Abstract

Jordan is one of the lowest countries in the world in terms of water resources. The reuse of treated wastewater is an important alternative to supply agricultural demands for water. In Jordan, Kherbet Al-Samra wastewater treatment plant (KSWWTP) is the largest and its effluent is mainly used for irrigation purposes. In this study, bacterial contamination and mutagenic potential were evaluated in six sites, beginning with KSWWTP and ending with King Tallal Dam. The results showed high contamination with many pathogenic bacteria and coliforms. The isolated pathogenic bacteria were Salmonella sp., Shigella sp., Bacillus cereus and Staphylococcus aureus. The isolated opportunistic pathogenic bacteria were Acinetobacter lwoffii, Elizabethkingia meningosepticum, Pseudomonas fluorescens and Bacillus licheniformis. These bacteria were found in all sampling sites without a specific prevalence pattern. Differences in temperature between seasons affect total coliform and other bacterial count. All water samples showed positive mutagenic activity and high bacterial pollution. Improving the disinfection efficiency in the wastewater treatment plant is important to minimize potential toxicity and exposure of public health to pathogenic bacteria, reduce water resources' contamination and environmental pollution. Increasing effluent sampling frequency from KSWWTP is required to monitor bacterial contamination and toxicity/mutagenicity level for water safety and public health risk assessments.

HIGHLIGHTS

  • Treated wastewater is an alternative water resource to supply agricultural demands.

  • High contamination with different types of pathogens and high levels of coliforms were reported.

  • Differences in temperature between seasons affect total coliform and other bacterial count.

  • Positive mutagenic activity and high bacterial pollution expose public health to high risk for bacterial infection.

Graphical Abstract

Graphical Abstract
Graphical Abstract

INTRODUCTION

Climate change, population growth, and rapid urbanization are the main reasons for the fresh water scarcity worldwide (Smith 2011). The rising demands for water and the limitation of water resources has led to the recycle and reuse of treated and untreated wastewater. Wastewater reuse has been developed and applied in many parts of the world (Jang et al. 2006). Countries with a per capita water production below 1,000 m3/year are classified as water-poor countries. The per capita water supply in Jordan is 145 m3/year, which makes Jordan one of the lowest countries in the world in water resources (Hadadin et al. 2010). In Jordan, treated wastewater has become an important water resource for restricted uses and has been actively incorporated into the strategic planning of water policymakers. Reclaimed wastewater is being used on an increasing scale for irrigation, primarily in the Jordan river valley (Seder & Abdel-Jabbar 2011). The largest wastewater treatment plant in Jordan is Khirbet Al-Samra (KSWWTP). It is located near Al-Hashmiya village, north of Zarqa city, about 60 km northeast of Amman (Fayyad & Al-Sheikh 2001). It collects domestic wastewater mainly from two heavily populated cities in Jordan, Amman and Al-Zarqa. The treated wastewater outflow is pumped directly into the Zarqa River which is then collected in King Tallal dam (Al-Zboon & Al-Ananzeh 2008). The KSWWTP plant treating capacity is about 267,000 m3/day using activated sludge system (Uleimat 2012).

Water pollution and water resources contamination is a worldwide public health problem (Egito et al. 2007). Pathogenic bacteria, viruses, and genotoxic micropollutants, consequences of anthropogenic impact (industry, urbanization, agriculture, food factories, hospitals, university laboratories, and scientific research centers), are contaminating the aquatic environment causing widespread public health concerns (Al-Mossawi 2014; Cevik et al. 2020). About 2.5 billion people do not have proper water sanitation and more than 5 million people die each year from water-borne diseases (Hanjra et al. 2012).

Wastewater carries many kinds of pollution, such as pathogens (bacteria, viruses, and protozoas), oxygen-demanding wastes (as decomposition of animal and plant matter by aerobic bacteria), water-soluble radioactive substances, water-soluble inorganic pollutants (acids, salts, and toxic metals), organic compounds (gasoline, oil, plastics, detergents, and pesticides) and anions and cations (nitrates, phosphate, sulfates, Ca+2, Mg+2 and F). Workers in wastewater treatment plants, farm workers exposed to wastewater during crop irrigation, people who live near water bodies that have been mixed with treated or untreated wastewater, and consumers who consume crops that were irrigated with wastewater are at greater risk from such pollutants. These substances can cause severe health problems to humans and other organisms if they exceed a limit value (Azizullah et al. 2011) and lead to potentially mutagenic activity that have serious public health consequences. The exposure routes for the pollutants are direct contact from contaminated surfaces, consumption of raw vegetables irrigated with reclaimed water, accidental ingestion of contaminated water, and long-term exposure to spray irrigation sites (Bitton 2005).

Various microorganisms such as total and fecal coliforms, fecal streptococci, anaerobic bacteria, helminth eggs, bacteriophages, yeasts and acid-fast organisms and bacterial spores have been proposed and used for indicating the occurrence of fecal contamination and treatment efficiency in water and wastewater treatment plants (Weiner & Matthews 2003). The evaluation of total coliform and bacterial contamination serves as an important indicator to assess and understand the profile and quality of treated wastewater effluents. Genotoxic chemicals cause DNA damage and increase the risk of cancer in humans and the potential presence of mutagenic and carcinogenic compounds in treated wastewater is a major public health concern (Žegura et al. 2009; Bianchi et al. 2015). Therefore, determination of the mutagenic and genotoxic potential of treated wastewater effluents is crucial for environmental protection and public health.

The aims of this study were to isolate and enumerate the different types of bacterial contamination in Zarqa River water (mixed with the Khirbet Al-Samra treatment plant effluents) and to evaluate the mutagenic potential in the collected water samples.

