira1, Amanda Pereira da Costa Araújo1, Guilherme Malafaia1# 1Biological
Research Laboratory, Post-graduation Program in Conservation of Cerrado Natural
Resources, Goiano Federal Institute – Urutaí Campus (Urutaí, GO, Brazil). #Corresponding Author: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil. CEP: 75790000. Phone number: +55 64 3465 1995. E-mail: [email protected]
ABSTRACT Although tannery effluent (TE) toxicity has already been demonstrated in different vertebrate models, our knowledge about their effects on birds remains significantly incipient. Thus, the aim of the current study was to evaluate the impact of ephemeral exposure of Gallus gallus domesticus eggs to environmental predictive TE dilutions (1.4% and 6.5%). Eggs at E6 developmental stage were opened in order to assess embryos’ external morphology and genotoxic biomarkers. Based on our data, embryos exposed in ovo to TE recorded higher mortality rate, lower biomass and different morphological abnormalities such as optic vesicle depigmentation, pericardial and encephalic edemas, as well as body rotation error. Embryos exposed to TE showed lower crownrump length head and anterior-posterior length, as well as reduced beak size. Embryos exposed to the highest TE dilution (6.5%) also showed greater lower/upper limb development, larger optic vesicle area and smaller crystalline lens area than the other groups. On the other hand, differences in mitotic index were not observed between groups; however, total erythrocyte chromosomal abnormalities, mainly in metaphase and anaphase, were higher in embryos exposed to TE. These phases presented chromosome fragments formed from typical chromosome breakage, laggard chromosome and chromosome bridge. higher Cr, Mn and Zn concentrations in embryos exposed to TE strongly suggest that the observed abnormalities were directly associated with the absorption of chemical constituents. The present study is pioneer in investigating the morphotoxic and genotoxic potential of TE (a complex mixture of various xenobiotics) in bird embryos in order to better understand the eco(toxicological) magnitude of this pollutant in aquatic ecosystems. 1
Journal Pre-proof Keywords: Aquatic pollution, agro-industrial wastes, developmental toxicity, reproduction. 1. INTRODUCTION Tanning is acknowledged as the activity responsible for the economy of different places; it is mainly driven by high and growing demands for products such as footwear, bags, clothing and upholstery, whose leather is the main raw material (Kibret & Tulu, 2014). However, the same economic development provided by the tannery sector raises environmental concerns, since the magnitude of impacts caused by inappropriate effluent disposal in watercourses have on the aquatic biota are yet to be fully understood (Ugya & Aziz, 2016; Ilyas et al., 2019; Sazena et al., 2020). The transformation of skin in-natura into leather in these companies generates large amounts of residue presenting a large variety of potentially toxic organic and inorganic compounds (Calheiros et al., 2007). Sulfides, sulfates, chlorides, as well as substantial amounts of sodium and calcium, are used in the skin cleaning stage. They generate high alkaline-content liquid residues, as well as solid residues such as hair, meat and fat (Joseph & Nithya, 2009). The drying stage generates residues rich in sodium chloride, mineral and organic acids, aluminum salts and in many toxic metals such as chrome, lead, arsenic, besides solvents used at the final leather-processing stage (Sabumon, 2016). Although several harmful effects of tannery effluent (TE) have been described in different aquatic species, studies about how these pollutants affect the health of terrestrial species have focused on mammalians (Souza et al., 2016). With respect to birds, for example, this knowledge is restricted to studies by Hoffman & Eastin (1981), Souza et al. (2017) and, more recently, by Sampaio et al. (2019). Hoffman & Eastin (1981) observed embryotoxic effects on Anas platyrhynchos, such as changes in individuals’ embryonic growth. According to Souza et al. (2017), the chronic intake-based exposure of
Melopsittacus undulates (budgerigars) to water contaminated with 5% TE induced increased micronucleus formation and other erythrocyte nuclear abnormalities in them. Samapaio et al. (2019) not only confirmed the mutagenic potential of these pollutants in Japanese quails (Coturnix coturnix japonica), but also observed that their exposure to TE induced neurotoxic and reproductive effects on them. Except for these studies, no other research has reported changes resulting from the exposure of avian species to TE, a fact that highlights a gap to be filled, mainly if one takes into consideration the ecological importance of birds to different ecosystems and the fact that they are excellent quality bioindicators. (Koskimies, 1989; Pilastro et al., 1993; O'Connell et al., 2000; Gubbay, 2004; Kalisinska, 2019). Thus, the impact caused by the ephemeral exposure to TE on the biology of birds, whose habitat and food source lie on aquatic environments, is a field to be 2
Journal Pre-proof explored. Bird species that build their grass-based nests on flooded lands, or that mainly use floating nests, may have their eggs exposed to TE-contaminated water, either through sudden and temporary flood events or through birds’ feathers. In such cases, TE chemical constituents can be transferred from the water or from birds’ plumage to eggs, and such transfer may have negative effects on the reproduction of these birds, as reported in previous studies comprising other pollutants [Albers (1980), Anas platyrhynchos; Bryan-Jr et al. (2003), Quiscalus quiscala; Verreault et al. (2006), Larus hyperboreus; Ackerman et al. (2016), Sterna forsteri, Himantopus
mexicanus and Recurvirostra americana; Vetter et al. (2017), Falco peregrinus and Ackerman et al. (2017), Troglodytes aedon and Tachycineta bicolor]. Thus, the aim of the current study was to test the hypothesis that the exposure of Gallus
gallus domesticus (model system) eggs to TE, even for a short period-of-time, induces embryonic development changes and has genotoxic effects on them. To the best of our knowledge, the present study is an insight about how these pollutants may affect reproductive aspects of waterfowl living in areas subjected to the disposal of this pollutant. Studies such as the present one can support pollution remediation and mitigation actions and, consequently, they can contribute to the conversation of animal biodiversity. 2. MATERIAL AND METHODS 2.1. Animals and experimental design In Total, 120 fertilized Gallus gallus domesticus, Label Rouge strain, (48.89 g ± 0.83; mean ± SEM) eggs were collected in a farm located in Urutaí County (GO, Brazil) and taken to the Biological Research Laboratory (Instituto Federal Goiano, Urutaí, GO, Brazil) where they were sanitized and left to rest for 24 h for proper internal structure orientation. Next, the eggs were distributed into groups exposed, or not, to different TE dilutions and placed (in horizontal position) in automatic incubator at 37.5o C and 52% humidity. Eggs exposed to water contaminated with 1.4% and 6.5% TE were allocated to groups TE-I and TE-II, respectively. Control eggs were exposed to pollutant-free drinking water. Eggs were incubated for 2 h to enable resuming the embryonic development process; the embryonic day (E) was taken as staging criterion and every 24 h corresponded to 1 day. Egg exposure to the pollutant took place at E0 and E3 and it consisted in full egg submersion in beaker filled with water or with the aforementioned TE dilutions for 30 s, based on Kertész et al. (2006). The exposure water was preheated to temperature similar to that of the incubator in order to avoid embryonic death by thermal shock. The exposure procedure was always carried out in the afternoon, between 4 pm and 6 pm. The adopted exposure protocol 3
