Evaluation of Genotoxic Effect of Photographic Effluent Using Allium cepa Assay

The increased need to keep graphic records of events has led to the use of photography in investigation and surveys and hence increased photographic activities. Toxicological survey of photographic effluents has received little attention in Nigeria, though the constituents have been shown to be very toxic. This study investigated the genotoxic effects of photographic effluents from selected locations in Lagos, Nigeria using Allium cepa assay, viability test, root length measurements and cytological studies. There was inhibition of root length development in addition to several chromosomal aberrations observed in the root of Allium cepa exposed to the effluents. Furthermore, there were statistical differences among the aberrations produced by the different concentrations of the effluent (p<0.05; p<0.01; p<0.001). It was inferred that photographic effluents have genotoxic and cytotoxic effects. Thus, there is need for biological detoxification of photographic effluents before disposal into the environment to remove or reduce the pollutant load in them.


Introduction
mixed with acid.The film-processing effluents are Industrial effluents have become one of the classified as hazardous waste since they may cause soil biggest problems in many developing and and water pollution, if not properly disposed of or developed countries.It is known that these treated (KODAK, 1996).effluents, when not treated properly, can cause The typical constituents of the photomutagenic or toxic effects directly or indirectly on processing wastewater streams are organic chemicals, humans, resulting in diseases such as cancer, chromium compounds, ferricyanide, silver, thiocyanate, congenital malformations, and cardiovascular ammonium compounds, sulfur compounds, phosphate diseases (Grover and Kaur, 1999).Although much and heavy metals (Bober et al., 2004).A wide variety of work has been done on the toxicity of some chemicals are used in black-and-white photographic effluents, little information exists on the toxicity of processing.These include developers, stop baths and photographic effluents.
For coloured processing, the industries' processing and activities generate chemicals include hydroquinone, ammonia, n-Butanol, photographic wastes such as spent fixers and Boron, 1,1,1-trichloroethane, methanol, 1,1,2,2bleaches (KODAK, 1996).Many photographic tetrachloroethane, 1,2-dichloropropane, silver and processes have been found to be hazardous and silver salts.produce toxic gases which may be released slowly Silver is one of the most toxic metals regulated into the environment.In addition, these gases are by regulatory bodies.The different health effects of silver usually generated at faster rates if the photo 1,1,1-trichloroethane, an ozone-depleting chemical chemicals are heated or if certain chemicals are whose production has been highly regulated was outlined by Drake and Hazelwood (2005).
To the best of our knowledge no information on Hydroquinone is a very toxic chemical with long the genotoxicity assay of the photographic effluents lasting carcinogenic effects on animals and humans exists.This is worrisome as much importance is (Enguita and Leitao, 2013).Dissolved borates in attached to photography as means of preserving high concentration show some toxicity on plants.
information.High vapour concentrations of n-butanol produce narcosis and death in animals (Stellman, 1998).

Some of the most predominantly used and
Sources and preparation of materials controversial chemicals in photographic developers The Onion bulbs (Allium cepa) of the purple are metol, hydroquinone, EDTA, DTPA, and NTA.
variety of average size (15-22 mm diameter) were Metol has been known for quite some time to cause purchased at Ile-Epo Market, Lagos.The photographic a dermatitis condition of the skin known as metol effluents were collected from three locations (i.e poisoning.Metol is also known to cause eye and Agbado-Oke, Ifako-Ijaiye, and Yaba areas) in Lagos respiratory tract irritation with unknown long term State, Nigeria.Each effluent was diluted into three effects.In addition to the human health concerns, concentrations including 50%, 25%, and 10%.Tap metol is extremely harmful to aquatic organisms water was used as the control (0%).The experimental (Andreozzi et al., 2000).There are different forms setup was done in duplicates of each concentration of of toxicity testing to detect the hazards posed by the photographic effluent (Rank and Nielsen, 1998).toxicants in the environment.One such toxicity testing methods is the Allium cepa test which is an Viability Test excellent model in vivo, where the roots grow in The onion bulbs were sun-dried for two weeks direct contact with the substance of interest (i.e. and the dried roots present at the base of the onion effluent or complex medicinal mix being tested) bulbs were carefully shaved off, with a sharp razor blade enabling possible damage to the DNA of eukaryotes to expose the fresh meristematic tissues.The rings of to be predicted (Nefic et al., 2013).
