Assessment of phytotoxicity of treated water of Tabuk wastewater plant by different technologies on seed germination of chick pea ( Cicer arietinum )

The use of reclaimed water as an alternative source is a sustainable way forward for an arid country like The Kingdom of Saudi Arabia. The sewage contains organic and inorganic pollutants from households and industrial sources that may not be removed during treatment. In this study, seeds of Cicer arietinum were germinated using six different concentrations of treated water from the Tabuk wastewater treatment plant and tap water was used as control. The physicochemical properties such as total dissolved solids, electrical conductivity, total suspended solids, and turbidity values of treated water were higher which gradually decreased on dilution with tap water. The amount of ammonia, nitrite, nitrate, and phosphate was in higher concentration in treated water as compared to control. The use of 40% treated water (T3) improved the germination percentage, speed of germination and germination index of C. arietinum . The phytotoxicity test reveals that undiluted treated water (T6) is not ﬁ t for direct use on plants. All the investigated treatments con ﬁ rmed that the use of more than 40% of treated water decreased the fresh weight and dry weight of the seedlings as compared to control. The results are encouraging and help in attaining water sustainability in the Tabuk region.

for harmful microorganism that could be a health hazard to farmers, farmworkers and users if not properly managed (Raja et al. 2015;Christou et al. 2017;Khalid et al. 2018;Diaz-Sosa et al. 2020;Ofori et al. 2021).
There is difference of opinions over the use of treated wastewater therefore, it is necessary to evaluate the toxicity of treated water before making any recommendations. Plant based bioassay using germination technologies are very popular due to its simplicity and easy application, it can be used in situ and in vitro condition. There is no constraint for major equipment and nominal maintenance costs is required. Seeds of higher plants can sustain without nutrients supply in the test water and are easily available. Moreover, small sample size is required that can be repeated multiple times in a short period (Mayer & Poljakoff-Mayber 1989;Wang & Freemark 1995;Salvatore et al. 2008;Rusan et al. 2015;Priac et al. 2017). Khaleel et al. (2013) evaluated the effect of wastewater treatments on seed germination and biochemical parameters of Abelmoschus esculentus and recommended its use after proper dilutions. In case of assessment of olive mill wastewater (OMW) use on seed germination of barley, Rusan et al. (2015) propose 1:3 (OMW:Tap water) dilution of OMW. Authors further reported that microfiltration followed by reverse osmosis in addition to solar fenton oxidation and Jacto reactor is the most effective method in reducing the phytotoxicity of OMW. Priac et al. (2017) applied germination and root elongation test parameters on Lactuca sativa to assess the phytotoxixcity of treated wastewater and reported differential sensitivity to treated wastewater among its cultivars. Shakir et al. (2017) highlighted the environmental and health hazards linked with the reuse of wastewater for irrigation. The poor socio-economic conditions of farmers compel them to use wastewater for irrigation and it leads to bioaccumulation of heavy metals in crops and vegetables (Raja et al. 2015). In view of the present scenario where opinions are divided about the use of treated wastewater, this study was carried out to screen the suitability of the use of treated water of Tabuk Waste Water Treatment Plant (TWWTP). The identification of degree of dilution of treated wastewater of TWTP as a non-conventional water resource by using different technologies of seed germination.

Site of work
Tabuk is among the top ten big cities of KSA in terms of population. In the last decade, it witnessed an increase of almost 150,000 people. The average daily wastewater generated in Tabuk city is around 186,000 m 3 . This water is treated at three levels in a central plant located at the outskirt of the city (Figure 1) around 35 km away from University of Tabuk.

Assessment of water quality parameters
The treated water was collected as per American Public Health Association (APHA 2012) from the outlet point of WWTP. The treated water was diluted as per the Table 1 and tap water was used as control. The physicochemical parameters such as pH, turbidity, total suspended solids (TSS) total dissolved solid (TDS), alkalinity, nitrite, nitrate, ammonia, chloride, sulphate, sulfide, phosphate and iron of all the water samples were measured. The standard instruments of Hach such as DR900 colorimeter and DR3900 benchtop spectrophotometer along with titration accessories and standard guidelines were used to measure the physicochemical properties of treated and tap water and compared with the standard guidelines of water use of KSA (Table 2).

