Nephrotoxic Effect of Sub-Acute Exposure of Treated Carbanaceous Effluent on Mice

Neurotoxicity tests in animals exposed to toxic substances could provide additional informat ion on possible neurotoxic effects. The aim of this study is to determine the nephrotoxic effect of wastewater discharged from a carbonaceous industry on mice (Mus musculus). The present study examined the nephrotoxic effects of carbonaceous wastewater in mice, the mice were exposed to five different concentrations of the waste water. Cyclophosphomide was used as the positive control and distilled water was used as a negative control, for a period of 35das. Creatinine and urea concentration in serum was used as kidney function. The weights of the animals were recorded weekly after which their kidneys were harvested. Organ weight was measured at post exposure and preserved afterwards for histology. The physical, chemical and heavy metal composition of the wastewater was also analysed. There was no significant (P>0.05) change in the kidney and body weight of the exposed mice or the negative control. The activities of urea and creatinine in the serum of exposed mice were significantly increased compared to the negative control mice and this increase was concentration dependent at P<0.05. The h istological lesions observed in the kidney showed generalised occlusion of the tubular lumen, general tubular necrosis, with protein casts in the tubular lumen and multip le foci of haemorrhage in the parenchyma were observed. The results of the study showed that the observed nephrotoxic effect in the exposed mice may be caused by the presence of heavy metal and other physical and chemical substances present in the waste water. Th is suggests a higher risk to kidney damage in humans and other organisms exposed to this waste water and may also be deleterious to the surrounding environment.


Introduction
Toxicology can be defined as a branch of science that deals with poisons. A poison is any substance that causes a harmful effect when ad min istered, either by accident or design, to a liv ing organism. The word 'poison' is a quantitative concept as almost any substance can be harmful at some doses but not harmful at others [1].
Neurotoxicity tests in animals exposed to toxic substances could provide additional informat ion on possible neurotoxic effects [2]. Hu mans are continuously exposed to different chemicals fro m water, air and soil everyday. The kidneys are important organs for metabolism, deto xification, storage and excret ion of these chemicals and their metabolites and thus are vulnerable to damage.
The s imilarity in body weight o f mice exposed to wastewater and control may be due to the ability of the mice to feed even wh ile being exposed. The increase in kidney may be due to inflammat ion of these chemicals present in waste water. Such observations were also observed in rats exposed to cadmiu m and alcohol [3]. Indiscriminate discharge of untreated or partially treated wastewaters directly or indirectly into aquatic bodies may render water resources unwholesome and hazardous to man and other living systems [4][5][6][7][8][9].
Several reports have demonstrated acute toxicity of industrial effluents to microalgae, fishes and bacteria. However reports on the nephrotoxicity are few and are mostly with microbial assays. More often than not, landfills of these effluents neither have a synthetic membrane liner at the bottom, nor a natural layer of co mpacted soil with the desired hydraulic conductivity, nor a run-off control system [9]. This is a potential source of leechates which ultimately find their way into mun icipal and local water supplies, adding to the toxic co mponents of domestic water we use every day [9].
An imperative need has been suggested to examine the toxic and nephrotoxic effects of these wastewaters in discharged from a carbonaceous industry on mice in order to generate information that will be useful to the environmental authorities for pro mu lgating laws that will guide the proper management of these waste waters [4][5][6][9][10][11].
Considering the high correlation between mutagenicity and carcinogenicity, it may be pertinent to mention that humans predisposed to cancer and exposed sewage systems which are heavy laden with imp roperly treated waste effluents may be at a very h igh risk for developing the disease [9]. This is because of the chemical content of industrial effluents that are active in in itiat ion, pro motion and progression and may work in concert to bring about neoplastic transformation [9,[12][13][14]. Thus, there are substances in the test sample that are capable of inducing genetic effects in mice which is relevant to human health because the toxico logical target is DNA, wh ich exists in all cellu lar forms [9,15].
The aim of this study is to determine the nephrotoxic effect of wastewater discharged fro m a carbonaceous industry on mice (Mus musculus). The aim would be achieved through examination of Kidney function test. These will include the monitoring of urea and creatin ine [16], histopathology of the kidney, body and organ weight index. The waste water will also be analysed for the presence of some physical, chemical and heavy metals. The findings fro m this study may be useful in the assessment of the to xic effects of waste water samp les on public health and environment. Th is may be a useful public marker in the promu lgation of stringent decisions as regards to the indiscriminate disposal of water.