the root primordial were left intact.The clean and The Allium cepa test is one of the few direct healthy onion bulbs were selected and suspended in methods for measuring damage in systems that are distilled water to determine viability as described by exposed to mutagens or potential carcinogens, and Fiskesjö (1997).After 24 hr, the onion bulbs with freshly enables the evaluation of the effects of these emerged roots were selected.As soon as the roots were damages through the observation of chromosomal about 1 cm long they were suspended in different concentrations of photographic effluent (50, 25 and alterations.An extensive review on the Allium cepa 10% respectively).test and its use in environmental contamination was carried out by Leme and Marin-Morales (2009), who Root Length Measurement reported that vascular plants are recognized as excellent genetic models for detecting The onion bulbs were removed from the environmental mutagens and are frequently used in distilled water and placed on a blotting paper to remove monitoring studies.excess water.At the end of the exposure period in effluent and in tap water (control), the roots of the Data from Allium cepa test can be onion bulbs with the best growth at each concentration extrapolated for all animal and plant biodiversity.
were removed with forceps and their lengths measured The Allium cepa test has been used by many (in cm) with a calibrated ruler.Growth inhibition was researchers mainly as a bioindicator of estimated as EC50 (the effective concentration of a environmental pollution by Leme and Marinchemical producing 50% of the total effect).The mean Morales ( 2009 et al., 2002;Lubini et al., 2008;Rashed et al., 2015; root length of the control was also calculated and the Tedesco et al., 2012) because it uses a model that is result plotted on a graph.adequately sensitive to detect innumerous substances that cause chromosomal alterations.

Cytological Study Evaluation of the ability of a compound to cause chromosomal damage is essential in the
The emerged root tips of the onion bulbs in the assessment of the potential genotoxicity of any different concentrations of the photographic effluents compound (Repetto et al., 2001).
were fixed in aceto-alcohol (1:3) after the exposure The aim of this work was to investigate the periods.The conventional feulgen-squash method cytotoxicity and genotoxicity of photographic (Sharma and DPhil, 1980) was used to prepare permanent slides of root meristems.The root tips were laboratory effluent using Allium cepa root tip cells.
put in 1-2 normal hydrochloric acid (HCl) for five cells divided by the number of dividing cells and minutes to soften the tissue.The tips were then multiplying the quotient with 100 (Bhatta and Sakya, macerated and stained with aceto-orcein (a mixture 2008) of acetic acid and orcein) stain for 20 minutes.Four root tip squashes were prepared for each treatment Statistical analyses and a minimum of 500 cells were examined for each The data obtained were statistically analysed concentration.
using the Graphpad Prsim 5.0 version software.The The slides were examined and counts were mean values were compared using two way analysis of observed for dividing cells, non-dividing cells, and variance followed with Bonferroni Posthoc test at cells at each mitotic phase and aberrant cells were P<0.5, P<0.1 and P<0.01 respectively.made (aberration test, bridges, fragments, laggards, and multipolar anaphases were scored) Results and Discussion using the method described by Adeyemo and Root length growth and inhibition Farinmade (2013).The mitotic index (MI) was The effects of the effluents on the root lengths determined by dividing the number of dividing cell of A. cepa are shown in Table 1.The effluents inhibited with the total number of cells counted as described the root length development in a dose dependent by Adeyemo and Farinmade (2013).
manner.The root length inhibition for each effluent had Characterization of mitosis and chromosomal a direct relationship with the concentration.Generally, aberrations were scored in 100 cells per slide.
the effluent collected from Ifako-Ijaye exhibited more Photomicrographs of some aberrant cells were inhibitory effects than others.Also, effluent collected taken in comparison with the normal cells.Mitotic from Agbado-Oke had the least inhibitory effect Index was estimated as number of dividing cells compared to the others.The median effective over the total number of cells counted expressed in concentrations (EC50) for samples from Agbado-Oke percentage.Similarly, the percentage abnormal and Ifako-Ijaye areas is 16% while that of sample from cells were calculated as the number of aberrant Yaba is 17%.

Table 1: Effect of photographic effluent on root growth of A. Cepa
was observed in 50% treatments.Also the mitotic index The effect of photolab effluent on the cell division and of the A. cepa exposed to effluents from those areas was mitotic index of A. Cepa 0. In addition, the mitotic indices generally decreased as The number of dividing cells decreased the concentration of the effluents increased.The total with the increase in the concentration of number of dividing cells from the control treatment was photographic effluent used (Table 2).Generally, A.
significantly higher than those from the roots treated cepa root exposed to effluents from Yaba had more with various concentrations of the effluents (P<0.05;dividing cells and mitotic index for each P<0.01).Significant differences in the number of concentration compared to the effluents from dividing cells were observed between cells of the roots Ifako-Ijaye and Agbado -Oke.For effluents from treated with various concentrations of the effluents.Ifako-Ijaiye and Agbado-Oke areas, no dividing cell  3. Generally all the effluents at -phase of the cell cycle, preventing the cell from different concentrations produced more sticky entering mitosis, are reasonable explanations for the chromosomes than other forms of aberrations.