Journal Pre-proof simulates natural conditions under which eggs can be ephemerally wetted with polluted water, either directly (when sudden movements of females near floating nests wet the eggs or in sudden and temporary flood events) or indirectly (via bird plumage). On the other hand, the option made for exposing early-stage eggs is justified by the fact that changes in early embryonic development may not only affect embryo development in the egg, but also chick birth and its survival (He et al., 2019). 2.2. Tannery effluent and tested dilutions The eggs in the present study were exposed to TE (previously characterized by Sampaio et al. (2019)) at the minimum (1.4%) and maximum (6.5%) environmentally relevant dilutions described in the current study. Chemical TE characterization revealed high concentration of heavy metals of known toxicity (Ba (2.4 mg/L), Cd (1.8 mg/L), Pb (0.6 mg/L), Cu (2.4 mg/L), Hg (0.0009 mg/L), Ni (1.8 mg/L), As (1.4 mg/L), Cr (479 mg/L), Sn 97.2 mg/L), and Si (2.9 mg/L)) and different mono-aromatic and polycyclic aromatic hydrocarbons, which presented complex molecular structures and were little identified in routine organic and chemical analyses. 2.3. Morphometry, cyto- and genotoxicity Eggs at E6 were opened and had their embryonic discs extracted. Next, embryos were weighed and fixed in formalin solution (10%) for further morphological evaluation, based on Dias & Müller (1998). Such evaluation consisted in classifying embryos as "normal" or "malformed", by taking into consideration that normal embryos presented: (i) arrangement of nervous system flexions resulting in dorsal body profile (broad curved line); (ii) full body rotation; (iii) welldefined brain vesicles and spinal cord in the neural tube; (iv) clear pigmented optic vesicle; (v) somite extension level reaching the tail end; (vi) limb length longer than limb width, and limbs ending in digital plates compatible with their developmental stage; (vii) caudal knob end facing the ventral region. In addition, embryo developmental stages were determined based on criteria suggested by Hamburger & Hamilton (1951). Different morphometric measurements of the embryos were taken in the ImageJ 1.52av software (https://imagej.nih.gov/ij/download.html), after they were photographed under stereoscopic microscope. Figure 1 specifies the morphometric measurements , whose results are presented in the form of indices, i.e., in comparison to other embryo measurements (e.g., nozzle length index = nozzle length/anterior – posterior head length x 100).
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Figure 1. Representative images of morphometric measurements of Gallus gallus domesticus embryos exposed in ovo, or not, to tannery effluent. CRL: crown-rump length; APL: anterior– posterior length of the head; OP: optic vesicle; CR: crystalline area; LLL: lower limb length; ULL: upper limb length. Cytotoxicity (mitotic index and some nuclear abnormalities) and genotoxicity (possible chromosomal aberrations) evaluations were carried out in two blood/embryo samples (approximately 10 µL), which were collected in blood vessels irrigating the embryonic disc in order to prepare blood smears on previously sanitized slides. Next, slides were dried, fixed in 100% (v/v) methyl alcohol (Dynamics®, São Paulo, Brazil) for 10 min and stained with Rapid® Panotic (Laborclin®, Paraná, Brazil), based on Araújo et al. al. (2020). Subsequently, they were analyzed under optical microscope equipped with immersion lens, based on Vieira et al. (2019). It is emphasized that blood was collected with the aid of a micropipette (previously sterilized) after opening of the eggs. Dividing cell rates (mitosis or interphase) were initially assessed in each slide; 100 red cells/slide (two slides/embryo) were analyzed, which totaled 4000 erythrocytes/experimental group. Then, the number of nuclear erythrocyte abnormalities in 2000 dividing/treatment cells was counted in order to evaluate chromosomal aberrations induced by TE at prophase, metaphase, anaphase and telophase. All slides were coded and blindly examined by a single researcher. Subsequently, the following indices were calculated (Eq. 1-3), based on the study by Montalvão et al. (2019): 5
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Micotic index =
TDC TC
(1)
x 100
Total abnormal cell index =
(2)
Tabn TDC Tabn per phase
Abnormal cells per mitotic/phase index = Tcell per phase x 100
(3)
Wherein, ‘TDC’ is the total n. of dividing cells; ‘TC’ is the total n. of observed cells; ‘Tabn’ is the total n. of abnormal cells; and ‘Tabn per phase’ is the total n. of abnormal cells observed in each phase (prophase, metaphase, anaphase or telophase). 2.4. Metal quantification Cr, Mn and Zn concentrations in embryos were measured based on procedures suggested by Niazi et al. (1993), with modifications, in order to evaluate the possible relationship between the observed effects and egg exposure to TE. These elements were defined as TE markers, based on their high concentrations in the effluent used in the current study [see chemical characterization by Sampaio et al. (2019)]. After embryos were weighed, they were digested in beaker filled with 2 ml of 65% nitric acid (HNO3) and kept in hot plate at 80° C, for 2 h. Next, the clock glass placed over the beaker was removed and the heating process continued until the sample was completely dry. Subsequently, 5 mL of aqueous solution [5% HNO3 diluted in purified water (via reverse osmosis)] was added to the beaker and stored at 4° C for further reading at atomic absorption spectrometer. Calibration standards 0.1, 1.0 and 10 mg/L of each chemical element were used for method validation purposes. 2.5. Statistical analysis All collected data were initially subjected to Shapiro-Wilk’s normality and Bartlett's variance homogeneity tests to validate the use of parametric statistics. Parametric data were subjected to one-way ANOVA, whereas non-parametric data were subjected to Kruskal-Wallis test, as well as to Tukey and Dunn’s post-tests, respectively, both at 5% probability level. Chisquare test was used to compare observed embryonic mortality rates between experimental groups. All statistical analysis and graphing were carried out in the GraphPad Prism software (version 7.00).
6
Journal Pre-proof 3. RESULTS Embryos whose eggs were exposed to ET recorded higher mortality rate; the ones presenting lack of heartbeat and whitish color were considered dead (control: 17.5%; TE-I group: 32%; TE-II group: 32% - χ2 = 7.61; p = 0.0223). In addition, embryos in groups exposed to TE presented reduced body biomass, although their developmental stages did not differ between experimental groups (Figures 2A and 2B, respectively). Embryos in the control group showed fully closed body wall; digits in the upper and lower limbs; large, well-pigmented optic vesicles; as well as developed brain vesicles positioned in a manner compatible with their staging phase [according to Hamburger & Hamilton (1951)]. However, there were different morphological abnormalities in embryos originating from eggs exposed to ET, with emphasis on optic (Figure 2C), pericardial (Figure 2D) and encephalic abnormalities such as mesoencephalic (Figure 2E) depigmentation, as well as on body rotation error (Figure 2F). With respect to morphometric parameters, embryos in groups TE-I and TE-II also presented smaller crown-rump length and anterior-posterior length (Figure 3A); as well as reduced nozzle size (Figure 3B). However, embryos exposed to the highest TE dilution (TE-II group) showed higher lower/upper limb development (Figure 3B), larger optic vesicle area (Figure 3C) and smaller crystalline lens area (Figure 3D) in comparison to other groups.
Figure 2. (A) Body biomass, (B) embryonic development stages and (C) main morphological abnormalities identified in Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. (C) Optic bladder depigmentation; (D) pericardial edema and (E) encephalic 7
Journal Pre-proof edema; (F) body rotation error. Bars in “A and B” indicate the mean + standard error of the collected data (n = 40 embryos/group). Data were subjected to the non-parametric KruskalWallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase letters indicate significant difference between experimental groups.