MATERIALS AND METHODS

Sampling locations

Between February and May, water samples from Zarqa River were collected monthly from six sites (Figure 1): Site 1: Kherbet Al-Samra-1(K1 W): the outflow (effluent) site of KSWWTP, at the geographic coordinate of 32°14′43, 71′′ north and 36°10′04,14′′ east; Site 2: Kherbet Al-Samra-2 (K2 W): about 4 km to the east from the first site, at the geographic coordinate of 32°08′36,91′′ north and 36°01′06,63′′ east; Site 3: Zarqa Farm (ZW): a farm that produces vegetables eaten raw, nearby the Zarqa River water stream at Qunaiah village, at the geographic coordinate of 32°09′16,39′′ north and 36°01′32,99′′ east; Site 4: Jarash river (JW): at Mittaba village near the Jerash water stream, at the geographic coordinate of 32°12′95,34′′ north and 35°52′95,09′′ east; Site 5: King Talal Dam-1 (D1 W): at the geographic coordinate of 32°11′36,11′′ north and 35°50′25, 44′′ east; Site 6: King Talal Dam-2 (D2 W): at the geographic coordinate of 32°10′31,65′′ north and 35°48′24,4.8′′ east.

Figure 1

Diagrammatic map of sampling sites for Zarqa River.

Figure 1

Diagrammatic map of sampling sites for Zarqa River.

Water sampling

Water samples were collected beneath the water surface in 50 mL sterile plastic containers. About 40 mL of water was collected directly from the running water effluent using sterile glass bottles across the river. Some air space was left in the bottles for aerobic bacteria survival.

Seasonal variations

The temperature during the study period ranged from 16 °C to 30 °C. All samples were preserved in ice boxes until processed in the laboratory for microbial analysis. Sterile distilled water was used to prepare a serial dilution from collected water samples.

Bacterial isolation, enumeration, and identification

Nutrient agar (Biolab, Hungary) and selective media: EMB agar, SS agar, and mannitol salt agar, were used to isolate the different types of bacteria. All media were incubated at 37 °C for 24 h. Enumeration of bacteria in all collected samples was done using a membrane filtration technique. 0.45 μm membrane filters were plotted on different types of media. After enumeration of colonies according to their morphological differences, pure cultures were obtained and sub-cultured on brain heart infusion agar. Identification of bacteria was done according to the standard procedures (Cown & Steel 1975). The following Microgen™ identification kits were used for further confirmation: Microgen Bacillus-ID kit (identification of mesophilic Bacillus sp.), Microgen GN-ID system (identification of the family Enterobacteriaceae and other non-fastidious Gram-negative bacilli, and Microgen™ Staph-ID identification kit (to identify medically important members of the genus Staphylococcus). All kits were used according to the manufacturers' instructions.

Mutagenicity test

Ames test was used in this study to detect any mutagenicity potential in Zarqa River water (Ames et al. 1973; Mortelmans & Zeiger 2002). The standard plate incorporation assay was applied by incubating Salmonella typhimurium AT100 with water samples directly on a minimal glucose agar. Test tubes containing 2 mL of molten agar medium with limited histidine and biotin with an equal amount of water sample were mixed together and incubated at 43–48 °C for 2–5 min. Then, tubes were poured on glucose minimal agar plates until the top agar was hardened. Inverted plates were then incubated at 37 °C for 48 h. Histidine-revertant colonies were counted and compared to those on the solvent (negative) control plates and the positive control (sodium azide). Tests were carried out in duplicate and the average was calculated. The mutagenicity activity in the samples was evaluated according to two calculation methods.

Mutagenicity percentage according to the positive control:
formula
Subtraction method using the negative control:
formula

Data statistical analysis

Each data point was expressed as the mean ± standard deviation (SD) of three independent experiments. The values were compared and analyzed by one-way analysis of variance (ANOVA) using least significant difference (LSD) multiple comparison tests on the means. Where differences are reported, they are at the 95% confidence level (P < 0.05). Data analysis was performed by using SPSS software version 15.0.

RESULTS

Site 1: Kherbet al-Samra 1(K1 W)

The following Gram-negative bacteria were isolated from water samples: Shigella sp., Acinetobacter lwoffii, Escherichia coli, Elizabethkingia meningosepticum, and Pseudomonas fluorescens. Cronobacter sakazakii and Klebsiella pneumonia were not isolated. The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 0.35 to 5.84. However, the lowest counts were in February and the highest counts were recorded in May, respectively, and Shigella sp. was the highest bacterial species isolated (Table 1). The isolated Gram-positive bacilli were: Bacillus firmus, Bacillus lentus, Bacillus subtilis, Brevibacillus brevis, Bacillus cereus, and Bacillus megaterium while Staphylococcus aureus was isolated as Gram-positive cocci. Bacillus licheniformis was not isolated. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.06 to 3.72, where the lowest counts were in February and the highest counts were recorded in March, respectively. However, the highest number of isolated bacteria was for B. lentus (Table 1). The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 1 (K1 W) ranged between 0.41 and 6.65, where the lowest counts were in February and the highest counts were recorded in March, respectively. The monthly average of coliform bacteria (×106 CFU/100 mL) ranged between 0.01 and 2.50, where the lowest were in April and the highest recorded for E. coli in March, respectively (Table 1).

