GROWTH DEVELOPMENT AND HEAVY METAL UPTAKE BY AMARANTHUS HYBRIDUS L. POLLUTED WITH PHARMACEUTICAL EFFLUENTS

ABSTRACT

Experiments were carried out to study the ability of Amaranthus hybridus to accumulate heavy metals and active pharmaceutical  ingredients  (APis) from two pharmaceutical  effluents samples (effluent P, containing Paracetamol and effluent Bcontaining Amoxicillin). The preliminary experiment was carried out in a screen house in completely  randomized  design (CRD), using sterilised  soil and controlled irrigation with the effluents while distilled water was the control. The main experiment involved natural attenuation using unsterilized soil,  poultry manure,  compost made from the leaves of Delonix  regia, access to natural sunlight,  air and rainwater. This was carried out outdoors in randomized  complete block  design  (RCBD).  Results  from analysis  of the  effluent  samples  showed  they  contained  high concentrations  of cadmium (3.002 ppm), zinc (3.11  ppm),  iron (4.02  ppm) and Paracetamol  (25.24 µg/ml) for effluent P and Cd (2.30 ppm), Zn (2.12 ppm), Fe (1.21  ppm) and Amoxicillin (60.82 µg/ml) for effluent B.  The screen house experiment resulted in poorly developed seedlings of A.  hybridus,in which treatment with effluent B showed the highest mean concentration of zinc (4.11±0.12 mg/kg) and of iron (5.20±0.11  mg/kg).   Results from the main experiment showed that the polluted plants did not grow significantly less than the pollution-free plants.  T (Soil + Poultry manure +effluent P) recorded

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the highest mean leaf area (68.98±2.15 cm2 ,  plant height (116.85±0.35 cm), stem girth (4.13±0.14 cm)

and root circumference  (4.45±0.18  cm)  at week  10. Also, T,  (Soil + Poultry manure + effluent B) recorded  the  highest  mean  fresh  weight  of  leaves  (23.41±1.06  g),  stem  (27.01±0.95  g)  and  root (7.41±0.40  g) at week7.  A.  hybridus  accumulated  up to 5.53±0.12mg/kg  of zinc  in the leaves and

5.49±0.19 mg/kg in its roots while 7.47±0.13 mg/kg of iron was recorded in the leaves and 8.16±0.13 mg/kg in its root.Only a minute quantity of cadmium was recorded in the plant parts (0.0047±0.0002 mg/kg in the leaves).  The highest  concentration of Paracetamol  (1.60±0.06 µg/ml) accumulated  in a plant part was recorded in the leaf of Ts  (Soil+ Compost + effluent P) while the highest concentration of Amoxicillin  (5.93±0.10  g/ml)  was recorded  in T,  stem (Soil + Poultry  manure +  effluent  B). Therefore, A.  hybriduswas able to withstand pharmaceutical pollution effects better when it is exposed to a natural environment and these findings should suggest caution in the use of effluents in agricultural practices.

CHAPTER  ONE

INTRODUCTION

A  manufacturing   industry is  an  entity   involved   in  the  manufacture   and

processing  of basic raw materials  into more refined and specialized  products,  or one that adds value to products  through  series of processes  (Akrani,  2011 ).  The products that  are  generated   during  the  processing   which  are  not  of further  value  to  the manufacturer   are  called  wastes.  An  industrial  waste  in  liquid  form  is  called  an effluent;  effluents  are  thus  liquid  wastes  generated  during  processing.  Idris  et al. (2013) refers to effluent as waste discharged  onto surface waters.  Effluent  is defined by Hossain  et al.  (2010)  as wastewater      treated  or untreated      that flows out of a treatment plant, sewer or industrial outfall.

Industrial wastewater effluents are usually highly variable, with variations both m  quantity  and  quality,  brought  about  by  industrial  routines  like bath  discharges, operation start-ups and shut-downs, working-hour  distribution, etc. (Munter, 2006). In developing  countries  like Nigeria,  many  industries  dispose  their  effluents  without treatment.  These industrial effluents have a hazardous  effect on water quality, habitat quality,  and have complex  effects  on flowing waters  (Idris et al.,  2013).     Effluents from industries  may still contain  heavy  metals  and persistent  organic  contaminants even after treatment.  The persistent  organic contaminants  accumulated  in soil transfer to organisms  through the food chains and cause adverse health  effects  on human  or biological  effects  on  soil  fauna  and  flora  after  long-term  application  (Islam  et al.,

2006). Presently, some 2.4 billion people lack adequate  sanitation and 3.4 million die each year in the world from water related diseases (Idris et al., 2013).

