Ecotoxicology and Environmental Safety 138 (2017) 98–104
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Aneugenicity and clastogenicity in freshwater ﬁsh Oreochromis niloticus exposed to incipient safe concentration of tannery eﬄuent
Abel Weldetinsaea, , Mekibib Dawitb, Abebe Getahunc, H.S. Patilb, Esayas Alemayehud, Melaku Gizawa, Moa Abatea, Daniel Aberaa a
Ethiopian Public Health Institute, Department of Environmental Public Health Research, Ethiopia Addis Ababa University, Center for Environmental Sciences, Ethiopia c Addis Ababa University, Department of Zoological Sciences, Ethiopia d National ﬁsh and other aquatic life research center, Ethiopia b
A R T I C L E I N F O
A BS T RAC T
Keywords: Micronuclei Nuclear abnormality Oreochromis niloticus Tannery eﬄuent Maximum tolerable concentration
Conventional eﬄuent bioassays mostly rely on overt responses or endpoints such as apical and Darwinian ﬁtness. Beyond the empirical observation, laboratory toxicity testing needs to rely on eﬀective detection of prognostic biomarkers such as genotoxicity. Indeed, characterization of tannery eﬄuent requires slotting in of genotoxic responses in whole eﬄuent toxicity testing procedures. Hence, the prime objective of the present experimental investigation is to apply the technique of biological assay as a tool of toxicity testing to evaluate the induction of micronuclei (MN) in peripheral erythrocytes, and exfoliated cells of gill and kidney of O.niloticus exposed to Maximum tolerable concentrations (MTCs) of composite Modjo tannery eﬄuent (CMTE) and to compare the sensitivity of each cells origin to the induction of MN. After 72 h of exposure, cellular aberrations were detected using MN and nuclear abnormality (NA) tests. The induction of MN was signiﬁcantly higher in exposed groups (P < 0.05) when compared to the control group; moreover the tissue speciﬁc MN response was in the order, gill cells > peripheral erythrocyte > kidney. Total NA was found to increase signiﬁcantly (P < 0.05), when compared to the non-exposed group. NA was also further ramiﬁed as blebbed (BL), bi-nucleated (BN), lobbed (LB) and notched (NT) abnormalities. The result of each endpoint measured has demonstrated that at a concentration of total chromium (0.1, 0.73 and 1.27 mg/L), a perceptible amount cellular aberration was measured, further implicating somber treat of genotoxicity to ﬁshes, if exposed to water contaminated with tannery eﬄuent. This further highlight that conventional eﬄuent monitoring alone cannot reveal the eﬀects expressed at cellular and genetic levels further demanding the incorporation of eﬄuent bioassays in risk assessment and risk management/abatement programs.
1. Introduction Toxic eﬀect of complex mixture of eﬄuents often with undetermined concentration and with possible synergistic, additive and/or antagonistic eﬀect can be detected only applying eﬄuent bioassay (Birkholz et al., 2000). However, conventional eﬄuent bioassay mostly relies on overt responses such as apical and Darwinian ﬁtness endpoints. Beyond the empirical observation, laboratory toxicity testing needs to rely on eﬀective detection of prognostic biomarkers i.e. molecular, cellular, physiological and behavior of reduced performance impeding pathology and damage to health (Moore et al., 2004). This is for a reason that chemicals may have other eﬀects not tested under current strategies, such as genotoxicity, that may, for example, lead to population decline (Walker, 2006). Even though ﬁrst developed for
mammalian genotoxicity studies, MN assay is used profusely in ecological biomonitoring, especially utilizing ﬁsh to detect any genotoxic activities in test species. A complete review of the use of ﬁsh MN assay for tests having ecotoxicological importance is presented by AlSabti and Metcalfe (1995) and Obiakor et al. (2012). According to Heddle et al. (1983), Al-Sabti and Metcalfe (1995) and Da Rocha et al. (2009), MNs are formed by condensation of chromosomal fragments or whole chromosomes that are not incorporated in the main nucleus following anaphase. In ﬁsh these anomalies are physically identiﬁed as 1/10- 1/30 lower than the main nucleus (Ayllon and Garcia- Vazquez, 2000). In addition processes such as problem in segregating tangled and attached chromosome (Shimizu et al., 1998; Seriani et al., 2012), aneuploidy (Fernandes et al., 2007) and cellular degradation (Ateeq et al., 2002) are speculated to cause NA.
Corresponding author. E-mail address: [email protected]
http://dx.doi.org/10.1016/j.ecoenv.2016.10.026 Received 30 June 2016; Received in revised form 17 October 2016; Accepted 22 October 2016 0147-6513/ © 2016 Published by Elsevier Inc.
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
composite tannery eﬄuent concentration (2%, 4%, 8%, 16% and 32%) was conducted to deﬁne the median lethal concentration. The concentration as calculated using online software LdP (http://www. ehabsoft.com/ldpline/) deemed to be 6.09% of the test concentrate. The second step was estimating the safe concentration. An application factor as measured in toxicity unit (TU) is a measure of strength of any toxicant and used in ﬁsh and pollution works as a way of predicting a safe level. The values of the toxicity units are sated based on the recommendations assigned by scientists through several experimental works. As recommended by Sprague (1971) toxicity unit values ranging from 0.01 to 0.4 can be applied depending on the constituent of the eﬄuent. For instance values ranging from 0.1 to 0.05 TU is used for eﬄuents composed of non persistent pollutants. For pollutants having cumulative and persistent toxic eﬀect, a value of 0.1 is recommendable. A higher value of 0.4 TU can cause a harmful eﬀect, therefore its uses shall be cautious. In the present experimental work, since the tannery eﬄuent is composed of both persistent and non persistent pollutants, which might have a cumulative eﬀect or antagonistic eﬀect, three values of TUs were applied. These are application factors of 0.01, 0.1 and 0.4 TU.