Table 1

The aerage number of CFU/(×106/100 mL) ± S.D. of water samples from site 1 at Kherbet al-Samra1 (K1 W)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Shigella sp. 0.15 ± 0.07 0.25 ± 0.07 0.16 ± 0.14 3.08 ± 0.00 
A. lwoffii 0.08 ± 0.00 0.13 ± 0.35 0.43 ± 0.25 1.25 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 
E. coli 0.11 ± 0.02 2.5 ± 0.70 0.01 ± 0.01 1.50 ± 0.15 
E. meningosepticum 0.01 ± 0.00 0.03 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.02 ± 0.01 0.02 ± 0.10 0.01 ± 0.00 
Gram-negative bacteria total number 0.35 ± 0.06 2.93 ± 0.82 0.62 ± 0.14 5.84 ± 1.08 
Gram-positive bacteria S. aureus 0.01 ± 0.00 0.02 ± 0.11 0.01 ± 0.71 0.02 ± 0.00 
B. firmus 0.00 ± 0.00 0.30 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 0.02 ± 0.01 2.75 ± 1.06 0.02 ± 0.01 0.02 ± 0.00 
B. subtilis 0.02 ± 0.01 0.01 ± 0.01 0.033 ± 0.00 0.03 ± 0.07 
B. brevis 0.00 ± 0.00 0.33 ± 0.10 0.00 ± 0.00 0.00 ± 0.00 
B. cereus 0.03 ± 0.01 0.3 ± 0.28 2.05 ± 0.12 0.25 ± 0.00 
B. megaterium 0.00 ± 0.00 0.01 ± 0.21 0.023 ± 0.35 0.00 ± 0.00 
B. freudenreichii 0.00 ± 0.00 0.01 ± 0.28 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.06 ± 0.01 3.72 ± 0.88 2.13 ± 0.68 0.32 ± 0.08 
Total bacterial count 0.41 ± 0.04 6.65 ± 0.83 2.75 ± 0.48 6.16 ± 0.81 
Total coliform 0.11 ± 0.06 2.50 ± 1.44 0.01 ± 0.01 1.50 ± 0.01 
Fecal coliform 0.11 ± 0.07 2.50 ± 1.76 0.01 ± 0.86 1.50 ± 1.05 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Shigella sp. 0.15 ± 0.07 0.25 ± 0.07 0.16 ± 0.14 3.08 ± 0.00 
A. lwoffii 0.08 ± 0.00 0.13 ± 0.35 0.43 ± 0.25 1.25 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 
E. coli 0.11 ± 0.02 2.5 ± 0.70 0.01 ± 0.01 1.50 ± 0.15 
E. meningosepticum 0.01 ± 0.00 0.03 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.02 ± 0.01 0.02 ± 0.10 0.01 ± 0.00 
Gram-negative bacteria total number 0.35 ± 0.06 2.93 ± 0.82 0.62 ± 0.14 5.84 ± 1.08 
Gram-positive bacteria S. aureus 0.01 ± 0.00 0.02 ± 0.11 0.01 ± 0.71 0.02 ± 0.00 
B. firmus 0.00 ± 0.00 0.30 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 0.02 ± 0.01 2.75 ± 1.06 0.02 ± 0.01 0.02 ± 0.00 
B. subtilis 0.02 ± 0.01 0.01 ± 0.01 0.033 ± 0.00 0.03 ± 0.07 
B. brevis 0.00 ± 0.00 0.33 ± 0.10 0.00 ± 0.00 0.00 ± 0.00 
B. cereus 0.03 ± 0.01 0.3 ± 0.28 2.05 ± 0.12 0.25 ± 0.00 
B. megaterium 0.00 ± 0.00 0.01 ± 0.21 0.023 ± 0.35 0.00 ± 0.00 
B. freudenreichii 0.00 ± 0.00 0.01 ± 0.28 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.06 ± 0.01 3.72 ± 0.88 2.13 ± 0.68 0.32 ± 0.08 
Total bacterial count 0.41 ± 0.04 6.65 ± 0.83 2.75 ± 0.48 6.16 ± 0.81 
Total coliform 0.11 ± 0.06 2.50 ± 1.44 0.01 ± 0.01 1.50 ± 0.01 
Fecal coliform 0.11 ± 0.07 2.50 ± 1.76 0.01 ± 0.86 1.50 ± 1.05 

Site 2: Kherbet al-Samra 2 (K2 W)

The following Gram-negative bacteria were isolated from water samples: Salmonella sp., Shigella sp., A. lwoffii, E. coli, and P. fluorescens. C. sakazakii, K. pneumonia, E. meningosepticum and Enterobacter cloacae were not isolated. The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 1.14 to 7.40, where the lowest counts were in April and the highest recorded in May, respectively. The highest number of isolated bacteria was for Shigella sp. (Table 2). The isolated Gram-positive bacilli were: B. firmus, B. lentus, B. subtilis, B. brevis, B. cereus, B. megaterium, and B. licheniformis, while S. aureus was isolated as Gram-positive cocci. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.44 to 29.91, where the lowest counts were in February and the highest counts recorded in March, respectively. The highest number of isolated bacteria was for B. firmus.