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1.1      Sources of industrial effluents

In most industries, wastewater effluents result from water uses such as sanitary wastewater,   cooling,   and   processing    wastewater.   The   most   important   water contaminants  created by human activities are microbial pathogens, nutrients, oxygen• consuming materials, heavy metals and persistent organic matter, as well as suspended sediments and dyes (Hanchang, 2011).  Generally in Nigeria, it is the common practice with  industrial waste  disposal  that  industries discharge  untreated  effluents  into the nearest  water  bodies  (Bichi,  2013).  In Kano,  many  of the  industries  do not  have wastewater  treatment   facilities  and  thus  discharge  their  untreated   effluents  into adjoining receiving water bodies.  In rural areas of Enugu,  drainage channels are not a common feature and so wastewater  is released onto run-off paths and erosion tracks through which they can travel into a water body.  In urban  areas where  gutters and drainages are constructed, they are sometimes blocked due to dumping of refuse in the gutters and poor construction of the gutters by construction companies. For instance in Enugu  industrial layout Emene, many of the manufacturing  industries  do not have properly constructed drainages to channel their wastewaters.

Many companies do not treat their effluents properly and often discharge them on the ground or in water bodies, and these discharges  accumulate  and cause water pollution,  diseases and death of plants  and animals through food chain (Idris et al.,

2013).  High  concentrations  of metals  from  improperly  disposed  wastes  exert  a negative influence on the development of plants, their use of nutrients and metabolism (Shon et al., 2011).  Sewage effluent irrigation contributes significantly to the heavy metal  content  of the  irrigated  soil leading  to accumulation  of metals  in the grown

plants  and further contamination  of the food chain,  thereby posing health risks to the organisms along the food chain (Cui et al.,  2004). Pathogenic organisms also give rise to  great  health  concerns  in  agricultural  use  of wastewaters.    Pathogenic  viruses, bacteria,  protozoa  and  helminths  may  be  present  in  raw  municipal  wastewater  at medium to high levels and will survive in the environment  for long periods (Shon et al., 2011).

The pharmaceutical  industry produces  chemical combatants  that may lead to the  elimination  of pathogenic  organisms  and  the  disruption  of their  detrimental activities  in human  beings,  plants  and  animals.  However,  these  chemicals  tend  to linger in the environment and because of the trace amounts in which they are usually found, their effects  are mostly  neglected.  Active pharmaceutical  ingredients  (APis) that are excreted or found in runoff from livestock manure  that is spread  on fields have the potential to pollute the soil, penetrate ground water from which the contamination is spread to linked water sources.  The presence of antibiotics in aquatic environments  is  of particular   concern  because  of fears  that  they  may  stimulate antibacterial resistance among native bacterial populations (Duff, 2007).

The  pharmaceutical   industry  selected  for  effluent  collection  is  located  in Emene,  Enugu State.  It is involved  in the production  of antibiotics,  anti-depressants, anti-amoebic,  antifungal and diuretics.  It has three on-site manufacturing  plants.  The first plant generates only particulate  dust as waste which is collected and channelled through  pipes,  into  an underground  septic  tank  where  it  mixes  with  water.  This particular waste is cleared out routinely by the State Waste Management Agency. The remaining two plants generate obtainable effluents which flow into separate channels

and they are involved in producing  drugs like Amoxil, Ampiclox, Cotrim, Flagyl, Paracetamol   and  Tetracycline.  It  was  observed  in  the  case  of this  pharmaceutical company  that  the  drainage  channels  they use  became  clogged  up  at some  points, thereby causing  the generated  effluents to spill onto land and surrounding  vegetation. Among  the plants present  in that vegetation  were a number  of Amaranths  grown to maturity. This is perhaps one of the motives propelling  this research.