8
Figure 3. (A) Crown-rump length and anterior-posterior length of the head, (B) length of lower/upper limbs and nozzle, (C) relative optic vesicle index and (D) relative crystalline area index (D) of Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. Bars indicate the mean + standard error of the data (n = 40 embryos/group). Data were subjected to the non-parametric Kruskal-Wallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase letters indicate significant difference between experimental groups.
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On the other hand, there were no differences in mitotic index between groups (Figure
3
4A). However, the total number of erythrocyte chromosomal abnormalities observed in dividing
4
cells (Figure 4B), mainly at metaphase and anaphase, were higher in embryos exposed to TE
5
(Figure 4C) than in the non-exposed ones. These phases presented chromosome fragments
6
(formed from typical chromosome breakage), laggard chromosome and chromosome bridge
7
(Figure 5).
Figure 4. (A) Mitotic index, (B) total number of erythrocyte chromosomal abnormalities (at all mitosis stages) and (C) metaphase and anaphase of Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. Bars indicate the mean + standard error of the collected data (n = 40 embryos/group). Different lowercase letters in “B” indicate significant difference between experimental groups. Data were subjected to the non-parametric KruskalWallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase and uppercase letters in “C” indicate significant differences between metaphase and anaphase, respectively, in the experimental groups. 8
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Figure 5. (A-B) Erythrocytes from Gallus gallus domesticus (E6) embryos during cell division process - (A) metaphase and (B) normal anaphase. (C-L) Examples of erythrocyte chromosomal abnormalities (arrows) at different stages of mitosis metaphase and anaphase. (C-F) Chromosomal fragments at metaphase and (G, I and K) anaphase. (H) Laggard chromosome at anaphase. (J) Chromosome bridge at anaphase. (L) Chromosomal fragment at the beginning of telophase. All chromosomal abnormalities were identified in G. gallus domesticus embryos exposed to tannery effluent. Slides were stained with Quick Panotic®. However, gray scale was applied to the images to better visualize erythrocyte’s chromosome structures. 9 10 11
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Data on the quantification of chemical elements in embryos suggest that TE chemical
13
components have penetrated the eggshell and accumulated in the embryos. Cr, Mn and Zn
14
concentrations in tissue (TE chemical markers) were higher in embryos belonging to groups TE-I
15
and TE-II; this outcome suggests that Cr, Mn and Zn accumulation can be associated with their
16
absorption and with the observed toxicological evidence.
17 18
4. DISCUSSION
19
The proposition of measures focused on mitigating and/or remediating environmental
20
pollution depends on basic information about the factors/aspects affecting the biota and/or the
21
landscape. According to Rodrigues & Castro (2008), the earlier changes caused by pollution
22
sources are identified, the faster measures can be adopted to remediate/mitigate their impacts.
23
The current study was pioneer in presenting evidence that the ephemeral contact (twice for 30
24
s/each time) of G. gallus domesticus eggs with TE-contaminated water can induce significant
25
changes in embryos. Although this pollutant did not induce embryonic developmental delay
26
(Figure 2B) - as observed by Zinabadinova et al. (2018), Kazemi et al., 2018 and Kohl et al. (2019)
27
who used technogenic pollutants, cadmium and carbamazepine, respectively - different changes
28
in the external morphology of animals were observed.
29
Embryos exposed in ovo to TE showed optic vesicle depigmentation, pericardial and
30
encephalic edema, and body rotation error (Figure 2). Such changes are relevant because they
31
can alter not only embryo topography sketches, but also characteristics concerning the species
32
itself. Complete optic vesicle depigmentation, for example, suggests changes in embryos’ ocular
33
morphogenesis, mainly in retinal and retinal pigment epithelium (RPE) formation processes.
34
Although retinal formation mechanisms are multifaceted (Fuhrmann, 2010), TE chemical
35
constituents may have influenced the complex interaction between inductive signals provided by
36
tissue-tissue interactions and intrinsic cellular factors that are critical to assure the proper
37
specification of ocular tissues, as well as the maintenance and fate of RPE cells. It is plausible to
38
assume the direct or indirect interference of TE in the synthesis or release of transcription factors
39
that play key roles in RPR formation and specification, such as mitf and orthodenticle homeobox
40
2 (Otx2). Evidence that heavy metals such as Cr (Jindal et al., 2019), Pb (Wu et al., 2019) and Cd
41
(Hou et al., 2017) may alter the production of transcription factors in other animal models
42
reinforces the present hypothesis. Since RPE is essential to enable eye growth, as well as to control
43
proper retinal lamination and regulate photoreceptor differentiation (Martinez-Morales et al.,
44
2004; Strauss, 2005; Bharti et al., 2006; Fuhrmann, 2010), it is possible inferring that even
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ephemeral embryo exposure in ovo to ET leads to poor vision formation, which has drastic
46
biological consequences after hatching.
47
Pericardial edema observed in embryos exposed to TE has also been reported in
48
vertebrate embryos exposed in ovo to different pollutants, such as pyraoxystrobin in zebrafish
49
(Danio rerio) (Li et al., 2018), polycyclic aromatic hydrocarbons in Japanese medaka (Oryzias
50
latipes) (Rhodes et al., 2005), tetrachlorodibenzo-p-dioxin (TCDD) in red seabream (Pagrus
51
major) (Yamauchi et al., 2006), as well as perfluorooctanoic acid (PFOA) (Brunström et al., 1991;
52
Jiang et al., 2016) and mono-ortho-chlorinated polychlorinated biphenyls (PCBs) (Brunström,
53
1990) in chicken embryo (G. gallus domesticus). In addition, it has been reported that aluminum
54
(ElMazoudy & Bekhet, 2016) and different chemical elements evaluated in separate (cadmium,
55
arsenic, cobalt, copper, indium, iron, manganese and molybdenum) (Gilani & Alibhai, 1990)
56
induced pericardium edema in chicken embryos exposed in ovo.
57
According to Incardona & Scholz (2016), fluid accumulation in extracellular spaces of the
58
heart is one of the most sensitive indicators of cardiac dysregulation in embryos. Therefore, the
59
current study suggests a cardiotoxic effect induced by TE, whose consequences for embryonic
60
development can be severe. Although it is too early to suggest any action mechanism to explain
61
pericardial edema formation, it is possible assuming that embryo exposure to TE affected the
62
interconnection of cardiovascular and osmoregulatory systems during early development, thus
63
leading to changes in tissue osmolarity, as proposed by Incardona & Scholz (2016). Similar
64
reasoning can be adopted for brain edema formation, which confirms the neurotoxic action of
65
TE already reported in other model systems who represented mammalians (Rabelo et al., 2016;
66
Guimarães et al., 2017; Chagas et al., 2018), fish (Chagas et al., 2019), amphibians (Amaral et al.,
67
2018) and birds (Sampaio et al., 2019).