Table 2

The average number of CFU (×106/100 mL) ± S.D. of water samples from site 2 at Kherbet al-Samra2 (K2 W)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.05 ± 0.07 0.00 ± 0.00 0.24 ± 0.18 0.65 ± 0.00 
Shigella sp. 0.45 ± 0.21 1.75 ± 0.35 0.27 ± 0.21 4.5 ± 0.71 
A. lwoffii 1.10 ± 0.141 2.0 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.90 ± 0.14 1.95 ± 0.78 0.63 ± 0.48 2.25 ± 2.47 
E. meningosepticum 0.00 ± 0.00 00.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 2.51 ± 0.44 5.70 ± 0.95 1.14 ± 0.22 7.40 ± 1.56 
Gram-positive bacteria S. aureus 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.00 ± 0.00 8.65 ± 8.98 0.00 ± 0.00 0.45 ± 0.70 
B. lentus 0.01 ± 0.06 15.0 ± 15.50 0.60 ± 0.14 2.30 ± 0.56 
B. subtilis 0.01 ± 0.01 0.31 ± 0.27 0.05 ± 0.07 0.00 ± 0.00 
B. brevis 0.04 ± 0.01 1.65 ± 1.90 0.00 ± 0.00 0.10 ± 0.00 
B. cereus 0.33 ± 0.11 4.0 ± 2.80 0.10 ± 0.14 1.70 ± 0.28 
B. megaterium 0.00 ± 0.00 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.25 ± 0.35 0.24 ± 0.19 0.00 ± 0.00 
Gram-positive bacteria total number 0.44 ± 0.11 29.91 ± 5.24 0.75 ± 0.20 4.55 ± 0.87 
Total bacterial count 2.95 ± 0.33 35.61 ± 3.9 1.89 ± 0.20 11.95 ± 1.24 
Total coliform 0.90 ± 0.52 1.95 ± 1.12 0.63 ± 0.36 2.25 ± 1.29 
Fecal coliform 0.90 ± 0.64 1.95 ± 1.37 0.63 ± 0.45 2.25 ± 1.59 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.05 ± 0.07 0.00 ± 0.00 0.24 ± 0.18 0.65 ± 0.00 
Shigella sp. 0.45 ± 0.21 1.75 ± 0.35 0.27 ± 0.21 4.5 ± 0.71 
A. lwoffii 1.10 ± 0.141 2.0 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.90 ± 0.14 1.95 ± 0.78 0.63 ± 0.48 2.25 ± 2.47 
E. meningosepticum 0.00 ± 0.00 00.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.01 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 2.51 ± 0.44 5.70 ± 0.95 1.14 ± 0.22 7.40 ± 1.56 
Gram-positive bacteria S. aureus 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.00 ± 0.00 8.65 ± 8.98 0.00 ± 0.00 0.45 ± 0.70 
B. lentus 0.01 ± 0.06 15.0 ± 15.50 0.60 ± 0.14 2.30 ± 0.56 
B. subtilis 0.01 ± 0.01 0.31 ± 0.27 0.05 ± 0.07 0.00 ± 0.00 
B. brevis 0.04 ± 0.01 1.65 ± 1.90 0.00 ± 0.00 0.10 ± 0.00 
B. cereus 0.33 ± 0.11 4.0 ± 2.80 0.10 ± 0.14 1.70 ± 0.28 
B. megaterium 0.00 ± 0.00 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.25 ± 0.35 0.24 ± 0.19 0.00 ± 0.00 
Gram-positive bacteria total number 0.44 ± 0.11 29.91 ± 5.24 0.75 ± 0.20 4.55 ± 0.87 
Total bacterial count 2.95 ± 0.33 35.61 ± 3.9 1.89 ± 0.20 11.95 ± 1.24 
Total coliform 0.90 ± 0.52 1.95 ± 1.12 0.63 ± 0.36 2.25 ± 1.29 
Fecal coliform 0.90 ± 0.64 1.95 ± 1.37 0.63 ± 0.45 2.25 ± 1.59 

The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 2 (K2 W) ranged between 1.89 and 35.61, where the lowest counts were in April and the highest counts recorded in March, respectively. The monthly average of coliforms bacteria (×106 CFU/100 mL) ranged between 0.63 and 1.95, where the lowest were in April and the highest recorded for E. coli in May, respectively (Table 2).

Site 3: Zarqa Farm (ZW)

The following Gram-negative bacteria were isolated from water samples: Salmonella sp., Shigella sp., A. lwoffii, E. coli and P. fluorescens. C. sakazakii, K. pneumonia, E. meningosepticum, and E. cloacae were not isolated (Table 3).

Table 3

The average number of CFU (×106/100 ml) ± S.D. of water samples from site 3 at Zarqa Farm (ZW)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.11 ± 0.07 0.01 ± 0.00 
Shigella sp. 0.01 ± 0.01 0.43 ± 0.52 0.73 ± 0.67 0.29 ± 0.29 
A. lwoffii 0.09 ± 0.06 0.23 ± 0.18 0.00 ± 0.00 0.20 ± 0.12 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.75 ± 0.49 2.10 ± 0.28 2.73 ± 0.31 0.66 ± 0.09 
E. meningosepticum 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.90 ± 0.25 2.75 ± 0.69 3.57 ± 0.90 1.15 ± 0.23 
Gram-positive bacteria S. aureus 0.25 ± 0.07 0.18 ± 0.35 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.17 ± 0.05 1.5 ± 0.70 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 1.15 ± 1.20 6.5 ± 0.70 1.00 ± 0.00 0.10 ± 0.00 
B. subtilis 0.02 ± 0.01 0.00 ± 0.00 0.65 ± 0.49 0.00 ± 0.00 
B. brevis 0.60 ± 0.28 0.35 ± 0.49 0.15 ± 0.21 0.10 ± 0 
B. cereus 0.45 ± 0.07 2.75 ± 0.35 0.00 ± 0.00 0.20 ± 0.14 
B. megaterium 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.01 ± 0 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.10 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 2.64 ± 0.39 11.38 ± 2.17 1.80 ± 0.37 0.40 ± 0.07 
Total bacterial count 3.54 ± 0.33 14.12 ± 1.64 5.37 ± 0.68 1.55 ± 0.17 
Total coliform 0.75 ± 0.43 2.10 ± 1.21 2.73 ± 1.57 0.66 ± 0.38 
Fecal coliform 0.75 ± 0.53 2.10 ± 1.48 2.73 ± 1.93 0.66 ± 0.46 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.11 ± 0.07 0.01 ± 0.00 
Shigella sp. 0.01 ± 0.01 0.43 ± 0.52 0.73 ± 0.67 0.29 ± 0.29 
A. lwoffii 0.09 ± 0.06 0.23 ± 0.18 0.00 ± 0.00 0.20 ± 0.12 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.75 ± 0.49 2.10 ± 0.28 2.73 ± 0.31 0.66 ± 0.09 
E. meningosepticum 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.90 ± 0.25 2.75 ± 0.69 3.57 ± 0.90 1.15 ± 0.23 
Gram-positive bacteria S. aureus 0.25 ± 0.07 0.18 ± 0.35 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.17 ± 0.05 1.5 ± 0.70 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 1.15 ± 1.20 6.5 ± 0.70 1.00 ± 0.00 0.10 ± 0.00 
B. subtilis 0.02 ± 0.01 0.00 ± 0.00 0.65 ± 0.49 0.00 ± 0.00 
B. brevis 0.60 ± 0.28 0.35 ± 0.49 0.15 ± 0.21 0.10 ± 0 
B. cereus 0.45 ± 0.07 2.75 ± 0.35 0.00 ± 0.00 0.20 ± 0.14 
B. megaterium 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.01 ± 0 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.00 ± 0.00 0.10 ± 0.14 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 2.64 ± 0.39 11.38 ± 2.17 1.80 ± 0.37 0.40 ± 0.07 
Total bacterial count 3.54 ± 0.33 14.12 ± 1.64 5.37 ± 0.68 1.55 ± 0.17 
Total coliform 0.75 ± 0.43 2.10 ± 1.21 2.73 ± 1.57 0.66 ± 0.38 
Fecal coliform 0.75 ± 0.53 2.10 ± 1.48 2.73 ± 1.93 0.66 ± 0.46 