Tannery eﬄuent is known to cause genotoxic eﬀect in various organisms including ﬁsh species; these were ascribed to the presence of chromium among others (Matsumoto et al., 2006; Al-Sabti et al., 1994). One good example to this assertion is the clastogenic and aneugenic eﬀect of water contaminated with tannery eﬄuent consisting of 0.01–0.05 mg/L of total chromium as reported by Matsumoto et al. (2006), whereas the discharge limit in the particular country was set to be 0.05 mg/L. This shows that the conventional method of tannery eﬄuent characterization which relies alone on physico-chemical analysis cannot depict the actual eﬀect of the eﬄuent. With speciﬁc to tannery eﬄuent there are still gaps in the use of MN in a comprehensive manner. For instance, the use of cells other than peripheral erythrocytes, like gill, liver and kidney cells altogether to appraise tissue speciﬁc responses is scanty. Thus, the present experimental study has attempted to evaluate the induction of micronuclei (MN) in peripheral erythrocytes, and exfoliated cells of gill and kidney and compared the sensitivity of each cell origin to the induced MN. In addition, the induction and use of NA in foil with MN was assessed. 2. Material and methods
MTC = 96 hrLC50 X AF (measured in TU (toxicity unit))… … … …
2.1. Eﬄuent sampling
Finally, the MN and NA test in the whole organism was conducted, at 0.06%, 0.6% and 2.4% of composite tannery eﬄuent. Prior to the genotoxicity testing, incipient MTC concentrations were tested for their potency of triggering mortality and/or behavioral alteration; to our beneﬁt none of the eﬀects were manifested. During the actual experiment the number of ﬁsh (N) per treatment was calculated using a prior experience sample size determination method. A one tailed t-value for an inﬁnite number of degree of freedom at the 95% conﬁdence level (i.e. P≤0.05) was considered. From other similar experiments the values of aberrated erythrocytes (d) frequency in ﬁsh blood i.e. 0.5–2.3% was considered in estimating the sample size. Likewise an examination of the control data from other studies shows that the sample standard deviation (S) is 0.825. Therefore, using an average aberration frequency of 1.4% the total number of ﬁsh samples was calculated as follows: -
For the experimental study eﬄuent from the Modjo Tannery Share Company, central Ethiopia was used. The eﬄuent is released from separate houses (processes) of the tannery, however it does not emerge as a composite eﬄuent; rather the eﬄuent is segregated into three ditches. The ﬁrst ditch is designed for the general eﬄuent, the second one is used to discharge eﬄuents enriched with sulphide and the last one is used to transfer chrome spent wash. Applying a composite sampling technique, three samples each from the inlets of the three ditches were taken in separate 10 L plastic containers. The samples were then transported to the Environmental Public Health Laboratory of the Ethiopian Public Health Institute and kept at 4 °C for the exposure assessment. Before the exposure assessment was commenced a proportional volume from each ditch was estimated and it was deemed to be 48% of general waste, 32% of sulphide waste and 20% of chrome spent wash. Based on this ratio, a mix was prepared in a 5 L plastic jar and was used for subsequent exposure studies.
(t1 + t2) 2 2 s …………………. d2
The t-value is taken from the t-table and the corresponding number is 1.645. Thus substituting the given values, number of ﬁshes stocked per aquarium becomes 5, and 10 ﬁshes per treatment were used in total. Further, Al-Sabti and Metcalf (1995) have reported that in most similar experimental studies the total number of ﬁsh in a single control group ranges between 3 and 12. Therefore, the total number of 5 samples is a reasonable estimate. After acclimatizing in well water, 12 cm long, O. niloticus were added to each aquarium for 72 h of exposure. Specimens were housed at a density of ﬁve specimens in 89-Liter aquarium under constant aeration and 12 h light/dark photo-period. A total of 4 exposure concentrations were prepared (0.06%, 0.6%, 2.4% eﬄuent) including well water and the test was carried out in duplicate with a total of 8 aquaria. At the beginning of each exposition period, water samples from each aquarium were taken and acidiﬁed using 3% HNO3 for subsequent analysis of trace metals.
2.2. Fish maintenance and transportation Healthy ﬁsh specimens were obtained from the Ethiopian Fishery Research Institute. Fishes, each having an average length of 12.75 ± 0.96 cm and an average body weight of 30.37 ± 2.77 g were transported to the laboratory in a big plastic bag half-ﬁlled with well water and compressed air. Prior to the experiment, the ﬁshes were acclimatized for 14 days in glass aquarium (89 L capacity) containing well water and in each aquarium seven ﬁshes were stocked. During acclimatization physico-chemical characteristics of the test environment were maintained at Temperature=27 ± 0.7 °C, pH=7.1 ± 0.24, Dissolved Oxygen=3.80 ± 1.13 mg/L, Total Hardness=6–49 mg/L (as CaCO3), and Conductivity=12–97 μS/cm, 12 h of exposure to light and 12 h of darkness. The ﬁsh were fed wheat bran amounting 3% of their body weight every day. 2.3. Experimental setup
2.4. Micronuclei and nuclear abnormality test
The genotoxicity of composite tannery eﬄuent was measured at maximum tolerable concentration (MTC) or safe concentration. As deﬁned by Hutchinson et al. (2009), MTC, “is the highest concentration that elicits a speciﬁc toxic eﬀects but not life threatening impairment in test animals”. Thus for the sake of analyzing genotoxic response in O.niloticus, the MTC was determined. In doing so, techniques of Sprague (1971), US EPA (2002), and Hutchinson et al. (2009) were consulted. First, a deﬁnitive acute toxicity study at ﬁve levels of
2.4.1. Blood and kidney erythrocytes To diagnose cellular aberrations among exposed and non-exposed ﬁsh specimen, whole blood samples were taken using a heart puncture and were collected in EDTA tubes. Kidney blood was taken using a capillary tube and two thin smears per blood samples were prepared and air dried for 24 h. Further, the slides were ﬁxed in absolute 99
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
methanol for 5 min and stained with 5% Giemsa for 15 min. A total of 2000 cells per each animal were scored under 1000 magniﬁcation microscope. Slides were coded and randomly analyzed by a single observer. The scoring was aimed at identifying cellular aberrations which are manifested with MN and NA. Scoring criteria was adopted from Carrasco et al. (1990) and Arkhipchuk and Garanko (2005). The criteria for the identiﬁcation of MN were as follows: (a) MN need to be smaller than one-third of the main nuclei, (b) MN clearly separated from the main nuclei, and (c) MN needs to be on the same plane of focus and cells with more than 4 MN were discarded to exclude apoptotic phenomena. NAs were also classiﬁed and scored as follows. Brieﬂy, cells with two nuclei were considered as binuclei (BN). Blebbed nuclei (BL) present a relatively small evagination of the nuclear membrane, which contains euchromatin. Evaginations larger than the blebbed nuclei which could have several lobes were classiﬁed as lobed nuclei (LB). Nuclei with vacuoles and appreciable depth into a nucleus that does not contain nuclear material were recorded as notched nuclei (NT).