Laboratory Mice
Forty two male alb ino mice were obtained fro m the animal breeding unit at the Institute for Advanced Medical Research and Training (IM RAT), Co llege of Medicine, University of Ibadan, UCH, Ibadan, Nigeria. M ice were acquired and quarantined in a pathogen-free, well ventilated room in order to enable the animals acclimatize to their environment. During the period of acclimat ization, the animals were supplied with food (pelleted foods) and drinking water on a daily basis. Their beddings were also changed daily (d isinfected and discarded). The mice were maintained in the depart mental animal and breeding unit at IMRAT where each cage contains six an imals.

Waste Water
The waste water sample was sourced from the drainage pipes of the Effluent Treat ment Plant of a beverage producing company in Lagos State, Nigeria. This co mpany is well known for the production of drin k beverages which are consumed nationwide.

Storage of Effluent
The collected effluents were stored in plastic bottles and refrigerated at 4°C until when needed. They were then brought out and diluted to various concentrations at room temperatures. The various concentrations were in-turn stored in plastic bottles and refrigerated all through the experiment.

Negati ve Control
Distilled water was used as a vehicular solvent for the dilution of the waste water used.

Positi ve Control
The drug cyclophosphamide was used. Administered dosage depends on the average body mass of the animal per kg, wh ich is 40mg/ kg. The value/informat ion in mg provided by the manufacturer was taken into consideration when the calcu lation was made. Average body weight of animal for positive control per kg is 24.5g; 40mg --- 1000g; X --- 24.5g; X = 40 x 24.5/ 1000 = 0.98mg of cyclophosphamide dissolved in 1000ml distilled water and administered orally for 35 days.

Exposure of Ani mals to the Samples
The animals were rando mly divided into seven groups. Each g roup was made up of six individuals. Five groups were each injected with a different d ilution of the effluent. One group was administered a positive control and another group the negative control sample. The dosage that was administered depends on the average body weight. 0.3ml of the sample was administered orally to each animal for a period of 35 consecutive days.

Serum Creatinine Es ti mation
This was carried out as described by Cheesbrough [16]. Creat inine in alkaline solution reacts with p icric acid to form a co loured co mplex. The amount of co mp lex fo rmed is directly proportional to the creatinine concentration.

Serum Urea Esti mati on
This was carried out as described by Cheesbrough [16]. Urea in the seru m is hydrolysed to ammon ia in the presence of urease. The ammonia is then measured photometrically by Berthelot's reaction.

Tests and Methodolog y
pH determination, alkalinity, total sulphate, total ch loride, total hardness, total dissolved solids, total suspended solids, biochemical o xygen demand (BOD), chemical o xygen demand (COD) and determination of metals.