The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 0.90 to 3.57, where the lowest counts were in February and the highest counts recorded in April. The highest number of isolated bacteria was for E. coli (Table 3). The isolated Gram-positive bacilli were B. firmus, B. lentus, B. subtilis, B. brevis, B. cereus, Bacillus freudenreichii, and B. licheniformis. S. aureus was isolated as Gram-positive cocci and B. megaterium was not isolated. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.40 to 11.38, where the lowest counts were in May and the highest counts recorded in March, respectively. However, the highest number of isolated bacteria was for B. lentus (Table 3). The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 3 (ZW) ranged between 1.55 and 14.13, where the lowest counts were in May and the highest counts recorded in March, respectively. The monthly average of coliforms bacteria (×106 CFU/100 mL) ranged between 0.66 and 2.73, where the lowest were in May and the highest recorded for E. coli in April, respectively (Table 3).

Site 4: Jarash river (JW)

The Gram-negative bacteria isolated from water samples were Salmonella sp., Shigella sp., A. lwoffii, Klebsiella ozaenae, E. cloacae, E. coli, and E. meningosepticum. C. sakazakii and P. fluorescens were not isolated. The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 0.76 to 2.28, where the lowest counts were in February and the highest counts recorded in May, respectively. The highest number of isolated bacteria was for Shigella sp. (Table 4). The isolated Gram-positive bacilli were B. firmus, B. lentus, B. brevis, B. cereus, B. freudenreichii, and B. licheniformis while S. aureus was isolated as Gram-positive cocci. B. subtilis, B. megaterium, and B. freudenreichii were not isolated. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.15 to 3.80, where the lowest counts were in May and the highest counts recorded in March, respectively. However, the highest number of isolated bacteria was for B. lentus (Table 4). The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 4 (JW) ranged between 1.74 and 5.85, where the lowest counts were in February and the highest counts recorded in March. The monthly average of coliform bacteria (×106 CFU/100 mL) ranged between 0.22 and 0.89, where the lowest were in February and the highest recorded for E. coli in April, respectively (Table 4).

Table 4

The average number of CFU (×106/100 mL) ± S.D. of water samples from site 4 at Jarash River (JW)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.04 ± 0.01 0.07 ± 0.09 0.02 ± 0.00 0.01 ± 0.00 
Shigella sp. 0.41 ± 0.55 0.60 ± 0.57 0.93 ± 0.88 1.08 ± 0.95 
A. lwoffii 0.08 ± 0.01 0.95 ± 0.07 0.00 ± 0.00 0.45 ± 0.07 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. ozaenae 0.00 ± 0.00 0.03 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.01 
E. coli 0.22 ± 0.11 0.40 ± 0.16 0.89 ± 0.79 0.75 ± 0.64 
E. meningosepticum 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.76 ± 0.14 2.05 ± 0.35 1.82 ± 0.40 2.28 ± 0.41 
Gram-positive bacteria S. aureus 0.10 ± 0.00 0.00 ± 0.00 0.01 ± 0.71 0.00 ± 0.00 
B. firmus 0.03 ± 0.01 0.55 ± 0.71 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 0.60 ± 0.57 1.70 ± 0.28 0.40 ± 0.14 0.05 ± 0.07 
B. subtilis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.00 ± 0.00 1.35 ± 0.92 0.05 ± 0.07 0.05 ± 0.07 
B. cereus 0.15 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.07 
B. megaterium 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.03 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.08 ± 0.03 0.20 ± 0.14 0.50 ± 0.71 0.00 ± 0.00 
Gram-positive bacteria total number 0.98 ± 0.19 3.8 ± 0.66 0.96 ± 0.20 0.15 ± 0.03 
Total bacterial count 1.74 ± 0.16 5.85 ± 0.52 2.77 ± 0.31 2.43 ± 0.31 
Total coliform 0.22 ± 0.13 0.43 ± 0.22 0.89 ± 0.51 0.76 ± 0.43 
Fecal coliform 0.22 ± 0.16 0.43 ± 0.26 0.89 ± 0.63 0.75 ± 0.53 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.04 ± 0.01 0.07 ± 0.09 0.02 ± 0.00 0.01 ± 0.00 
Shigella sp. 0.41 ± 0.55 0.60 ± 0.57 0.93 ± 0.88 1.08 ± 0.95 
A. lwoffii 0.08 ± 0.01 0.95 ± 0.07 0.00 ± 0.00 0.45 ± 0.07 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. ozaenae 0.00 ± 0.00 0.03 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.01 
E. coli 0.22 ± 0.11 0.40 ± 0.16 0.89 ± 0.79 0.75 ± 0.64 
E. meningosepticum 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.76 ± 0.14 2.05 ± 0.35 1.82 ± 0.40 2.28 ± 0.41 
Gram-positive bacteria S. aureus 0.10 ± 0.00 0.00 ± 0.00 0.01 ± 0.71 0.00 ± 0.00 
B. firmus 0.03 ± 0.01 0.55 ± 0.71 0.00 ± 0.00 0.00 ± 0.00 
B. lentus 0.60 ± 0.57 1.70 ± 0.28 0.40 ± 0.14 0.05 ± 0.07 
B. subtilis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.00 ± 0.00 1.35 ± 0.92 0.05 ± 0.07 0.05 ± 0.07 
B. cereus 0.15 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.07 
B. megaterium 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. freudenreichii 0.03 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. licheniformis 0.08 ± 0.03 0.20 ± 0.14 0.50 ± 0.71 0.00 ± 0.00 
Gram-positive bacteria total number 0.98 ± 0.19 3.8 ± 0.66 0.96 ± 0.20 0.15 ± 0.03 
Total bacterial count 1.74 ± 0.16 5.85 ± 0.52 2.77 ± 0.31 2.43 ± 0.31 
Total coliform 0.22 ± 0.13 0.43 ± 0.22 0.89 ± 0.51 0.76 ± 0.43 
Fecal coliform 0.22 ± 0.16 0.43 ± 0.26 0.89 ± 0.63 0.75 ± 0.53 