68
On the other hand, neural tube rotation errors are a clear evidence of teratological effect
69
induced by TE. Such errors can lead to scoliosis formation and to other torsion defects, as
70
observed in the present study (Figure 2F). According to Fujinaga et al. (1995), axial rotation is an
71
interesting developmental event not only because it is a dynamic process but also because it is
72
one of the earliest morphological signs of body asymmetry. The causes of this condition can be
73
complex, as can be the chemical constitution of TE; however, its consequences for individuals
74
comprise abnormal body formation (culminating in asymmetrical conditions) with severe
75
implications for biomechanical aspects of locomotion. Changes in the axial body rotation in mice
76
can be associated with rotating protein deficiency, which is considered the novel player in the
77
mice’s left-right specification cascade and represents a genetic link between axial rotation
78
processes and processes leading to organ asymmetry (Faisst et al., 2002). Therefore, the 13
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hypothesis that TE chemical constituents have some influence on biochemical and molecular
80
mechanisms associated with the synthesis and release of this protein cannot be ruled out.
81
Another important aspect lies on the negative effects of TE on the morphometric
82
parameters evaluated for embryos. These effects were evidenced by the shorter crown-rump
83
length and anterior-posterior length of the head, by the beak and lens area observed in embryos
84
exposed to the TE as well as by increased optic vesicle area, and lower and posterior limbs, of
85
embryos in group TE-II (Figure 2).
86
Although the current study is pioneer in demonstrating these effects on embryos exposed
87
in ovo to TE, other studies have reported teratogenic effects on G. gallus domesticus eggs exposed
88
to pesticides (Abbas et al., 2018), plasticides (Wang et al., 2019 ), drugs (Ertekin et al., 2019),
89
nanoparticles (Patel et al., 2019), as well as to different heavy metals, much of which were
90
identified in the effluent tested in the current study (Birge & Roberts, 1976; Gilani & Alibhai,
91
1990); Asmatullah et al., 1998; Asmatullah & Shakoori, 1998; Papaconstantinou et al., 2003).
92
Obviously, the action mechanisms suggested by the aforementioned studies vary and depend on
93
the tested pollutant. Multiple mechanisms may have induced the morphological changes
94
observed in the present study, given the chemical complexity of TE. Such mechanisms may
95
include biochemical and molecular changes capable of causing endocrine disruption and of
96
inducing cytotoxicity, as well as genomic mutagenicity, which was reinforced by outcomes
97
observed in erythrocytes of embryos exposed to TE.
98
The increased frequency of chromosomal aberrations in embryos exposed to the
99
pollutant (Figure 4B) indicates how genotoxic TE can be to embryos. The incidence of
100
chromosomal fragments in metaphase and anaphase suggests clastogenic action of pollutants,
101
whereas chromosomal losses (laggard, Figure 5) suggest their aneugenic effect. According to
102
Fiskesjö (1993), chromosomal fragments may result from breaks in chromosome bridges that
103
may originate from either translocations or cohesive chromosome terminations. Chromosomal
104
losses often take place when –pollutants have negative interference on mitotic spindle, which
105
prevents one or more chromosomes from attaching to the spindle fibers during anaphase (Kirsch-
106
Volders et al., 2002).
107
Thus, it is possible noticing that changes identified in the present study are just the “tip”
108
of an “iceberg” that represents the toxicological magnitude of TE effects on embryonic
109
development. Based on developmental, morphometric and genotoxic biomarkers, it was possible
110
demonstrating that even the ephemeral exposure to these pollutants at low dilutions can cause
111
effects that may compromise the embryonic development of birds, as well as affect their post-
112
hatching health. However, the fact that the toxic action mechanisms of TE components remain 14
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unknown should be taken into consideration. The effects of different xenobiotics found in TE
114
may derive from synergistic, antagonistic and additive interactions or from interactions capable of
115
enhancing the toxic action of a given agent that acts along with a non-toxic agent or with an agent
116
presenting other toxicity types. In addition, studies focused on identifying chemical constituents
117
of major toxicological influence may be useful to support pollution remediation measures and to
118
help mitigating component-specific impacts. Therefore, overcoming these limitations and
119
expanding the use of more sensitive and early toxicity biomarkers are good prospects for future
120
studies.
121 122
5. CONCLUSION
123
The initial hypothesis in the current study was confirmed by morphological changes in,
124
and by genotoxic effects on, erythrocyte cells of G. gallus domesticus embryos exposed to TE,
125
even for a short period-of-time and at low dilutions. Thus, the present research was pioneer in
126
demonstrating that aquatic ecosystems contaminated with TE are a risk factor for the embryonic
127
development of waterfowl living in polluted areas, as well as that they can significantly affect the
128
dynamics of these populations. If, on the one hand, future studies can help better understanding
129
the biological mechanisms affected by this pollutant, on the other hand, the present data provide
130
sufficient evidences to reinforce the need of taking measures aimed at depolluting habitats
131
contaminated with TE, at preserving the environment and/or at mitigating impacts caused by
132
these pollutants. These measures should certainly include the effective implementation of stricter
133
environmental laws, whose legal devices can be, and are, enforced through sound and
134
comprehensive enforcement actions.
135 136
6. ACKNOWLEDGMENT
137
The authors are grateful to the Brazilian National Research Council (CNPq) (Process n.
138
426531/2018-3) and to Instituto Federal Goiano for the financial support (Process n.
139
23219.001291/2019-11). Moreover, they are grateful to Coordenação de Aperfeiçoamento de
140
Pessoal de Nível Superior (CAPES, Brazil) and to Fundação de Amparo à Pesquisa do Estado
141
de Goiás (FAPEG, Brazil) for granting the scholarship to the student who developed the research.
142
Malafaia G. thanks CNPq for the research productivity fellowship (Proc. N. 307743/2018-7).
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Journal Pre-proof INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS Alex Rodrigues Gomes1, Julya Emmanuela de Andrade Vieira1, Amanda Pereira da Costa Araújo1, Guilherme Malafaia1# 1Biological
Research Laboratory, Post-graduation Program in Conservation of Cerrado Natural
Resources, Goiano Federal Institute – Urutaí Campus (Urutaí, GO, Brazil).