reduction in the mitotic activity (Sudha et al., 2010).However, the photographic effluent from Yaba Reduction in the mitotic activity is accompanied by produced more chromosomes with multipolar decreased amounts of DNA, which could be due to anaphase (at 50% concentration) than the other inhibition of DNA synthesis or blocking in the G2 phase forms of aberration observed.Although, no of the cell cycle, preventing the cell from entering aberration was recorded generally for the roots mitosis (Türkoðlu, 2008;Türkoðlu, 2009).Decline of treated with 50% concentration of the effluents mitotic index below 22% in comparison to negative from Agbado-Oke and Ifako-ijaye areas due to none control can cause lethal effects on the organism dividing cells, the 50% concentration of effluent (Antonise-Wiez, 1990).from Yaba produced the highest percentages The decrease in mitotic index caused by the aberration of 61.70%.
photographic laboratory effluent treatments was The lowest percentage aberration (9.5%) associated with failed chromosome segregation in was produced in roots treated with 25% anaphase and the appearance of micronuclei as concentration of effluents from Yaba.The total suggested by the results of the mitotic indices.This also, number of aberrations in cell of roots treated with suggest that the effluent has cytotoxic effect which is 10% effluents from Agbado-Oke and Yaba and not lethal at the concentrations used in the study.The 25% effluent from Ifako-Ijaye was significantly decrease in the mitotic index (MI) with increasing higher than the vagrant cell at P<0.001 and for the concentrations of photographic effluents as well as the 10% effluent from Ifako-ijaye (P<0.05).Also, the formation of chromosomal aberrations such as vagrant, total chromosomal aberrations were significantly sticky chromosomes, binucleus and severe reduction in higher than the bridged, and the binucleated cells root length can be attributed to the presence of some for cells treated with 10% effluents from Agbado-cytotoxic and genotoxic compounds in the effluent.Oke and Yaba and 25% effluent from Ifako-Ijaye Root length is an important parameter which reflects (P<0.001).In the case of roots treated with 10% the toxicity of substances.The elongation zone of the effluent from Ifako-Ijaye, the bridged chromosomes onions may serve as a sensitive external signal of and the binuleated cells are significantly lower than ongoing internal cellular events (Adeyemo and the total aberration at P<0.05.Similar difference Farinmade, 2013).Plant roots interact with each of the was noticed between the total aberrations and the physical factors in their environment and this interaction multipolar cells and cells with chromosome breaks.
can lead to modification of the roots.In the A. cepa The cytotoxicity level of environmental assay, inhibition of root growth has been shown to pollutants can be determined by a decreased rate indicate retardation of growth and cytotoxicty of mitotic index (MI); this is an acceptable measure (Odeigah, et al., 1997;Grant, 1982).Also, growth inhibition can be caused by reduction in mitotic lead to aneuploidy and cell death (Kwon et al., 2008).activities and occurrence of various chromosomal Multipolar spindle, aneuploidy, disruption of cell polarity aberrations (Adeyemo and Farinmade, 2013).
and failure of asymmetric cell division can occur due to According to Rank (2003), vagrant chromosomes failure to properly control centrosome number and are also indicators of spindle poisoning and the function (Nigg, 2002).induction of vagrant chromosomes is a The bridges noticed in the cells are probably consequence of spindle disturbances.Thus the formed by breakage and fusion of chromosomes and presence of vagrant chromosomes noticed in this chromatids.Such chromosome bridges were also work, suggests that photographic effluents have reported to be induced by other chemicals (Donbak et the ability to poison and disturb the mitotic spindles. al., 2002).Chromosome bridges may also be due to Such can lead to arrest of mitosis and cell death chromosomal stickiness and/or unsuccessful separation (Matson and Stukenberg, 2011).
of chromosomes at anaphase, otherwise, it may be The different forms of aberration observed attributed to unequal translocation or inversion of in this study are similar to the findings of chromosome segments (Gomurgen, 2000).Akinsemolu, et al. (2015) who reported various According to Onyemaobi et al. (2012), bridges arise from joined ends of broken sister chromatids while chromosomal aberrations when Allium cepa roots lagging results from failure of chromosome movement were exposed to tobacco industrial effluents.