Model plant
The plant of our study was Cicer arietinum (Chick pea) of family Fabaceae. It is known for its high protein value and is one of the oldest cultivated legume of human civilization. It is very popular in Mediterranean, Middle East and Indian cuisine. Hummus, falafel, chana masala and chhole are the popular dishes enjoyed by the large population of these countries.
In each petri dishes fifteen seeds of C. arietinum were placed, and three pertidishes were used for each water sample. It results in 45 seeds for each treatment distributed into three dishes. The whole set-up was irrigated with respective concentration of water at regular interval throughout the course of the experiment. The seeds were germinated under control conditions of incubator and following parameters of seed germination were recorded to prepare the results.

Seed germination
The seed germination is defined as the first emergence of the radicle (Redondo et al. 2004) and newly-germinated seeds were recorded after two, three, four and, five days and percentage seed germination was calculated according to Ruan et al. (2002).
where N T ¼ Number of seedlings emerging on day; N ¼ Total Number of seeds.

Speed of germination
It was calculated as per the formula given by (Krishnaswamy & Seshu (1990).
Speed of Germination ¼ Number of seed germinated at 72 hrs Number of seed germinated at 72 hrs Â 100

Germination index
It was calculated as per the formula given by Tao & Zheng (1990).

Germination Index ¼ % Seed germination Germination days
Phytotoxicity index (PI) The phytotoxicity bioassay rests on calculating the root elongation in the germinating seeds as per the formula given by Mekki et al. (2007).
Where R LT ¼ Root length I the treated seed; R LC ¼ Root length in the control seeds. The values of the PI ranges between 0 and 1, a higher value means a toxic effects and the lower values signifies a stimulatory effects of the treated water.

Fresh weight and dry weight of seedlings
After 10 days of growth (Figure 3) fresh weight of each seedling was recorded. The samples were then placed in an oven maintained at 60°C for 48 hrs and thereafter dry weight of seedlings were recorded.

Statistical analysis
In one treatment, a petri dish was treated as one replicate and all the treatments were repeated five times. The data were expressed as means + standard error, and analysed statistically with IBM SPSS Statistics 20 software (SPSS Inc., Chicago, IL, USA). Means were statistically compared by Duncan's multiple-range test (DMRT) at the p,0.05% level.