Table 1 Levels of heavy metals present at different proportion of composite tannery effluent as analyzed by Atomic absorption spectroscopy. Physicochemical parameter
pH Cd Co Cr Cu Fe Ni Pb Zn
Concentration in mg/L 0.01AF
96 h LC50
7.28 0.22 0.03 0.1 0.01 0.85 0.06 0.18 0.01
7.20 0.23 0.10 0.73 0.09 1.20 0.30 0.22 0.40
7.16 0.43 0.12 1.27 0.30 1.30 1.12 0.42 0.47
– 0.89 0.26 7.28 0.71 2.55 3.21 0.92 1.18
one way ANOVA was used. Further to analyze variations among test concentration and tissue speciﬁc responses, a post hoc comparison test (Tukey HSD and Games-Howell) was used; these were applied based on the levene homogeneity test carried out prior; When the Levene test was not signiﬁcant the tukey HSD test was used as post hoc comparison, where as when the Levene test was signiﬁcant the Games-Howell is applied. Moreover, Pearson correlation was used to evaluate the correlation status of concentration with cellular aberration.
2.4.2. Gill epithelial cells Processing gills and scoring for induced MN in gill cells of O. niloticus was conducted according to Cavas and Ergene-Gozukara, (2003). Gills were removed and treated with a 15% acetic acid solution for 10 min. Epithelial cells were then gently scraped oﬀ the gill arches using forceps. Free cells were collected by centrifugation and treated with distilled water for 10 min. Cells were then ﬁxed in three successive changes of freshly prepared methanol–acetic acid (3:1) solution. Fixed cells were spread on clean slides and stained with a 5% Giemsa Solution for 30 min. Only the epithelial cells isolated from surrounding cells were scored. Micronuclei were determined as non-refractive small nuclei lying near the main nucleus.
3. Results 3.1. Physico-chemical characteristics of composite tannery eﬄuent The physico-chemical analysis of composite Modjo Tannery eﬄuent was analyzed using standardized protocol and the results are presented in Table 1. Accordingly, selected metals (chromium, cadmium, copper, cobalt, iron, nickel, lead and zinc) were quantiﬁed. The results shows that a signiﬁcant concentration of chromium was detected in all treatment groups, when compared to the control (less than the detection level (LDL)). The concentrations of metals have increased as the application factor (AF) increases. Concentration of chromium was found to be lower than the Ethiopian standard for chromium in tannery eﬄuent (2 mg/L) in each treatment. Unfortunately, the standard has not speciﬁed a limit for the rest of the elements; therefore comparison was not made; though their importance in subsequent appraisal of genotoxic response was not disregarded.
2.5. Water sample analysis The levels of cadmium (Cd), lead (Pb), zinc (Zn), chromium (Cr), copper (Cu), nickel (Ni), cobalt (Co) and iron (Fe) were determined by atomic absorption spectrometry according to APHA (1992). Immediately after collection, the water samples were acidiﬁed with 3% nitric acid. During sample digestion, 100 mL of eﬄuent sample was digested with the addition of 5 mL of nitric acid and boiling chips on a hot plate until the sample becomes clear, with the lowest volume possible (about 10–20 mL). To make the digestion more eﬃcient, as needed conc. nitric acid was added. This was followed by diluting the digested sample to a 100 mL volumetric ﬂask. Finally samples were then submitted to acid digestion and subsequent concentration quantiﬁcation of metals was performed using ﬂame based (acetylene and air) atomic absorption spectrometer (Nova 400A). The analysis was performed with hollow cathode lamp, single beam, and current, depending on the type of element analyzed 2–5 mA; slit width 0.2– 1.2 ηm. The wave length used for each elements is Cd (228.8 ηm), Cr (357.9 ηm), Cu (324.8 ηm), Ni (232.0 ηm), Zn (213.9 ηm), Co (240.7 ηm) and Pb (283.3 ηm).
3.2. Micronuclei in Oreochromis niloticus The values obtained for the MN analysis in erythrocyte, gill and kidney cells of O.niloticus are shown in Table 2. The induction of MN in Table 2 Mean frequency of Micronuclei in peripheral erythrocyte, gill and kidney cells of Oreochromis niloticus exposed for 72 h to three levels of Maximum tolerable concentration of composite Modjo tannery eﬄuent. Treatment groups
2.6. Data analysis Statistical analyses were performed using SPSS 12.0. To consider for either parametric or non parametric test, three basic assumptions were tested. First whether the value of one observation is not related to any other observation is critical. And this was proofed that each observational values are independent of each other. Second the normality of the test frequency was tested for normality using Kolmogorov-Smirnov Test and deemed to be normally distributed. The third check point is the test for homogeneity of variances, detected using Leven test. Since all assumptions are fulﬁlled a parametric test was carried out. Therefore to compare means of genotoxic response,
Control group 0.06% 0.6% 2.4% a
MN frequency (%) Erythrocyte
0.03 ± 0.03 0.49 ± 0.07a,b 1.23 ± 0.43a 2.08 ± 0.87a,b
0.06 ± 0.03 0.71 ± 0.12a,b 1.61 ± 0.22a,b 3.50 ± 0.52a,b
0.03 ± 0.04 0.44 ± 0.05a,b 0.35 ± 0.06a,b 0.48 ± 0.14a,b
Indicates signiﬁcant diﬀerence (P < 0.05) between mean frequency of MN % of treatment groups with respect to negative control. Indicates signiﬁcant diﬀerence (P < 0.05) of mean frequency of MN % amongst treatment groups. Two slides per fish blood, kidney and gill sample was prepared, in each slide 2000 of cells are scored, 4000 per fish and this was done in duplicate.