Site 5: King Tallal Dam-1 (D1 W)

The following Gram-negative bacteria were isolated from water samples: Salmonella sp., Shigella sp., A. lwoffii, K. pneumoniae, E. coli, E. meningosepticum, and P. fluorescens. C. sakazakii and Elizabethkingia meningosepticum were not isolated. The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 0.25 to 6.01, where the lowest counts were in February and the highest counts recorded in May, respectively. The highest number of isolated bacteria was for E. coli (Table 5). The isolated Gram-positive bacilli were: B. firmus, B. lentus, B. subtilis, B. brevis, B. cereus, B. freudenreichii, and B. megaterium. S. aureus was isolated as Gram-positive cocci. B. licheniformis was not isolated. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.15 to 28.60, where the lowest counts were in February and the highest counts recorded in May, respectively. However, the highest number of isolated bacteria was for B. cereus (Table 5).

Table 5

The average number of CFU (×106/100 mL) ± S.D. of water samples from site 5 at King Tallal Dam-1 (D1 W)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.04 
Shigella sp. 0.12 ± 0.11 0.55 ± 0.07 0.53 ± 0.38 0.45 ± 0.07 
A. lwoffii 0.07 ± 0.04 0.35 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.005 ± 0.007 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.06 ± 0.04 0.14 ± 0.06 0.83 ± 0.80 5.50 ± 0.70 
E. meningosepticum 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.25 ± 0.45 1.09 ± 0.20 1.36 ± 0.31 6.01 ± 1.81 
Gram-positive bacteria S. aureus 0.00 ± 0.00 0.00 ± 0.00 0.04 ± 0.14 0.00 ± 0.00 
B. firmus 0.02 ± 0.01 0.00 ± 0.00 0.05 ± 0.07 1.15 ± 1.62 
B. lentus 0.11 ± 0.01 0.90 ± 0.14 0.85 ± 0.21 8.05 ± 4.31 
B. subtilis 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.00 ± 0.00 0.40 ± 0.14 0.25 ± 0.07 2.75 ± 0.35 
B. cereus 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 15.85 ± 8.27 
B. megaterium 0.01 ± 0 00.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.07 
B. freudenreichii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.75 ± 0.35 
B. licheniformis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.15 ± 0.03 1.30 ± 0.31 1.19 ± 0.28 28.6 ± 5.41 
Total bacterial count 0.40 ± 0.04 2.39 ± 0.25 2.55 ± 0.29 34.61 ± 4.12 
Total coliform 0.06 ± 0.03 0.14 ± 0.08 0.83 ± 0.49 5.51 ± 3.17 
Fecal coliform 0.06 ± 0.042 0.14 ± 0.10 0.83 ± 0.59 5.51 ± 3.88 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.04 
Shigella sp. 0.12 ± 0.11 0.55 ± 0.07 0.53 ± 0.38 0.45 ± 0.07 
A. lwoffii 0.07 ± 0.04 0.35 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.005 ± 0.007 
E. cloacae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. coli 0.06 ± 0.04 0.14 ± 0.06 0.83 ± 0.80 5.50 ± 0.70 
E. meningosepticum 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.05 ± 0.07 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 0.25 ± 0.45 1.09 ± 0.20 1.36 ± 0.31 6.01 ± 1.81 
Gram-positive bacteria S. aureus 0.00 ± 0.00 0.00 ± 0.00 0.04 ± 0.14 0.00 ± 0.00 
B. firmus 0.02 ± 0.01 0.00 ± 0.00 0.05 ± 0.07 1.15 ± 1.62 
B. lentus 0.11 ± 0.01 0.90 ± 0.14 0.85 ± 0.21 8.05 ± 4.31 
B. subtilis 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.00 ± 0.00 0.40 ± 0.14 0.25 ± 0.07 2.75 ± 0.35 
B. cereus 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 15.85 ± 8.27 
B. megaterium 0.01 ± 0 00.00 ± 0.00 0.00 ± 0.00 0.05 ± 0.07 
B. freudenreichii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.75 ± 0.35 
B. licheniformis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.15 ± 0.03 1.30 ± 0.31 1.19 ± 0.28 28.6 ± 5.41 
Total bacterial count 0.40 ± 0.04 2.39 ± 0.25 2.55 ± 0.29 34.61 ± 4.12 
Total coliform 0.06 ± 0.03 0.14 ± 0.08 0.83 ± 0.49 5.51 ± 3.17 
Fecal coliform 0.06 ± 0.042 0.14 ± 0.10 0.83 ± 0.59 5.51 ± 3.88 

The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 5 (D1 W) ranged between 0.40 and 34.61, where the lowest counts were in February and the highest counts recorded in May. The monthly average of coliforms bacteria (×106 CFU/100 mL) ranged between 0.06 and 5.51, where the lowest were in February and the highest recorded for E. coli in May, respectively (Table 5).