Alex Rodrigues Gomes: Conceptualization, Methodology. Julya Emmanuela de Andrade Vieira: Methodology. Amanda Pereira da Costa Araújo: Conceptualization, Methodology Guilherme Malafaia: Management and coordination responsibility for the research activity planning and execution and Writing- Reviewing and Editing. #Corresponding Author: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil. CEP: 75790000. Phone number: +55 64 3465 1995. E-mail: [email protected]
1
Journal Pre-proof Highlights INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS
Tannery effluent (TE) induces morphological changes in G. gallus domesticus embryos
Ephemeral exposure of G. gallus domesticus eggs to TE cause mutagenic effect
Cr, Mn and Zn concentrations are higher in embryos exposed to TE
Alex Rodrigues Gomes, Julya Emmanuela de Andrade Vieira, Amanda Pereira da Costa Araújo, Guilherme Malafaia PII:
S0045-6535(19)32643-8
DOI:
https://doi.org/10.1016/j.chemosphere.2019.125403
Reference:
CHEM 125403
To appear in:
Chemosphere
Received Date:
03 November 2019
Accepted Date:
17 November 2019
Please cite this article as: Alex Rodrigues Gomes, Julya Emmanuela de Andrade Vieira, Amanda Pereira da Costa Araújo, Guilherme Malafaia, INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS, Chemosphere (2019), https://doi. org/10.1016/j.chemosphere.2019.125403
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS Alex Rodrigues Gomes1, Julya Emmanuela de Andrade Vieira1, Amanda Pereira da Costa Araújo1, Guilherme Malafaia1# 1Biological
Research Laboratory, Post-graduation Program in Conservation of Cerrado Natural
Resources, Goiano Federal Institute – Urutaí Campus (Urutaí, GO, Brazil). #Corresponding Author: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil. CEP: 75790000. Phone number: +55 64 3465 1995. E-mail: [email protected]
ABSTRACT Although tannery effluent (TE) toxicity has already been demonstrated in different vertebrate models, our knowledge about their effects on birds remains significantly incipient. Thus, the aim of the current study was to evaluate the impact of ephemeral exposure of Gallus gallus domesticus eggs to environmental predictive TE dilutions (1.4% and 6.5%). Eggs at E6 developmental stage were opened in order to assess embryos’ external morphology and genotoxic biomarkers. Based on our data, embryos exposed in ovo to TE recorded higher mortality rate, lower biomass and different morphological abnormalities such as optic vesicle depigmentation, pericardial and encephalic edemas, as well as body rotation error. Embryos exposed to TE showed lower crownrump length head and anterior-posterior length, as well as reduced beak size. Embryos exposed to the highest TE dilution (6.5%) also showed greater lower/upper limb development, larger optic vesicle area and smaller crystalline lens area than the other groups. On the other hand, differences in mitotic index were not observed between groups; however, total erythrocyte chromosomal abnormalities, mainly in metaphase and anaphase, were higher in embryos exposed to TE. These phases presented chromosome fragments formed from typical chromosome breakage, laggard chromosome and chromosome bridge. higher Cr, Mn and Zn concentrations in embryos exposed to TE strongly suggest that the observed abnormalities were directly associated with the absorption of chemical constituents. The present study is pioneer in investigating the morphotoxic and genotoxic potential of TE (a complex mixture of various xenobiotics) in bird embryos in order to better understand the eco(toxicological) magnitude of this pollutant in aquatic ecosystems. 1
Journal Pre-proof Keywords: Aquatic pollution, agro-industrial wastes, developmental toxicity, reproduction. 1. INTRODUCTION Tanning is acknowledged as the activity responsible for the economy of different places; it is mainly driven by high and growing demands for products such as footwear, bags, clothing and upholstery, whose leather is the main raw material (Kibret & Tulu, 2014). However, the same economic development provided by the tannery sector raises environmental concerns, since the magnitude of impacts caused by inappropriate effluent disposal in watercourses have on the aquatic biota are yet to be fully understood (Ugya & Aziz, 2016; Ilyas et al., 2019; Sazena et al., 2020). The transformation of skin in-natura into leather in these companies generates large amounts of residue presenting a large variety of potentially toxic organic and inorganic compounds (Calheiros et al., 2007). Sulfides, sulfates, chlorides, as well as substantial amounts of sodium and calcium, are used in the skin cleaning stage. They generate high alkaline-content liquid residues, as well as solid residues such as hair, meat and fat (Joseph & Nithya, 2009). The drying stage generates residues rich in sodium chloride, mineral and organic acids, aluminum salts and in many toxic metals such as chrome, lead, arsenic, besides solvents used at the final leather-processing stage (Sabumon, 2016). Although several harmful effects of tannery effluent (TE) have been described in different aquatic species, studies about how these pollutants affect the health of terrestrial species have focused on mammalians (Souza et al., 2016). With respect to birds, for example, this knowledge is restricted to studies by Hoffman & Eastin (1981), Souza et al. (2017) and, more recently, by Sampaio et al. (2019). Hoffman & Eastin (1981) observed embryotoxic effects on Anas platyrhynchos, such as changes in individuals’ embryonic growth. According to Souza et al. (2017), the chronic intake-based exposure of
Melopsittacus undulates (budgerigars) to water contaminated with 5% TE induced increased micronucleus formation and other erythrocyte nuclear abnormalities in them. Samapaio et al. (2019) not only confirmed the mutagenic potential of these pollutants in Japanese quails (Coturnix coturnix japonica), but also observed that their exposure to TE induced neurotoxic and reproductive effects on them. Except for these studies, no other research has reported changes resulting from the exposure of avian species to TE, a fact that highlights a gap to be filled, mainly if one takes into consideration the ecological importance of birds to different ecosystems and the fact that they are excellent quality bioindicators. (Koskimies, 1989; Pilastro et al., 1993; O'Connell et al., 2000; Gubbay, 2004; Kalisinska, 2019). Thus, the impact caused by the ephemeral exposure to TE on the biology of birds, whose habitat and food source lie on aquatic environments, is a field to be 2
Journal Pre-proof explored. Bird species that build their grass-based nests on flooded lands, or that mainly use floating nests, may have their eggs exposed to TE-contaminated water, either through sudden and temporary flood events or through birds’ feathers. In such cases, TE chemical constituents can be transferred from the water or from birds’ plumage to eggs, and such transfer may have negative effects on the reproduction of these birds, as reported in previous studies comprising other pollutants [Albers (1980), Anas platyrhynchos; Bryan-Jr et al. (2003), Quiscalus quiscala; Verreault et al. (2006), Larus hyperboreus; Ackerman et al. (2016), Sterna forsteri, Himantopus
mexicanus and Recurvirostra americana; Vetter et al. (2017), Falco peregrinus and Ackerman et al. (2017), Troglodytes aedon and Tachycineta bicolor]. Thus, the aim of the current study was to test the hypothesis that the exposure of Gallus
gallus domesticus (model system) eggs to TE, even for a short period-of-time, induces embryonic development changes and has genotoxic effects on them. To the best of our knowledge, the present study is an insight about how these pollutants may affect reproductive aspects of waterfowl living in areas subjected to the disposal of this pollutant. Studies such as the present one can support pollution remediation and mitigation actions and, consequently, they can contribute to the conversation of animal biodiversity. 2. MATERIAL AND METHODS 2.1. Animals and experimental design In Total, 120 fertilized Gallus gallus domesticus, Label Rouge strain, (48.89 g ± 0.83; mean ± SEM) eggs were collected in a farm located in Urutaí County (GO, Brazil) and taken to the Biological Research Laboratory (Instituto Federal Goiano, Urutaí, GO, Brazil) where they were sanitized and left to rest for 24 h for proper internal structure orientation. Next, the eggs were distributed into groups exposed, or not, to different TE dilutions and placed (in horizontal position) in automatic incubator at 37.5o C and 52% humidity. Eggs exposed to water contaminated with 1.4% and 6.5% TE were allocated to groups TE-I and TE-II, respectively. Control eggs were exposed to pollutant-free drinking water. Eggs were incubated for 2 h to enable resuming the embryonic development process; the embryonic day (E) was taken as staging criterion and every 24 h corresponded to 1 day. Egg exposure to the pollutant took place at E0 and E3 and it consisted in full egg submersion in beaker filled with water or with the aforementioned TE dilutions for 30 s, based on Kertész et al. (2006). The exposure water was preheated to temperature similar to that of the incubator in order to avoid embryonic death by thermal shock. The exposure procedure was always carried out in the afternoon, between 4 pm and 6 pm. The adopted exposure protocol 3