Discussion
The present study examined the nephrotoxic effects of carbonaceous wastewater in mice, the mice were exposed to five d ifferent concentrations of the waste water. The present study showed that heavy metals concentrations were on the high side. This finding is therefore useful in further assessment of the toxic effects of chemicals present in the wastewater samples on the environment if dumped indiscriminately. Co mpared to the allowable limits [9,[17][18][19] most of the parameters analyzed, especially the heavy metals were present in high concentrations. This is comparable to the findings of previous studies [6,9,[20][21][22][23]. Ubalua [20] stated that the claim that cassava wastewater can cause problems in some crops is based on anecdotal informat ion.
Also in the present study, there was a concentration-dependent increase in serum urea and creatinine when compared to the control at the p<0.05 level. The kidney damage probably contributed to their increase. Orisakwe et al. [24] reported similar conditions in rats exposed to Hibiscus Sabdariffa Caly x.
The histological lesions observed in the kidney showed generalised occlusion of the tubular lu men, general tubular necrosis, with protein casts in the tubular lu men and mu ltip le foci of haemo rrhage in the parenchyma were observed. The results of the study showed that the observed nephrotoxic effect in the exposed mice may be caused by the presence of heavy metal and other physical and chemical substances present in the waste water. This is in line with similar observation made in other related studies by some authors. Freund [25] observed small hemorrhages in the bronchi and trachea of a person who died from accidental exposure to vapors of Nitrosodimethylamine (NDMA). Gastrointestinal hemo rrhage was observed in a person who died from accidental exposure to vapors of NDMA [2,25]. Jenkins et al. [26] also observed mortality in rats that received NDMA for 9 weeks. According to ATSDR/USEPA [2] reports, mice that received NDMA in drinking water for 224 days did not experience significantly decreased survival [27].
In a study by Maduagwu and Bassir [28], daily gavage exposure to NDMA for 30 days caused decreased survival in cats. According to Ungar [29] and ATSDR/USEPA [2] reports, daily ad min istration of the same substance in drinking water for 8, 12, or 16 weeks resulted in occasional moribundity in hamsters, while no lethality resulted fro m daily ad ministration of the same substance for 28 days [2,30]. In a study by Nishie [31], pregnant and nonpregnant rats were treated with a single NDMA dose. An unspecified number of deceased animals had distal tubule necrosis two days following treat ment, and surviving rats had normal kidneys. Macroscopic congestion was noted in kidneys of rats that were administered with NDMA in the diet for l-12 weeks [2,32]. Moderate tubule congestion with other effects (glo meru lus dilatation, slightly thickened Bowman's capsule) were observed in mink that ingested the same substance via diet [2,33].
In a related study, Olorunfemi et al. [23] reported that heavy metals-cyanide interaction in the cassava waste waters was responsible for the anomalies in cell division process and chromosome aberration induction in the Allium cepa root meristem. Also in another related study conducted by Adeyemo [34] to assess the haematological and histopathological effects of cassava effluent on adult female African catfish, Clarias gariepinus, the fish was found to show signs of gill and liver damage. Similarly, histopathological examination of the kidney, gill and liver of the fingerlings of the Nile Tilap ia, Oreochromis niloticus treated with cassava effluent indicated damage [35].
Ivanova et al. [36] and Staykova et al. [37] have established the genotoxic and mutagenic effects of open water contaminated with heavy metals and cyanide, further confirming the results of the inhibitory effects of these effluents in previous studies [9,[21][22][23]. In another study by Agunbiade et al. [9], the genotoxic and histopathological effects of the effluents from carbonaceous bottling plant were established. The study indicates that the effluents contain toxic substances which may constitute a risk to the environment and human health, more especially as the waste generated from effluents from carbonaceous bottling processing plant is not properly managed [9].
In mammals, several sub-chronic exposures of rats, mice and monkeys to substances such as perfluorooctanesulfonate (PFOS) have resulted in effects on body weight gain in females and in males [38][39][40][41][42][43][44] as reported in this present study with carbonaceous effluent. The results of the study by Agunbiade et al. [44] showed that the observed hepatotoxic effect in the exposed mice may be caused by the presence of heavy metal and other physical and chemical substances present in the wastewater. It suggested a higher risk to liver damage in hu mans and other organisms exposed to this wastewater and may also be deleterious to the surrounding environment [44].
However, limited informat ion is available regard ing renal effects of orally ad ministered wastewater d ischarged fro m a carbonaceous industry in animals.

Conclusions
In this study, there was no significant (P>0.05) change in the kidney and body weight of the exposed mice or the negative control. However, the activit ies of urea and creatinine in the seru m o f exposed mice were significantly increased as compared to the negative control mice. Our study showed that this increase in the activities of urea and creatinine in the seru m of exposed mice was concentration dependent (P<0.05).
The findings of this study also showed that the histological lesions observed in the kidney were characterized by generalized occlusion of the tubular lu men, general tubular necrosis, with protein casts in the tubular lu men and multip le foci of haemo rrhage in the parenchyma.
The carbonaceous wastewater caused kidney dysfunction in mice at various concentrations. This suggests that exposure to these waste may pose risk to human health and will pollute the aquatic environ ment, contaminating the source of water supply for both domestic and commercial uses.
Our results showed that the observed nephrotoxic effect in the exposed mice may be caused by the presence of heavy metal and other physical and chemical substances present in the wastewater. This suggests a higher risk to kidney damage in hu mans and other organisms exposed to this wastewater and may also be deleterious to the surrounding environment.