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
concentration, however these drop oﬀs were not signiﬁcant when compared to the preceding medium concentration. A signiﬁcant (P < 0.05) diﬀerence of LB abnormalities was reported only between lower and medium concentrations, whereas this was reported to be between lower and higher concentration in the case of BL abnormalities. Accordingly the order of sensitivity was stipulated to be in the following order; BN > NT > BL > LB. More to the ﬁndings as shown in Table 3, the overall nuclear aberration was recapitulated as total nuclear abnormality (TNA), and this sum up was found to increase signiﬁcantly (P < 0.05), when compared to non-exposed group. In addition, TNA increased concentration dependently; correlation of abnormalities with eﬄuent concentration ratio is calculated—the highest correlation coeﬃcient is calculated in the case of BN abnormalities (r=87.1) and the least was reported for LB (r=46). This correlation coeﬃcient implies the sensitivity of the abnormalities as a predicator to speciﬁc toxicity. Moreover a strong correlation was recorded between MN and NT abnormalities (r=89) followed by lobed and blebbed.
erythrocyte, gill and kidney cells of O.niloticus exposed to three levels of MTCs of composite Modjo Tannery eﬄuent indicates a signiﬁcant (P < 0.05) diﬀerence (ANOVA) when compared to control group; the diﬀerence was tissue speciﬁc and concentration reliant. In gill cells, the frequency of MN increased with concentration while signiﬁcant diﬀerence among test concentrations was observed (ANOVA, P < 0.05). Similarly, concentration dependent rise in MN frequency was also observed in peripheral erythrocytes, however the diﬀerence among medium (0.6%) and higher (2.4%) treatment groups was found to be insigniﬁcant (P > 0.05). Despite the fact that a signiﬁcant increment in contrast to the control group reported, kidney cells encompasses comparatively poor association between concentration and frequency of MN. This was owing to a lower frequency scored at medium (0.6%) concentration when compared to lower (0.06%) and higher (2.4%) eﬄuent concentrations. Moreover, the correlation coeﬃcient (r) was found to be signiﬁcant (p < 0.01) for gill (r=95) and peripheral erythrocyte (r=86), whereas at p < 0.05 moderate consequence reported for kidney cells (r=54). In none technical terms a speciﬁc toxicity (i.e. cellular aberration) was detected in peripheral erythrocyte, gill and kidney cells of O.niloticus exposed to MTCs of composite tannery eﬄuents. Hence, gill cells were reported to be the most aﬀected followed by peripheral erythrocyte and kidney cells. A positive correlation coeﬃcient accounted for all cell types as well indicates that a higher concentrations induce a higher frequency of MN, denoting concentration dependent increment of induced MN. This interdependency was found to be higher in gill cells pursued by peripheral erythrocyte and poor association detected in kidney cells.
4. Discussion The scoring of MN in ﬁsh has been a potential assay being adhered in proliferating cells, hitherto, peripheral erythrocytes and exfoliated erythrocytes of gill and kidney were among choices of interest (Bolognesi and Hayashi, 2011). However, frequency of MN varies depending on toxicokinetics and cell proliferation kinetics, as this bioindicator merely shows a discrepancy according to the exposition of ﬁsh species to diﬀerent classes of genotoxicants (Manna and Sadhukan, 1986; Hayashi et al., 1998; Palhares and Grisolia, 2002; Cavas and Ergene-Gozukara, 2003, 2005b; Ali et al., 2008). With respect to tannery eﬄuent, thus far, a paucity of information on the topic of comparative use in the midst of kidney, gill and peripheral erythrocyte exist; however few studies show that peripheral erythrocyte (Al-sabti et al., 1994 and Matsumoto et al., 2006) and kidney cells (Walia et al., 2013) respond positively. Therefore, which of the three origins of peripheral erythrocyte in O.niloticus are suitable and sensitive to score frequency of induced MN when O.niloticus is exposed to MTC of tannery eﬄuent is yet not explored. In view of that, one has to consider tissue speciﬁc as well as concentration dependent response of MN in gill, kidney and peripheral erythrocyte of O.niloticus. The MN test in all origins of erythrocyte has shown a positive response, though gill cells were the most aﬀected, followed by peripheral erythrocyte and kidney cells. A higher frequency of MN as demonstrated in gill cells when compared to the rest is in accordance with Hayashi et al., (1998) and Cavas and Ergene- Gozukara (2003, 2005b), indicating that, gills are under continuous exposure to pollutants when compared to blood and kidney. It is plausible that the higher frequency as noted in gills of O.niloticus was also due to, at least in part to, the presence of diﬀerent kinds of heavy metals in composite eﬄuent (Table 1). Given particular emphasis to heavy metals, Shukla et al. (2007), suggested this waterborne species initially bound to the gills, main site of water movement and subsequently deposited in other tissues, even the eﬀect might persist in these organs, after the heavy metals are siphoned. In addition, since cell proliferation kinetics is a determinant to MN scoring (Al-sabti and Metcalfe, 1995), a higher mitotic index in gill cells could be considered as one of the reasons to a higher frequency of MN scored in erythrocytes exfoliated from this organ. Furthermore, this might be because some of the MN scored in gill cells might not be transported back to kidney (Ali et al., 2008). There seems to be only two case studies, (Al-sabti et al., 1994) and (Matsumoto et al., 2006), who reported response of MN in peripheral erythrocyte of ﬁshes exposed to water contaminated with tannery eﬄuent. In general terms the present investigation is in substantial agreement with the ﬁndings of these two experimental investigations, although, there are slight diﬀerences. According to both studies