or acentric fragments.Bridges and fragments are Previous study by Bhatta and Sakya (2008) also clastogenic effects, both resulting from chromosomal reported chromosomal aberrations in a study of the and chromatid breaks Kovalchuk et al., 1998).Bridges mitotic activity and chromosomal behavior in root probably occur by the interruption and joining meristem of A. cepa treated with magnesium chromosomes or chromatids (Turkoglu, 2007), as a sulphate.Such aberrations have some significance result of chromosome stickiness, or due to unequal to the development and wellbeing of organisms.
translocation or inversion of chromosome segments Our results also corroborates that of (Gömürgen, 2005).This may suggest that the effluents Dragoeva et al.( 2009) who evaluated genotoxic can cause chromatid break.Chromosome stickiness is potential of agricultural soil and reported various classified as a physiological effect exerted by the chromosomal abnormalities like vagrant chromosomes, wastewater in A. cepa, which alter the proteins of the chromosomal fragments at anaphase and telophase chromosomes.Stickiness is irreversible and reflects and multipolar anaphases.Binucleate cells are due high toxicity of substances, while acentric fragments in to a defect in cytokinesis.Similar inhibitions and anaphase are the result of chromosome or chromatids formation of binucleated cells were reported by interruptions, indicating interference with DNA (Jensen et al., 1996;Yasui et al., 2015).These cells (Olorunfemi et al., 2012).can arise in a culture as a result of an incomplete Sticky chromosomes represent poisoned process of cell division, i.e. karyokinesis with chromosomes with sticky surface and probably lead to incomplete cytokinesis or as a result of the mitotic cell death (Fiskesjö, 1997).Chromosome stickiness can division of a pre-existent binucleate cell.The mitotic also lead to apoptosis due to toxic effects of the division of a binucleate cell can give rise to different wastewater.These results are in line with other studies types of daughter cells (Rodilla, 1993).The on the effects of different chemicals on plants and binucleation is the result of nuclear division not human cells (Rencuzogullarý et al., 2001; Gomurgen, followed by cytoplasmic division (Uetake and 2000;Shahin and El-Amoodi,1991;Nwangburuka and Sluder, 2004).This could imply that photographic Oyelana, 2011).Sticky bridges might be as a result of effluents inhibit or interfere with cytokinesis.
incomplete replication of chromosomes by defective or Binucleate formation therefore may be a way of less active replication enzymes (Sinha, 1979).Sticking doubling up the haploid genome, to produce diploid of chromosomes probably occurs due to degradation or cells of androgenetic origin as seen in placental depolymerization of chromosome DNA (Grant, 1982) or mesenchymal dysplasia, a rare condition associated as a result of DNA condensation and stickiness of interwith intrauterine growth restriction, prematurity chromosome fibers (Schneiderman et al., 1971).and intrauterine death.
This may imply that the sticky chromosomes All multipolar anaphases contain extra observed in this study could have been caused by either centrosome.Multipolar anaphase might be a result depolymerization of the chromosome DNA or due to of partial or incomplete C-mitosis (Grant, 1982).
DNA condensation.Stickiness is a highly toxic and Thus, our data demonstrate that multipolar mitoses irreversible effect, generally leading to cell death can activate the spindle assembly checkpoint (SAC) (Fiskesjö, 1997) and the folding of chromosome fibres in S2 cells.Multiple centrosomes in tumor cells into single chromatids (Klásterská et al., 1976).The create the potential for multipolar divisions that can statistically higher number of stickiness compared photographic effluents are highly toxic and that most to other aberrations may suggest that the cells may die if exposed to photographic effluents.
);Bagatini et al. (2009), testing crude root length of each onion in each concentration was extracts of cyanobacteria, as well as to evaluate the calculated by dividing the total root length for each genotoxic potential of medicinal plants (Camparoto concentration by the number of roots measured.The

Table 3 : Chromosomal Aberration in A. cepa due to exposure to photolab effluent Conclusion
. This study revealed that photographic Bhatta, P. and Sakya, S. R. 2008: Study of Mitotic effluent has genotoxic effects on Allium cepa and Activity and Chromosomal Behaviour in Root Meristem can pose a potential health risk to plants, animals of Allium cepa L. Treated with Magnesium sulphate, and humans.The results suggest that photographic effluent is of ecotoxicological importance and has Ecoprint 15: 83-88.the ability to interfere with the DNA biosynthesis.A. cepa may be a sensitive biosensor for screening the Bober, T.W., Vacco, D., Dagon, T.J. and Fowler, H.E. genotoxicity of photographic laboratory effluents.2004: Treatment of Photographic Processing Wastes.In: Handbook of Industrial and Hazardous Wastes