Journal Pre-proof simulates natural conditions under which eggs can be ephemerally wetted with polluted water, either directly (when sudden movements of females near floating nests wet the eggs or in sudden and temporary flood events) or indirectly (via bird plumage). On the other hand, the option made for exposing early-stage eggs is justified by the fact that changes in early embryonic development may not only affect embryo development in the egg, but also chick birth and its survival (He et al., 2019). 2.2. Tannery effluent and tested dilutions The eggs in the present study were exposed to TE (previously characterized by Sampaio et al. (2019)) at the minimum (1.4%) and maximum (6.5%) environmentally relevant dilutions described in the current study. Chemical TE characterization revealed high concentration of heavy metals of known toxicity (Ba (2.4 mg/L), Cd (1.8 mg/L), Pb (0.6 mg/L), Cu (2.4 mg/L), Hg (0.0009 mg/L), Ni (1.8 mg/L), As (1.4 mg/L), Cr (479 mg/L), Sn 97.2 mg/L), and Si (2.9 mg/L)) and different mono-aromatic and polycyclic aromatic hydrocarbons, which presented complex molecular structures and were little identified in routine organic and chemical analyses. 2.3. Morphometry, cyto- and genotoxicity Eggs at E6 were opened and had their embryonic discs extracted. Next, embryos were weighed and fixed in formalin solution (10%) for further morphological evaluation, based on Dias & Müller (1998). Such evaluation consisted in classifying embryos as "normal" or "malformed", by taking into consideration that normal embryos presented: (i) arrangement of nervous system flexions resulting in dorsal body profile (broad curved line); (ii) full body rotation; (iii) welldefined brain vesicles and spinal cord in the neural tube; (iv) clear pigmented optic vesicle; (v) somite extension level reaching the tail end; (vi) limb length longer than limb width, and limbs ending in digital plates compatible with their developmental stage; (vii) caudal knob end facing the ventral region. In addition, embryo developmental stages were determined based on criteria suggested by Hamburger & Hamilton (1951). Different morphometric measurements of the embryos were taken in the ImageJ 1.52av software (https://imagej.nih.gov/ij/download.html), after they were photographed under stereoscopic microscope. Figure 1 specifies the morphometric measurements , whose results are presented in the form of indices, i.e., in comparison to other embryo measurements (e.g., nozzle length index = nozzle length/anterior – posterior head length x 100).
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Figure 1. Representative images of morphometric measurements of Gallus gallus domesticus embryos exposed in ovo, or not, to tannery effluent. CRL: crown-rump length; APL: anterior– posterior length of the head; OP: optic vesicle; CR: crystalline area; LLL: lower limb length; ULL: upper limb length. Cytotoxicity (mitotic index and some nuclear abnormalities) and genotoxicity (possible chromosomal aberrations) evaluations were carried out in two blood/embryo samples (approximately 10 µL), which were collected in blood vessels irrigating the embryonic disc in order to prepare blood smears on previously sanitized slides. Next, slides were dried, fixed in 100% (v/v) methyl alcohol (Dynamics®, São Paulo, Brazil) for 10 min and stained with Rapid® Panotic (Laborclin®, Paraná, Brazil), based on Araújo et al. al. (2020). Subsequently, they were analyzed under optical microscope equipped with immersion lens, based on Vieira et al. (2019). It is emphasized that blood was collected with the aid of a micropipette (previously sterilized) after opening of the eggs. Dividing cell rates (mitosis or interphase) were initially assessed in each slide; 100 red cells/slide (two slides/embryo) were analyzed, which totaled 4000 erythrocytes/experimental group. Then, the number of nuclear erythrocyte abnormalities in 2000 dividing/treatment cells was counted in order to evaluate chromosomal aberrations induced by TE at prophase, metaphase, anaphase and telophase. All slides were coded and blindly examined by a single researcher. Subsequently, the following indices were calculated (Eq. 1-3), based on the study by Montalvão et al. (2019): 5
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Micotic index =
TDC TC
(1)
x 100
Total abnormal cell index =
(2)
Tabn TDC Tabn per phase
Abnormal cells per mitotic/phase index = Tcell per phase x 100
(3)
Wherein, ‘TDC’ is the total n. of dividing cells; ‘TC’ is the total n. of observed cells; ‘Tabn’ is the total n. of abnormal cells; and ‘Tabn per phase’ is the total n. of abnormal cells observed in each phase (prophase, metaphase, anaphase or telophase). 2.4. Metal quantification Cr, Mn and Zn concentrations in embryos were measured based on procedures suggested by Niazi et al. (1993), with modifications, in order to evaluate the possible relationship between the observed effects and egg exposure to TE. These elements were defined as TE markers, based on their high concentrations in the effluent used in the current study [see chemical characterization by Sampaio et al. (2019)]. After embryos were weighed, they were digested in beaker filled with 2 ml of 65% nitric acid (HNO3) and kept in hot plate at 80° C, for 2 h. Next, the clock glass placed over the beaker was removed and the heating process continued until the sample was completely dry. Subsequently, 5 mL of aqueous solution [5% HNO3 diluted in purified water (via reverse osmosis)] was added to the beaker and stored at 4° C for further reading at atomic absorption spectrometer. Calibration standards 0.1, 1.0 and 10 mg/L of each chemical element were used for method validation purposes. 2.5. Statistical analysis All collected data were initially subjected to Shapiro-Wilk’s normality and Bartlett's variance homogeneity tests to validate the use of parametric statistics. Parametric data were subjected to one-way ANOVA, whereas non-parametric data were subjected to Kruskal-Wallis test, as well as to Tukey and Dunn’s post-tests, respectively, both at 5% probability level. Chisquare test was used to compare observed embryonic mortality rates between experimental groups. All statistical analysis and graphing were carried out in the GraphPad Prism software (version 7.00).
6
Journal Pre-proof 3. RESULTS Embryos whose eggs were exposed to ET recorded higher mortality rate; the ones presenting lack of heartbeat and whitish color were considered dead (control: 17.5%; TE-I group: 32%; TE-II group: 32% - χ2 = 7.61; p = 0.0223). In addition, embryos in groups exposed to TE presented reduced body biomass, although their developmental stages did not differ between experimental groups (Figures 2A and 2B, respectively). Embryos in the control group showed fully closed body wall; digits in the upper and lower limbs; large, well-pigmented optic vesicles; as well as developed brain vesicles positioned in a manner compatible with their staging phase [according to Hamburger & Hamilton (1951)]. However, there were different morphological abnormalities in embryos originating from eggs exposed to ET, with emphasis on optic (Figure 2C), pericardial (Figure 2D) and encephalic abnormalities such as mesoencephalic (Figure 2E) depigmentation, as well as on body rotation error (Figure 2F). With respect to morphometric parameters, embryos in groups TE-I and TE-II also presented smaller crown-rump length and anterior-posterior length (Figure 3A); as well as reduced nozzle size (Figure 3B). However, embryos exposed to the highest TE dilution (TE-II group) showed higher lower/upper limb development (Figure 3B), larger optic vesicle area (Figure 3C) and smaller crystalline lens area (Figure 3D) in comparison to other groups.