3.3. Nuclear abnormality in O. niloticus Results of nuclear abnormality (NA) frequency in peripheral erythrocytes of O.niloticus exposed to composite tannery eﬄuent are presented in Table 3. The NA was presented as blebbed (BL), binucleated (BN), lobbed (LB) and notched (NT) abnormalities (Fig. 1). The induction of NT nuclear abnormality at all concentrations signiﬁcantly (P < 0 0.05) increased when compared to control group. On the other hand, frequencies of BN and LB nuclear abnormality showed statistically signiﬁcant (P < 0.05) diﬀerence only at lower (0.06%) and higher (2.4%) eﬄuent concentration ratios. Frequency of blebbed (BL) nuclear abnormality was also found to be inconsistent, a signiﬁcant (P < 0.05) diﬀerence between exposed and control group was found only at lower (0.06%) and higher (2.4%) eﬄuent concentration ratios. Both BN and NT abnormalities increased concentration dependently, though no signiﬁcant (P > 0.05) diﬀerence was recorded between medium and higher concentrations, respectively. On the other hand BL and LB nuclear abnormality increased concentration independently. In both cases the induction decreased at higher eﬄuent Table 3 Mean frequency of NA in peripheral erythrocytes, gill and kidney cells of O.niloticus exposed to three Levels of MTC of composite Modjo tannery eﬄuent for 72 h. NA frequency (%)
Notched Blebbed Lobed Bi-nucleated Total NA
Treatment groups Control group
0.28 ± 0.09 0.11 ± 0.06 0.25 ± 0.13 0.03 ± 0.02 0.67 ± 0.11
0.70 ± 0.15a,b,c 0.49 ± 0.08a,b 0.60 ± 0.17a,b 0.40 ± 0.22b,c 2.16 ± 0.31a,b,c
2.04 ± 0.44a,b 1.18 ± 0.68a 1.50 ± 0.27b 1.80 ± 0.51a,b 6.48 ± 0.91a,b
2.7 ± 0.53a,c 1.07 ± 0.24a,b 1.18 ± 0.68a 2.69 ± 0.37a,c 7.63 ± 1.38a,c
indicates signiﬁcant diﬀerence (P < 0.05) between mean frequency of MN % of treatment groups with respect to negative control. Other similar letters across treatment groups indicates a signiﬁcant diﬀerence (P < 0.05) between mean frequency of MN % of treatment groups. Two slides per blood sample, in each slide 2000 of cells are scored i.e. 4000 per ﬁsh.
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
Fig. 1. Demonstrating nuclear abnormality and micronuclei; (1) normal cells. Blebbed nuclei (2) present a relatively small evagination of the nuclear membrane, which contains euchromatin. Evaginations larger than the blebbed nuclei which could have several lobes are classiﬁed as lobed nuclei (4). Nuclei with vacuoles and appreciable depth into a nucleus that does not contain nuclear material were recorded as notched nuclei (3). Micronuclei (5). * Represents artifacts.
longest exposure duration, representing a slow absorption of Cr (III) found in the chemical composition of the eﬄuent. It is reported that heavy metals such as Cd, Cu and Zn antagonistically and synergistically with other metals, may exert a strong inhibitory eﬀect on cell division (Barbosa et al., 2009). It has also been reported by Koca, et al. (2008) who conﬁrmed the presence of Pb, Ni, and Zn, among other metals in water did not bring a change of MN frequency in peripheral erythrocyte. In view of the current investigation, elevated amount of Cd, Co, Cr, Cu, Fe, Ni, Pb and Zn have been demonstrated when compared to well water (LDL) used for the experiment. More or less the concentration of these metals in higher eﬄuent concentration treatment group was calculated to be 1.5–47 folds higher than lower eﬄuent concentration treatment groups. Thus, a diminishing marginal change of MN in peripheral erythrocyte of test species exposed to higher eﬄuent concentration might be in part due to an elevated concentration of heavy metals. Therefore, the presence of these metals might have an inhibition of the cell division and hindrance of MN transportation into peripheral circulation. The latter is accounted to enunciation of Das and Nanda (1986), who described that MN, tends to persist in their sites of production (kidney and spleen) and later that at least some of them enter the peripheral blood circulation. Thus, in this study there might have been a delay in the transportation of induced MN from the hemopoitic cells. One reason can be also the opposite damaged cells might have been discarded from the cellular environment in higher rates than undamaged cells to maintain healthy tissue capable of carrying on normal physiological functions (De Flora et al., 1993; Mersch et al., 1996). However, this proposition needs to be investigated further. Another approach that might give a clear image is to appraise responses of the lesions in kidney erythrocyte. Therefore, a decline phenomenon in MN frequency of kidney erythrocytes and a rise of MN frequency in peripheral erythrocyte in each treatment groups, especially at medium concentration could be attributable to transportation of MN from the kidney through peripheral erythrocytes. Yet, to
chromium is pointed out as the prime cause of the clastogenic response. Even though chromium has a signiﬁcant importance in genotoxicity of tannery eﬄuent, considering it as the only genotoxicant in tannery eﬄuent must be taken cautiously. Here, consideration of water chemistry, presence of other heavy metals and their interactions also need to be considered separately. One of the techniques used in both investigations was analysis of total chromium in samples taken from three courses of a river, namely upper, point of discharge and lower course of the river. The approach has legitimacy and somewhat works for their conclusion; however both reports have disregarded the role of other heavy metals and organic compounds that might be present in total mixture of the eﬄuent. One good example is the concentration of heavy metals listed in the present investigation is higher than those presented by Matsumoto et al. (2006). In addition to this, diﬀerent class of surfactants and biocides used in leather processing as reported by Labunska et al. (2011), are worth considering for any potential eﬀects. It is also evident, as the concentration