1.2     Amaranthus  hybridus Linn. Classification

Kingdom Division Class Order Family Genus

Species

Plantae Angiospermae Dicotyledonae Caryophyllales Amaranthaceae Amaranthus

A.  hybridus    (Farrukh et al., 2003).

(Common names: green amaranth or smooth amaranth).

Amaranthus       hybridus      L. is      a      cosmopolitan       species       In        the family Amaranthaceae which in some parts of the world is classified as a weed, but in West  Africa  is  cultivated   for  its  edible  leaves  and  cereal  from  the  ground  seeds (Farrukh et al., 2003).  It is an annual crop, often growing to 0.7 m (Plate 1).  The peak

of its vegetative  season  is April;  flowering  takes place  from July to September,  and

the  seeds  ripen  from  August  to  October.  The  flowers  are  monoecious  (individual flowers are either male or female,  but both sexes can be found on the same plant) and are pollinated by wind and by self (Farru kh et al., 2003 ).

Economic importance ofA.  hybridus.

•  It is cultivated as a food plant for green leafy vegetable,  and for animal fodder in the tropics.  It is a very good source of vitamins  including vitamin A,  B6, and C,  and riboflavin.

•  It is also a good source of dietary minerals including calcium,  iron, magnesium, phosphorus,  potassium,  zinc,  copper,  and manganese.  Its  seeds  have  been shown to contain protein (Atayese et al., 2009).

•  Yellow  and  green  dyes  can  be  obtained  from  the  whole  plant  and  it  is traditionally used for treatment  of constipation, inflammation, eczema, bronchitis, anaemia, and leprosy (Atayese et al., 2009).

•  The medicinal root juice  is used to treat inflammation  during urination. Some studies have shown that amaranth seeds or oil may benefit people with hypertension  and cardiovascular  disease; hence regular consumption  reduces blood pressure and cholesterol levels (Atayese et al., 2009).

•  No  members  of this  genus  are known  to be poisonous, but when  grown on nitrogen-rich soils they are known to concentrate nitrates in the leaves. This is especially noticeable  on land where chemical fertilizers are used and it is not advisable,  therefore,  to eat this plant  if it is grown inorganically (Atayese et al., 2009).

Amaranthus  hybridus  was labelled a suitable phytoremediant by Orwa (2014) because  it was  able  to  accumulate  Cu,  Zn and  Cd.  In  a remediation  experiment conducted at a polluted riverside, the metal concentration in the water samples (37.61 mg/L) were found to be low compared to that in the plants (43.01  mg/Kg). According to Orwa (2014), determination of heavy metal pollution in any water body by direct analysis of water samples may not be accurate because it will not reflect the real bio• available pollutant  level in the water due to removal of heavy metals from polluted waters by green plants.  This is because  most of the heavy metals will be removed from the water by biota  and sediment resident in the same water;  hence the use of bio-indicators  e.g. A.  hybridus  would be more  accurate  (Orwa,  2014).  The visible anatomical changes seen in A.  hybridus  grown in crude oil contaminated  soil could also be employed as an index of phytomonitoring  crude oil pollution (Omosun et al., 2008).

Amaranthus  hybridus has been used in phytoremediation  studies on crude oil polluted sites (Odjegba  and Sadiq,  2002;  Omosun et al.,  2008);  on effluent polluted river  banks  (Orwa,  2014);  and  on  heavy  metal  polluted  roadside  (Atayese  et al.,

2009).  Under  field conditions,  effects  of crude  oil on A.  hybridus  are likely to be short-term because the plants were seen to recover once residual oil was removed by rainfall (Omosun et al., 2008).

1.3      Aims and objectives of the study

•     To assess the effects of pharmaceutical  effluents on the germination,  growth and development ofAmaranthus hybridus;

•     To investigate the absorption and accumulation of heavy metals from the effluents by A.  hybridus;

•     To   investigate   the   absorption   and   accumulation   of  active   pharmaceutical ingredients from the effluents,  and the manifestation of morphological  variations; and

•     To determine the potential  of the use of compost and organic manure in natural attenuation and remediation of pharmaceutical effluent pollution.

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