Site 6: King Tallal Dam-2 (D2 W)

The following Gram-negative bacteria were isolated from water samples: Salmonella sp., Shigella sp., A. lwoffii, E. coli, E. cloacae, and E. meningosepticum (Table 6). C. sakazakii and K. pneumoniae were not isolated. The monthly average of the total number of Gram-negative bacteria (×106 CFU/100 mL) ranged from 3.19 to 8.01, where the lowest counts were in February and the highest counts recorded in March, respectively. The highest number of isolated bacteria was for Shigella sp. (Table 6). The isolated Gram-positive bacilli were B. firmus, B. lentus, B. brevis, B. cereus, B. megaterium, B. freudenreichii, and B. licheniformis. S. aureus was isolated as Gram-positive cocci. B. subtilis was not isolated. The monthly average of the total number of Gram-positive bacteria (×106 CFU/100 mL) ranged from 0.34 to 29.05, where the lowest counts were in February and the highest counts recorded in March, respectively. However, the highest number of isolated bacteria was for B. lentus (Table 6). The monthly total bacterial count (×106 CFU/100 mL) in all water samples analyzed from site 6 (D2 W) ranged between 3.53 and 37.06, where the lowest counts were in February and the highest counts recorded in March, respectively. The monthly average of coliforms bacteria (×106 CFU/100 mL) ranged between 0.69 and 2.19, where the lowest were in May and the highest recorded for E. coli in March, respectively (Table 6).

Table 6

The average number of CFU (×106/100 mL) ± S.D. of water samples from site 6 at King Tallal Dam-2 (D2 W)

Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 1.61 ± 1.90 2.025 ± 1.16 0.00 ± 0.00 0.00 ± 0.00 
Shigella sp. 0.40 ± 0.14 0.70 ± 0.42 1.8 ± 1.69 2.35 ± 0.21 
A. lwoffii 0.08 ± 0.04 1.60 ± 1.97 0.00 ± 0.00 0.95 ± 0.07 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.02 ± 0. 01 0.00 ± 0.00 0.00 ± 0.00 
E. coli 1.10 ± 0.14 2.17 ± 0.24 1.74 ± 1.85 0.69 ± 0.62 
E. meningosepticum 0.00 ± 0.00 1.50 ± 2.12 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 3.19 ± 0.60 8.01 ± 0.93 3.54 ± 0.78 3.99 ± 0.80 
Gram-positive bacteria S. aureus 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.03 ± 0.01 2.00 ± 1.41 0.15 ± 0.07 1.95 ± 0.07 
B. lentus 0.10 ± 0.01 23.50 ± 23.30 0.85 ± 0.21 7.70 ± 2.40 
B. subtilis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.02 ± 0.01 0.80 ± 0.28 0.25 ± 0.07 1.50 ± 0.71 
B. cereus 0.13 ± 0.11 2.75 ± 1.06 0.10 ± 0.14 13.25 ± 2.47 
B. megaterium 0.02 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.15 ± 0.21 
B. freudenreichii 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.28 
B. licheniformis 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.34 ± 0.04 29.05 ± 7.67 1.35 ± 0.28 24.75 ± 4.64 
Total bacterial count 3.53 ± 0.44 37.06 ± 5.43 4.89 ± 0.58 28.74 ± 3.44 
Total coliform 1.10 ± 0.64 2.19 ± 1.24 1.74 ± 1.00 0.69 ± 0.40 
Fecal coliform 1.10 ± 0.78 2.17 ± 1.53 1.74 ± 1.23 0.69 ± 0.49 
Types of bacteriaFebruaryMarchAprilMay
Gram-negative bacteria Salmonella sp. 1.61 ± 1.90 2.025 ± 1.16 0.00 ± 0.00 0.00 ± 0.00 
Shigella sp. 0.40 ± 0.14 0.70 ± 0.42 1.8 ± 1.69 2.35 ± 0.21 
A. lwoffii 0.08 ± 0.04 1.60 ± 1.97 0.00 ± 0.00 0.95 ± 0.07 
C. sakazakii 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
K. pneumoniae 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
E. cloacae 0.00 ± 0.00 0.02 ± 0. 01 0.00 ± 0.00 0.00 ± 0.00 
E. coli 1.10 ± 0.14 2.17 ± 0.24 1.74 ± 1.85 0.69 ± 0.62 
E. meningosepticum 0.00 ± 0.00 1.50 ± 2.12 0.00 ± 0.00 0.00 ± 0.00 
P. fluorescens 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-negative bacteria total number 3.19 ± 0.60 8.01 ± 0.93 3.54 ± 0.78 3.99 ± 0.80 
Gram-positive bacteria S. aureus 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. firmus 0.03 ± 0.01 2.00 ± 1.41 0.15 ± 0.07 1.95 ± 0.07 
B. lentus 0.10 ± 0.01 23.50 ± 23.30 0.85 ± 0.21 7.70 ± 2.40 
B. subtilis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
B. brevis 0.02 ± 0.01 0.80 ± 0.28 0.25 ± 0.07 1.50 ± 0.71 
B. cereus 0.13 ± 0.11 2.75 ± 1.06 0.10 ± 0.14 13.25 ± 2.47 
B. megaterium 0.02 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.15 ± 0.21 
B. freudenreichii 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.28 
B. licheniformis 0.02 ± 0.01 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 
Gram-positive bacteria total number 0.34 ± 0.04 29.05 ± 7.67 1.35 ± 0.28 24.75 ± 4.64 
Total bacterial count 3.53 ± 0.44 37.06 ± 5.43 4.89 ± 0.58 28.74 ± 3.44 
Total coliform 1.10 ± 0.64 2.19 ± 1.24 1.74 ± 1.00 0.69 ± 0.40 
Fecal coliform 1.10 ± 0.78 2.17 ± 1.53 1.74 ± 1.23 0.69 ± 0.49 

Seasonal variations for the water samples

Among all sampling sites, water samples which were collected in March from King Tallal Dam-2 (D2 W) recorded the highest total bacterial count (37.06 × 106 CFU/100 mL), and the lowest was collected in February from King Tallal Dam-1 (D1 W) (0.40 × 106 CFU/100 mL) (Tables 5 and 6). In addition, the highest coliform number was recorded in May for the water sample collected from King Tallal Dam-1 (D1 W) (5.51 × 106 CFU/100 mL). However, the lowest coliform number was recorded in April from Kherbet al-Samra-1 (K1 W) (0.01 × 106 CFU/100 mL (Table 1).