of the eﬄuent used increases, sensitivity of detecting MN in peripheral erythrocyte decreases. This accounted for no signiﬁcant diﬀerence of MN frequency scored between medium and higher concentrations (‘diminishing marginal increment’). Indeed, no consistent pattern of MN response is seen in peripheral erythrocyte as concentration of genotoxicants increases. Interspecies diﬀerences in xenobiotics metabolism, DNA repair, and cell proliferation in the target organ are known to be the prominent factors that aﬀect the sensitivity of ﬁsh species to genotoxicity (Al-sabti and Metcalfe 1995). According to Das and Nanda (1986), who reported similar phenomena in Heteropneustes fossilis, exposed to paper mill eﬄuent, suggested that this might happen in relation to the inhibitory eﬀect of the eﬄuent on cell division and subsequent hindrance to the passage of the aﬀected cells into peripheral circulation. On the contrary, Cavas and Ergene-Gozukara (2005b) observed MN increases in O.niloticus exposed to chromium factory eﬄuent at highest and
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
formation of nuclear anomalies induced in Lobio rohito exposed to tannery eﬄuent. They have also referred to propositions of Ateeq et al. (2002) that toxicants creates hypoxic conditions which result in depression of ATP that leads to abnormal shape of erythrocytes. Further, toxicants interrupted the lipid solubility of membranes of erythrocytes resulting in vacuolated and echinocytic cells and ultimately leading to apoptosis. In addition, blocking of cytokinesis can cause BN and BL nuclei and these anomalies are suggested as bioindicators of abnormal cell division (Serrano-Garcia and MonteroMontoya, 2001). Although the mechanisms underlying the formation of NAs have not been fully explained, one way of understanding the essence of recording them would be appraisal of their co-occurrence with MN. According to the present investigation a signiﬁcant association between frequency of MN and NA was recorded. In view of the Pearson correlation analysis the co-occurrence of MN with each abnormalities was found to be in the order of NT (r=89) > BN(r=86) > LB(r=78) > BL (r=60). Therefore, the variation of the frequencies in the present study might be due to the sensitivity of these frequencies along time variations. According to Walia et al. (2013) NAs are precursors of MN formation. It is also known to be a fact that the induction of MN is highly dependent on cell proliferation kinetics. According to Al-Sabti and Metcalfe (1995), MN induction typically occurred within 1–5 days post-exposure; for most ﬁsh this is known to be between 2 and 3 h. In addition, formations of BL and LB abnormalities are related to nuclear budding process during interphase (Shimizu et al., 1998; Seriani et al., 2012). Besides blocking of cytokinesis can cause BN and BL nuclei and these anomalies are suggested as bio-indicators of abnormal cell division (Serrano-Garcia and Montero-Montoya, 2001). Therefore, one might conclude that like the formation of MN in ﬁsh treated with genotoxicants is associated with cell proliferation kinetics, the reason for the inconsistent response of NA reported in the present investigation is unlikely to be a diﬀerent phenomenon. Particularly, when considering the link among cell kinetics and induction of NA, reports of Walia et al. (2013), would be one of the potential implications, however this also needs to be replicated in O.niloticus. Therefore, one possible explanation for the presence of a lower frequency of NA identiﬁed at higher eﬄuent concentration, especially the induction of BL and LB abnormalities might be due to the inhibition of the exocytosis process responsible for the elimination of aﬀected chromatin. This might be triggered due to the presence of elevated concentration of chromium and heavy metals; metals inhibit cell division exerting their aneugenic eﬀect to cause tublin failure and mitotic fuse. However, this hypothesis requires further investigation.
reach at in no doubt assertion of the fact that such transportation breach can cause a lower frequency of MN in either of the cell origins, future experiments need to consider a time based investigation. Even though the mechanisms underlying the formation of NAs have not entirely been understood, several studies have demonstrated that these nuclear lesions might potentially be a foil for the scoring of MNs under in vivo experimental conditions. In attendance to these, a number of studies have proposed the use of NA to complement MN assay as a prospective biomarker of exposure in ﬁsh species exposed to diﬀerent types of chemicals and industrial eﬄuents (Ayllon and GarciaVazquez, 2000; Cavas and Ergene-Gozukara, 2003, 2005a, 2005b; Da Silva Souz and Fontanetti 2006). The current study as well provides additional evidence of positive responses of nuclear lesion in peripheral erythrocyte of O.niloticus exposed to MTC of composite tannery eﬄuent. The classes of NAs as identiﬁed in this experimental study fall accordingly as blebbed (BL), bi-nucleated (BN), lobbed (LB) and notched (NT), which are aggregated as total nuclear abnormality (TNA). When analyzed separately the abnormalities have a variable response with respect to the control group and along the concentration gradient. In view of that, the sensitivity was set in the order of BN > NT > BL > LB. Both NT and BN have increased concentration dependently, while the frequency of BL and LB NAs decreased at higher eﬄuent concentrations. Comparatively, the occurrence of such responses along a concentration gradient seems not to be the only trend with exposition to sub-lethal concentration of tannery eﬄuent. At the same exposure period, Walia et al. (2013) has also presented the variation of the responses in peripheral erythrocytes of kidney with respect to concentration and in advance with time variation. Accordingly, Walia et al. (2013) emphasized the presence of chromium and heavy metals are contributory