RESULTS AND DISCUSSION
The use of reclaimed water has several advantages and disadvantages at environmental level along with issues of public health. It depends upon the quality of the treated effluent and other external factors. The quality of treated wastewater is determined by various water quality parameters like pH, turbidity, TDS, conductivity etc. and other factors include the frequency of irrigation, climatic conditions and soil properties (Elgallal et al. 2016;Ofori et al. 2021). In our study water quality parameters of all the water samples were measured and compared with standard guidelines of KSA for reuse of water. It was found that the values of TDS, conductivity, turbidity and chloride were more than the permissible limits (Table 2). Due to Uncorrected Proof dilution, these values were decreased and found to be near optimum in T4 treatments. The amount of nitrate, iron, total suspended solids and the value of pH was recorded within the permissible limits. Our results showed due to dilution almost all studied parameters were in the permissible limits of the standard guidelines of KSA for reuse of water. In another study, Balkhair et al. (2013) conducted a field study in KSA and conserved 60% of ground water by using six different wastewater qualities of treated wastewater for irrigation.
The seed germination is a very complex process and begins with water uptake and transition of a quiescent dry seed to a metabolically active state. It is the basis that forecast plant growth, development and productivity. In the present work, different technologies of seed germinations were assessed to determine the efiiciency of different concentration of treated wastewater as an alternative source for water management. The results shows that after five days of treatment maximum seed germination was recorded in T3 (96%) and minimum was in T6 (65%) (Figure 4). The negative effect of the T6 water on seed germination of Cicer may be attributed to the high TDS, conductivity and chloride content. There are reports that  Uncorrected Proof suggests seed germination is negatively affected by salinity through osmotic effects (Zhang et al. 2010;Krmz & Bell 2012), by ion toxicity (Hampson & Simpson 1990) or by a combination of the two (Huang & Redmann 1995).
The speed of germination and germination index can be used as an indicator of phytotoxicity. The speed of germination was fast in the initial 72 hrs as compared to 120 hrs in all the treatments except control. The speed of germination in T6 was lowest after 120 hrs ( Figure 5) After 2 days the germination index was higher in T3 whereas in T6 it reached maximum only after three days ( Figure 6). Naseri et al. (2012) reported that due to salinity germination rate index, root length and root weight reduce significantly. Osmotic pressure of the effluent increases at higher concentrations of total salts making imbibition more difficult and retard germination efficiencies (Nagda et al. 2006;Khan et al. 2011). In another report, Rahman et al. (2008) argues salinity disturb the  Uncorrected Proof imbibition process and water intake by seeds decreases it is followed by osmotic and ion toxic effects that prevent the seed germination and reduces germination rate. Further, salt concentration reduces the germination rate (Sabir & Ashraf 2005;Akbari et al. 2007) which confirms our results of % of seed germination, speed of germination and germination index.
The phytotoxicity index measures the difference of values in root elongation of treated and control water during seed germination. The PI values after 5 days were lower in all treatments thereafter it gradually increased in seedlings. It was lowest in T3 (0.133) and maximum in T6 (0.90) after 10 days of treatments (Figure 7). The nutrient uptake by plant is motivated by the root system and concentration of nutrients at the root surface. The nutrients are generally taken up in their ionic form ( Jones & Olson-Rutz 2016;Ofori et al. 2021). Our results showed that in the root system, the harmonious concentration of nutrients were resulted in low PI value in T3 treatment as compared to T6. The fresh weight and dry weight of the seedlings were significantly different in the treatments (Figure 8). The maximum fresh weight was recorded in T3 (48.93 g/45 seedlings) and  Uncorrected Proof minimum in T6 (34.712 g/45 seedlings). Similarly, the maximum dry weight was recorded in T3 (14.99 g/45 seedlings) and minimum in T6 (11.65 g/45 seedlings).
Germination is a very sensitive and vulnerable plant process. It is affected by water availability, salinity and to the toxic compounds of the growth medium (Khan et al. 2002). In T6 water sample, TDS, Conductivity, Ammonia, and chloride concentrations were higher than the permissible values. There are reports that suggest salt stress results in reduced cell turgor and depressed rate of root and leaf elongation (Fricke et al. 2006). It was visible in the fresh and dry weight of the Cicer seedlings.
As stated earlier, use of treated wastewater has both positive and negative implications on plant growth and development (Ofori et al. 2021). It was clearly visible in our study; in different concentrations of treated water, the nitrogen was available in the form of nitrates, nitrite and ammonia whereas, phosphate is the source of phosphorus in the treated water. This makes the accessibility of nitrogen and phosphorus uptake very easy for plant and it improved the growth and yield (Aziz & Farissi 2014;Vergine et al. 2017) by providing nutrients in easy accessible form upto T3 treatments. However, at higher concentrations of treated water (T4 onwards) a sharp decrease in the growth and yield was recorded. The possible reasons are the excess nutrients (Mccauley et al. 2011), heavy metals (Becerra-Castro et al. 2015, trace elements (Galavi et al. 2010), organic xenobiotic compounds like pharmaceuticals, personal care products, endocrine disrupting compounds (Fatta-Kassinos et al. 2011). The accumulation of these compounds affects biochemical processes of plants and inhibits growth and development and results in reduce yield.

CONCLUSION
In the present study, it was found that use of treated wastewater for irrigation purposes require caution and management. The phytotoxicity test reveals that undiluted treated water (T6) is not fit for direct use on plants. All the investigated treatments confirmed that the use of more than 40% of treated water decreased the fresh weight and dry weight of the seedlings as compared to control. To our best knowledge, this is the first report of the use of reclaimed water of Tabuk wastewater treatment plant on a seed germination experiment. The results are encouraging and help in attaining water sustainability in the Tabuk region. It also decreases the load of desalination of Red Sea and helps to fulfill the sustainable development goals of VISION 2030 of KSA.