Figure 2. (A) Body biomass, (B) embryonic development stages and (C) main morphological abnormalities identified in Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. (C) Optic bladder depigmentation; (D) pericardial edema and (E) encephalic 7
Journal Pre-proof edema; (F) body rotation error. Bars in “A and B” indicate the mean + standard error of the collected data (n = 40 embryos/group). Data were subjected to the non-parametric KruskalWallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase letters indicate significant difference between experimental groups.
8
Figure 3. (A) Crown-rump length and anterior-posterior length of the head, (B) length of lower/upper limbs and nozzle, (C) relative optic vesicle index and (D) relative crystalline area index (D) of Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. Bars indicate the mean + standard error of the data (n = 40 embryos/group). Data were subjected to the non-parametric Kruskal-Wallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase letters indicate significant difference between experimental groups.
9
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On the other hand, there were no differences in mitotic index between groups (Figure
3
4A). However, the total number of erythrocyte chromosomal abnormalities observed in dividing
4
cells (Figure 4B), mainly at metaphase and anaphase, were higher in embryos exposed to TE
5
(Figure 4C) than in the non-exposed ones. These phases presented chromosome fragments
6
(formed from typical chromosome breakage), laggard chromosome and chromosome bridge
7
(Figure 5).
Figure 4. (A) Mitotic index, (B) total number of erythrocyte chromosomal abnormalities (at all mitosis stages) and (C) metaphase and anaphase of Gallus gallus domesticus embryos from eggs exposed, or not, to tannery effluent. Bars indicate the mean + standard error of the collected data (n = 40 embryos/group). Different lowercase letters in “B” indicate significant difference between experimental groups. Data were subjected to the non-parametric KruskalWallis test (multiple comparisons were performed through the Dunn’s test at 5% probability level). Different lowercase and uppercase letters in “C” indicate significant differences between metaphase and anaphase, respectively, in the experimental groups. 8
10
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Figure 5. (A-B) Erythrocytes from Gallus gallus domesticus (E6) embryos during cell division process - (A) metaphase and (B) normal anaphase. (C-L) Examples of erythrocyte chromosomal abnormalities (arrows) at different stages of mitosis metaphase and anaphase. (C-F) Chromosomal fragments at metaphase and (G, I and K) anaphase. (H) Laggard chromosome at anaphase. (J) Chromosome bridge at anaphase. (L) Chromosomal fragment at the beginning of telophase. All chromosomal abnormalities were identified in G. gallus domesticus embryos exposed to tannery effluent. Slides were stained with Quick Panotic®. However, gray scale was applied to the images to better visualize erythrocyte’s chromosome structures. 9 10 11
11
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Data on the quantification of chemical elements in embryos suggest that TE chemical
13
components have penetrated the eggshell and accumulated in the embryos. Cr, Mn and Zn
14
concentrations in tissue (TE chemical markers) were higher in embryos belonging to groups TE-I
15
and TE-II; this outcome suggests that Cr, Mn and Zn accumulation can be associated with their
16
absorption and with the observed toxicological evidence.
17 18
4. DISCUSSION
19
The proposition of measures focused on mitigating and/or remediating environmental
20
pollution depends on basic information about the factors/aspects affecting the biota and/or the
21
landscape. According to Rodrigues & Castro (2008), the earlier changes caused by pollution
22
sources are identified, the faster measures can be adopted to remediate/mitigate their impacts.
23
The current study was pioneer in presenting evidence that the ephemeral contact (twice for 30
24
s/each time) of G. gallus domesticus eggs with TE-contaminated water can induce significant
25
changes in embryos. Although this pollutant did not induce embryonic developmental delay
26
(Figure 2B) - as observed by Zinabadinova et al. (2018), Kazemi et al., 2018 and Kohl et al. (2019)
27
who used technogenic pollutants, cadmium and carbamazepine, respectively - different changes
28
in the external morphology of animals were observed.
29
Embryos exposed in ovo to TE showed optic vesicle depigmentation, pericardial and
30
encephalic edema, and body rotation error (Figure 2). Such changes are relevant because they
31
can alter not only embryo topography sketches, but also characteristics concerning the species
32
itself. Complete optic vesicle depigmentation, for example, suggests changes in embryos’ ocular
33
morphogenesis, mainly in retinal and retinal pigment epithelium (RPE) formation processes.
34
Although retinal formation mechanisms are multifaceted (Fuhrmann, 2010), TE chemical
35
constituents may have influenced the complex interaction between inductive signals provided by
36
tissue-tissue interactions and intrinsic cellular factors that are critical to assure the proper
37
specification of ocular tissues, as well as the maintenance and fate of RPE cells. It is plausible to
38
assume the direct or indirect interference of TE in the synthesis or release of transcription factors
39
that play key roles in RPR formation and specification, such as mitf and orthodenticle homeobox
40
2 (Otx2). Evidence that heavy metals such as Cr (Jindal et al., 2019), Pb (Wu et al., 2019) and Cd
41
(Hou et al., 2017) may alter the production of transcription factors in other animal models
42
reinforces the present hypothesis. Since RPE is essential to enable eye growth, as well as to control
43
proper retinal lamination and regulate photoreceptor differentiation (Martinez-Morales et al.,
44
2004; Strauss, 2005; Bharti et al., 2006; Fuhrmann, 2010), it is possible inferring that even
12
Journal Pre-proof 45
ephemeral embryo exposure in ovo to ET leads to poor vision formation, which has drastic
46
biological consequences after hatching.
47
Pericardial edema observed in embryos exposed to TE has also been reported in
48
vertebrate embryos exposed in ovo to different pollutants, such as pyraoxystrobin in zebrafish
49
(Danio rerio) (Li et al., 2018), polycyclic aromatic hydrocarbons in Japanese medaka (Oryzias
50
latipes) (Rhodes et al., 2005), tetrachlorodibenzo-p-dioxin (TCDD) in red seabream (Pagrus
51
major) (Yamauchi et al., 2006), as well as perfluorooctanoic acid (PFOA) (Brunström et al., 1991;
52
Jiang et al., 2016) and mono-ortho-chlorinated polychlorinated biphenyls (PCBs) (Brunström,
53
1990) in chicken embryo (G. gallus domesticus). In addition, it has been reported that aluminum
54
(ElMazoudy & Bekhet, 2016) and different chemical elements evaluated in separate (cadmium,
55
arsenic, cobalt, copper, indium, iron, manganese and molybdenum) (Gilani & Alibhai, 1990)
56
induced pericardium edema in chicken embryos exposed in ovo.
57
According to Incardona & Scholz (2016), fluid accumulation in extracellular spaces of the
58
heart is one of the most sensitive indicators of cardiac dysregulation in embryos. Therefore, the
59
current study suggests a cardiotoxic effect induced by TE, whose consequences for embryonic
60
development can be severe. Although it is too early to suggest any action mechanism to explain
61
pericardial edema formation, it is possible assuming that embryo exposure to TE affected the
62
interconnection of cardiovascular and osmoregulatory systems during early development, thus
63
leading to changes in tissue osmolarity, as proposed by Incardona & Scholz (2016). Similar
64
reasoning can be adopted for brain edema formation, which confirms the neurotoxic action of
65
TE already reported in other model systems who represented mammalians (Rabelo et al., 2016;
66
Guimarães et al., 2017; Chagas et al., 2018), fish (Chagas et al., 2019), amphibians (Amaral et al.,
67
2018) and birds (Sampaio et al., 2019).