to the nuclear anomalies in Lobio rohito. Their conclusion strengthens the hypothesis of Von Sonntag (1987) and Steenken (1989). According to their reports an intracellular reduction of Chromium (VI) to Chromium (III) triggers the production of reactive oxygen species which freely can react with DNA and are later responsible for the formation of NA. In fact, Al-Sabti et al. (1994) also reported somewhat equal frequency of MN in ﬁsh species treated with Cr (VI) and Cr (III) but also leather processing waste, indicating a possible reduction of Cr (VI), which is likely to occur in the digestive tract. On the other hand, the current study also shares their proposition, because the concentration of chromium and other heavy metals are reportedly found to be higher than the control group. It is also likely that the oxidative stress caused with heavy metals lead to lipid peroxidation process increasing permeability of genotoxicants through cellular membrane making DNA more susceptible (Seriani et al., 2012). One or the other way these genotoxicants might have an opportunity to interfere in the DNA synthesis of an exposed organism, which gives rise to NA and later MN (Da Silva Souza and Fontanetti, 2006). It has also been suggested that gene ampliﬁcation via the Breakage– Fusion–Bridge cycle could cause LBs or BLs during the elimination of ampliﬁed DNA from the nucleus. This is proposed due to the fact that the nucleus may have a capacity to ‘sense’ excess DNA that does not ﬁt well with in its envelop. As a result the ampliﬁed DNA is localized to speciﬁc sites at the periphery of the nucleus and eliminated thorough nuclear budding to form MN (Shimizu et al., 1998). Seriani et al. (2012) has also recapitulated the hypothesis of (Shimizu et al., 1998) portraying the process at cellular level. According to their presentation, cells detect aﬀected regions of the cell and begin a process of repair and eliminate aﬀected chromatin by dragging it to the periphery of the nucleus through the process of exocytosis. However, when the exocytosis process becomes ineﬃcient or interrupted, the nuclear membrane becomes imperfect and as a consequence higher number of NA stagger. Moreover, aneuploidy is another abnormality that resulted due to tubulin failure and mitotic fuses caused by aneugenic actions of toxicants. According to Fernandes et al. (2007), BN and NT nuclei might be indicators of aneugenicity of toxicants, while Walia et al. (2013) too suggested this mechanism as one form of mechanism for the
5. Conclusion Lethality is an overt response in ecotoxicological protocols. This study has shown that relying on a mere response that inculcates an apical endpoint and Darwinian ﬁtness for toxicity testing in industrial eﬄuents is inadequate. Eﬄuent genotoxicity testing is a better alternative in whole eﬄuent toxicity testing. The result of this study showed a positive response of genotoxicity in O.niloticus as measured using MN and NA assay. The result of each endpoint measured in the present investigation has demonstrated that at a concentration of chromium (0.1, 0.73 and 1.27 mg/L), a perceptible amount of cellular aberration endpoints are measured, further implying that conventional eﬄuent monitoring alone cannot reveal the eﬀects occurring at genetic and cellular levels. 6. Recommendation One of the limitations that hampered giving a full inference on the principal component that triggers the observed genotoxic response was an incomplete physicochemical analysis performed in this experimental study. It is therefore vital to perform detailed characterization of 103
Ecotoxicology and Environmental Safety 138 (2017) 98–104
A. Weldetinsae et al.
67–71. De Flora, S., Vigano, L., D’Agostini, F., Camoirano, A., Bagnasco, M., Bennicelli, C., Melodia, F., Arillo, A., 1993. Multiple genotoxicity biomarkers in ﬁsh exposed in situ to polluted river water. Mutat. Res. 319, 167–177. Fernandes, T.C.C., Mazzeo, D.E., Marin-Morales, M., 2007. Mechanism of micronuclei formation in poliploidizated cells of Allium cepa exposed to triﬂuralin herbicida. Pestic. Biochem. Phys. 88, 252–259. Hayashi, M., Ueda, T., Wada, K., Kinae, N., Saotome, K., Tanaka, N., Takai, A., Sasaki, Y.F., Asano, N., Sofuni, T., Ojima, Y., 1998. Development of genotoxicity assay systems that use aquatic organisms. Mutat. Res. 399, 125–133. Heddle, J.A., Hite, M., Kirkhart, B., Mavourin, K., Mac-Gregor, J.T., Newell, G.W., Salamon, M.F., 1983. The induction of micronuclei as a measure of genotoxicity. Mutat. Res. 123, 61–118. Hutchinson, T.H., Bögi, C., Winter, M.J., Owens, J.W., 2009. Beneﬁts of the maximum tolerated dose (MTD) and maximum tolerated concentration (MTC) concept in aquatic toxicology. Aquat. Toxicol. 91, 197–202. Koca, S., Koca, Y.B., Yildiz, S., Gurcu, B., 2008. Genotoxic and histopathological eﬀects of water pollution on two ﬁsh species, Barbus capito pectoralis and Chondrostoma nasus in the Buyuk Menderes. Biol. Trace Elem. Res. 122, 276–291. Labunska, I., Brigden, K., Santillo, D., Johnston P., 2011. Heavy metal and organic chemical contaminants in wastewater discharged from leather tanneries in the Lanús district of Buenos Aires, Argentina, April 2011. Greenpeace Research Laboratories Technical Note 07‐2011. 〈http://www.greenpeace.to/greenpeace/wp-content/ uploads/2012/03/Argentina-tanneries-Technical-Note-07-2011-ﬁnal.pdf〉 Manna, G.K., Sadhukhan, A., 1986. Use of cells of gill and kidney of Tilapia ﬁsh in micronucleus test (MNT). Curr. Sci. 55, 498–501. Matsumoto, T.S., Mantovani, S.M., Malaguttii, A.I.M., Dias, L.A., Fonseca, C.I., MarinMorales, A.M., 2006. Genotoxicity and mutagenecity of water contaminated with tannery eﬄuents, as evaluated by the micronucleus test and comet assay using the ﬁsh Oreochromis niloticus and chromosome aberrations in onion root-tips. Genet. Mol. Biol. 29 (1), 148–158. Mersch, J., Beauvais, M.N., Nagel, P., 1996. Induction of micronuclei in haemocytes and gill cells of zebra mussels, Dreissena poly-morpha, exposed to clastogens. Mutat. Res. 371, 47–55. Moore, M.N., Depledge, M.N., Readman, J.W., Leonard, D.R.P., 2004. An integrated biomarker-based strategy for ecotoxicological evaluation of risk in environmental management. Mutat. Res. 552, 247–268. Obiakor, M.O., Okonkwo, J.C., Nnabude, P.C., Ezeonyejiaku, C.D., 2012. EcoGenotoxicology: micronucleus assay in ﬁsh erythrocytes as in Situ Aquatic Pollution Biomarker: a Review. J. Anim. Sci. Adv. 2, 123–133. Palhares, D., Grisolia, C.K., 2002. Comparison between the micronucleus frequencies of kidney and gill erythrocytes in Tilapia ﬁsh, following Mitomycin C treatment. Genet. Molec. Biol. 25, 281–284. Seriani, R., Abessa, D.M.S., Kirschbaum, A.A., Pereira, C.D.S., Ranzani-Paiva, M.J.T., Assunção, A., Silveira, F.L., Romano, P., MuccI, J.L.N., 2012. Water toxicity and cyto-genotoxicity biomarkers in the ﬁsh Oreochromis niloticus (Cichlidae). J. Braz. Soc. Ecotoxicol. 7 (2), 67–72. Serrano-Garcia, L., Montero-Montoya, R., 2001. Micronuclei and chromatid buds are the result of related genotoxic events. Environ. Mol. Mutagen. 38, 38–45. Shimizu, N., Itoh, N., Utiyama, H., Wahl, G.M., 1998. Selective entrapment of extrachromosomally ampliﬁed DNA by nuclear budding and micronucleation during S phase. J. Cell Biol. 140, 1307–1320. Shukla, V., Dhankhar, M., Prakash, J., Sastry, K.V., 2007. Bioaccumulation of Zn, Cu and Cd in Channa punctatus. J. Environ. Biol. 28 (2), 130–159. Sprague, J.B., 1971. Measurement of pollutant toxicity to Fish—III, sub lethal eﬀects and "safe" concentrations. Water Res. 5, 245–266. Steenken, S., 1989. Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reaction of their radical cations and e and OH adducts. Chem. Rev. 89, 503–520. US EPA, 2002. Methods for Measuring the Acute Toxicity of Eﬄuents and Receiving Waters to Freshwater and Marine Organisms, 5th ed. Von Sonntag, C., 1987. New aspects in the free-radical chemistry of pyrimidine nucleobases. Free Radic. Res. Commun. 2, 217–224. Walia, G.K., Handa, D., Kaur, H., Kalotra, R., 2013. Erythrocyte abnormalities in a freshwater ﬁsh, Labeo rohita exposed to tannery industry eﬄuent. Int. J. Pharm. Biol. Sci. 3 (1), 287–295. Walker, C.H., 2006. Ecotoxicity testing of chemicals with particular reference to pesticides. Pest Manag. Sci. 62, 571–583.
tannery eﬄuent for classes of organic pollutants especially those identiﬁed qualitatively and partly quantitatively by Labunska et al. (2011). In addition we have attempted to analyze only for concentration dependent responses of genotoxicity at MTC. However, the induction of MN is also time dependent phenomena, thus, time based studies are recommended. Conﬂict of interest The authors declare that they have no conﬂict of interest. Ethical consideration All applicable international guidelines for the care and use of animals were followed. References Ali, D., Nagpure, N.S., Kumar, S., Kumar, R., Kushwaha, B., 2008. Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater ﬁsh Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Chemosphere 71, 1823–1831. Al-Sabti, K.M., Metcalfe, C.D., 1995. Fish micronuclei for assessing genotoxicity in water. Genet Toxicol. 343, 121–135. Al-Sabti, K.M., Franko, B., Andrijani, S.K., Stegnar, P., 1994. Chromium induced micronuclei in ﬁsh. J. Appl. Toxicol. 14, 333–336. APHA, 1992. Standard Method for the Examination of Water and Waste Water, 18th ed., Washington: American Public Health Association Arkhipchuk, V.V., Garanko, N.N., 2005. Using the nucleolar biomarker and the micronucleus test on in vivo ﬁsh ﬁn cells. Ecotoxicol. Environ. Saf. 62, 42–52. Ateeq, B., Abul farah, M., Niamat, A.M., Ahmad, W., 2002. Induction of micronuclei and erythrocyte alterations in the catﬁsh Clarias batrachus by 2, 4dichlorophenoxyacetic acid and butachlor. Mutat. Res. 518, 135–144. Ayllon, F., Garcia-Vazquez, E., 2000. Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and Mollie Poecilia latipinna: an assessment of the ﬁsh micronucleus test. Mutat. Res. 467, 177–186. Barbosa, J.S., Cabral, T.M., Ferreira, D.N., Agnez-Lima, L.F., De Medeiros, S.R.B., 2009. Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals. Ecotoxicol. Environ. Saf. 73, 320–325. Birkholz, D.A., Headley, J.V., Ongley, E.D., Goudeya, S., 2000. Toxicity assessment and remediation of industrial wastewater. Can. Water Resour. J. 25 (4), 361–385. Bolognesi, C., Hayashi, M., 2011. Micronucleus assay in aquatic animals. Mutagenesis 26, 205–213. Carrasco, K.R., Tilbury, K.L., Mayers, M.S., 1990. Assessment of the piscine micronuclei test as an in situ biological indicator of chemical contaminants eﬀects. Can. J. Fish. Aquat. Sci. 47, 2123–2136. Cavas, T., Ergene-Gozukara, S., 2003. Micronuclei, nuclear lesions and interphase silverstained nucleolar organizer regions (AgNORs) as cyto-genotoxicity indicators in Oreochromis niloticus exposed to textile mill eﬄuent. Mutat. Res. 538, 81–91. Çavas, T., Ergene-Gozukara, S., 2005a. Induction of micronuclei and nuclear abnormalities in Oreochromis niloticus following exposure to petroleum reﬁnery and chromium processing plant eﬄuents. Aquat. Toxicol. 74, 264–271. Çavas, T., Ergene-Gozukara, S., 2005b. Micronucleus test in ﬁsh cells: a bioassay for in situ monitoring of genotoxic pollution in marine environment. Environ. Mol. Mutagen. 46, 64–70. Da Rocha, M.A.C., Dos Santos, A.R., De Marcelo, B.O., Da Cunha, A.L., Ribeiro, F.H., Burbano, R.M.R., 2009. The micronucleus assay in ﬁsh species as an important tool for xenobiotic exposure risk assessment—a brief review and an example using neotropical ﬁsh exposed to methylmercury. Rev. Fish. Sci. 17 (4), 478–484. Da Silva Souza, T., Fontanetti, C.S., 2006. Micronucleus test and observation of nuclear alterations in erythrocytes of Nile tilapia exposed to waters aﬀected by reﬁnery eﬄuent. Mutat. Res. 605, 87–93. Das, R.K., Nanda, N.K., 1986. Induction of micronuclei in peripheral erythrocytes of ﬁsh Heteropneustes fossilis by mitomycin C and paper mill eﬄuent. Mutat. Res. 175,