68
On the other hand, neural tube rotation errors are a clear evidence of teratological effect
69
induced by TE. Such errors can lead to scoliosis formation and to other torsion defects, as
70
observed in the present study (Figure 2F). According to Fujinaga et al. (1995), axial rotation is an
71
interesting developmental event not only because it is a dynamic process but also because it is
72
one of the earliest morphological signs of body asymmetry. The causes of this condition can be
73
complex, as can be the chemical constitution of TE; however, its consequences for individuals
74
comprise abnormal body formation (culminating in asymmetrical conditions) with severe
75
implications for biomechanical aspects of locomotion. Changes in the axial body rotation in mice
76
can be associated with rotating protein deficiency, which is considered the novel player in the
77
mice’s left-right specification cascade and represents a genetic link between axial rotation
78
processes and processes leading to organ asymmetry (Faisst et al., 2002). Therefore, the 13
Journal Pre-proof 79
hypothesis that TE chemical constituents have some influence on biochemical and molecular
80
mechanisms associated with the synthesis and release of this protein cannot be ruled out.
81
Another important aspect lies on the negative effects of TE on the morphometric
82
parameters evaluated for embryos. These effects were evidenced by the shorter crown-rump
83
length and anterior-posterior length of the head, by the beak and lens area observed in embryos
84
exposed to the TE as well as by increased optic vesicle area, and lower and posterior limbs, of
85
embryos in group TE-II (Figure 2).
86
Although the current study is pioneer in demonstrating these effects on embryos exposed
87
in ovo to TE, other studies have reported teratogenic effects on G. gallus domesticus eggs exposed
88
to pesticides (Abbas et al., 2018), plasticides (Wang et al., 2019 ), drugs (Ertekin et al., 2019),
89
nanoparticles (Patel et al., 2019), as well as to different heavy metals, much of which were
90
identified in the effluent tested in the current study (Birge & Roberts, 1976; Gilani & Alibhai,
91
1990); Asmatullah et al., 1998; Asmatullah & Shakoori, 1998; Papaconstantinou et al., 2003).
92
Obviously, the action mechanisms suggested by the aforementioned studies vary and depend on
93
the tested pollutant. Multiple mechanisms may have induced the morphological changes
94
observed in the present study, given the chemical complexity of TE. Such mechanisms may
95
include biochemical and molecular changes capable of causing endocrine disruption and of
96
inducing cytotoxicity, as well as genomic mutagenicity, which was reinforced by outcomes
97
observed in erythrocytes of embryos exposed to TE.
98
The increased frequency of chromosomal aberrations in embryos exposed to the
99
pollutant (Figure 4B) indicates how genotoxic TE can be to embryos. The incidence of
100
chromosomal fragments in metaphase and anaphase suggests clastogenic action of pollutants,
101
whereas chromosomal losses (laggard, Figure 5) suggest their aneugenic effect. According to
102
Fiskesjö (1993), chromosomal fragments may result from breaks in chromosome bridges that
103
may originate from either translocations or cohesive chromosome terminations. Chromosomal
104
losses often take place when –pollutants have negative interference on mitotic spindle, which
105
prevents one or more chromosomes from attaching to the spindle fibers during anaphase (Kirsch-
106
Volders et al., 2002).
107
Thus, it is possible noticing that changes identified in the present study are just the “tip”
108
of an “iceberg” that represents the toxicological magnitude of TE effects on embryonic
109
development. Based on developmental, morphometric and genotoxic biomarkers, it was possible
110
demonstrating that even the ephemeral exposure to these pollutants at low dilutions can cause
111
effects that may compromise the embryonic development of birds, as well as affect their post-
112
hatching health. However, the fact that the toxic action mechanisms of TE components remain 14
Journal Pre-proof 113
unknown should be taken into consideration. The effects of different xenobiotics found in TE
114
may derive from synergistic, antagonistic and additive interactions or from interactions capable of
115
enhancing the toxic action of a given agent that acts along with a non-toxic agent or with an agent
116
presenting other toxicity types. In addition, studies focused on identifying chemical constituents
117
of major toxicological influence may be useful to support pollution remediation measures and to
118
help mitigating component-specific impacts. Therefore, overcoming these limitations and
119
expanding the use of more sensitive and early toxicity biomarkers are good prospects for future
120
studies.
121 122
5. CONCLUSION
123
The initial hypothesis in the current study was confirmed by morphological changes in,
124
and by genotoxic effects on, erythrocyte cells of G. gallus domesticus embryos exposed to TE,
125
even for a short period-of-time and at low dilutions. Thus, the present research was pioneer in
126
demonstrating that aquatic ecosystems contaminated with TE are a risk factor for the embryonic
127
development of waterfowl living in polluted areas, as well as that they can significantly affect the
128
dynamics of these populations. If, on the one hand, future studies can help better understanding
129
the biological mechanisms affected by this pollutant, on the other hand, the present data provide
130
sufficient evidences to reinforce the need of taking measures aimed at depolluting habitats
131
contaminated with TE, at preserving the environment and/or at mitigating impacts caused by
132
these pollutants. These measures should certainly include the effective implementation of stricter
133
environmental laws, whose legal devices can be, and are, enforced through sound and
134
comprehensive enforcement actions.
135 136
6. ACKNOWLEDGMENT
137
The authors are grateful to the Brazilian National Research Council (CNPq) (Process n.
138
426531/2018-3) and to Instituto Federal Goiano for the financial support (Process n.
139
23219.001291/2019-11). Moreover, they are grateful to Coordenação de Aperfeiçoamento de
140
Pessoal de Nível Superior (CAPES, Brazil) and to Fundação de Amparo à Pesquisa do Estado
141
de Goiás (FAPEG, Brazil) for granting the scholarship to the student who developed the research.
142
Malafaia G. thanks CNPq for the research productivity fellowship (Proc. N. 307743/2018-7).
143 144
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Journal Pre-proof INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS Alex Rodrigues Gomes1, Julya Emmanuela de Andrade Vieira1, Amanda Pereira da Costa Araújo1, Guilherme Malafaia1# 1Biological
Research Laboratory, Post-graduation Program in Conservation of Cerrado Natural
Resources, Goiano Federal Institute – Urutaí Campus (Urutaí, GO, Brazil).
Alex Rodrigues Gomes: Conceptualization, Methodology. Julya Emmanuela de Andrade Vieira: Methodology. Amanda Pereira da Costa Araújo: Conceptualization, Methodology Guilherme Malafaia: Management and coordination responsibility for the research activity planning and execution and Writing- Reviewing and Editing. #Corresponding Author: Biological Research Laboratory, Goiano Federal Institute – Urutaí Campus, GO, Brazil. Rodovia Geraldo Silva Nascimento, 2,5 km, Zona Rural, Urutaí, GO, Brazil. CEP: 75790000. Phone number: +55 64 3465 1995. E-mail: [email protected]
1
Journal Pre-proof Highlights INSIGHTS ABOUT THE TOXICITY OF TANNERY EFFLUENT ON CHICKEN (Gallus gallus domesticus) EMBRYOS
Tannery effluent (TE) induces morphological changes in G. gallus domesticus embryos
Ephemeral exposure of G. gallus domesticus eggs to TE cause mutagenic effect
Cr, Mn and Zn concentrations are higher in embryos exposed to TE