In the tested water samples, the isolated pathogenic bacteria were Salmonella sp., Shigella sp., B. cereus, and S. aureus. It was observed that those pathogenic bacteria were isolated in all months during the study period, but not at all sampling sites. Salmonella sp., as an example, was found during all the study period with month variations in all sampling sites except for site 1: Kherbet al-Samra-1 (K1 W) (Table 1).

Mutagenicity potential of the water samples

Using the mutagenicity percentage calculation method according to the positive control (Table 7) and the subtraction method using the negative control (Table 8), the results showed that water samples from all sampling sites had potential mutagenic activity in all months. The highest mutagenic activity was recorded in May (Table 8).

Table 7

Mutagenic activity degree according to the negative control revertant colony number

Mutagenicity degree
≤0 – 
1–19 
20–39 ++ 
40–59 ++ + * 
60––79 ++ + +* 
80–100 ++ + ++ * 
>100 ++ + ++ + * 
Mutagenicity degree
≤0 – 
1–19 
20–39 ++ 
40–59 ++ + * 
60––79 ++ + +* 
80–100 ++ + ++ * 
>100 ++ + ++ + * 

(*P-value < 0.05).

Table 8

The mutagenic activity of water samples using the subtraction method

SiteMonth
FebruaryMarchAprilMay
Kherbet alsamra-1 (K1W) ++ + * ++ + * ++ ++ + ++ + * 
Kherbet alsamra 2 (K2W) ++ + ++ + * ++ ++ ++ + ++ + * 
Zarqa farm (ZW) ++ ++ + * ++ + +* ++ + +* 
Jarash water stream (JW) ++ + * ++ + +* 
King's Tallal dam-1 (D1W) ++ ++ + +* ++ ++ + ++ * 
King's Tallal dam 2 (D2W) − − 
SiteMonth
FebruaryMarchAprilMay
Kherbet alsamra-1 (K1W) ++ + * ++ + * ++ ++ + ++ + * 
Kherbet alsamra 2 (K2W) ++ + ++ + * ++ ++ ++ + ++ + * 
Zarqa farm (ZW) ++ ++ + * ++ + +* ++ + +* 
Jarash water stream (JW) ++ + * ++ + +* 
King's Tallal dam-1 (D1W) ++ ++ + +* ++ ++ + ++ * 
King's Tallal dam 2 (D2W) − − 

(*P-value < 0.05).

All water samples collected from all sites during the study period, showed a positive mutagenic activity except the samples that were collected from King Tallal Dam-2 site in February and May, as shown in Figure 2. The highest positive mutagenicity percentage among all water samples was recorded in May from Kherbet al-Samra-2 (58%) followed by Kherbet al-Samra-1 (56%) (Figure 2).

Figure 2

Mutagenicity percentage for water samples from all sampling sites (K1 W: Kherbet Al-Samra-1 water; K2 W: Kherbet Al-Samra-2 water; ZW: Zarqa Farm water; JW: Jarash River water; D1 W: King Talal Dam-1 water; D2 W: King Talal Dam-2 water) in four months (February, March, April, and May) ± S.E. Within sampling sites, columns with different letter scripts statistically differ (P < 0.05).

Figure 2

Mutagenicity percentage for water samples from all sampling sites (K1 W: Kherbet Al-Samra-1 water; K2 W: Kherbet Al-Samra-2 water; ZW: Zarqa Farm water; JW: Jarash River water; D1 W: King Talal Dam-1 water; D2 W: King Talal Dam-2 water) in four months (February, March, April, and May) ± S.E. Within sampling sites, columns with different letter scripts statistically differ (P < 0.05).

DISCUSSION

Bacterial contamination

In Jordan, there are 26 wastewater treatment plants (WWTP) that serve most of the cities in the country, in which their effluents are used indirectly for agriculture (Al-Zboon & Al-Ananzeh 2008; Uleimat 2012). Different systems are used for wastewater treatment and they are divided into activated sludge, trickling filters, and waste stabilization ponds (Uleimat 2012). The Zarqa River in Jordan is seriously polluted with wastewater leakage from nearby factories, car wash stations, flooding manholes and sewer systems particularly in wintertime when the sewer system overflows due to rising water levels.

Our results showed that Zarqa River is highly contaminated with total and coliform bacteria beginning from Kherbet Al-Samra1 until the King Tallal Dam. Different types of Gram-positive and Gram-negative bacteria were isolated (Tables 16). The importance of E. coli contamination comes from the fact that E. coli causes a variety of infections, such as enteritis, septicemia, peritonitis, urinary tract infections, and meningitis in humans and animals. It can be isolated from water, soil, and food. E. coli and other coliforms were isolated from all sampling sites collected in this study. On the other hand, some serotypes can cause serious food poisoning in humans (Vogt & Dippold 2005). E. coli strains that bear virulence factors and cause diarrhea include enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and enteroaggregative (EAggEC) (Levine et al. 1987; Karmali 2004). Approximately 2–8% of the E. coli present in water was found to be enteropathogenic E. coli, which causes traveler's diarrhea (Lundgren et al. 2013).

The distribution of coliforms in the Zarqa River was studied by Qublan (2001), who found that the average of coliform level was (0.05 × 106 CFU/100 mL), which is less than our findings (90 × 106 CFU/100 mL). The Qublan (2001) study was performed 15 years before our study. This indicates the increased level of pollution despite the renewal and the improvements that were added to the KSWWTP in 2008. Salmonella is one of the most predominant pathogenic bacteria in wastewater and its number in wastewater may reach 8,000 organisms/100 mL (Jimenez & Chavez 2000). It was isolated from site 2 during February and March and its count was rather high, 1–2 × 106 CFU/100 mL (Table 2).

Shigella sp. was also isolated from all sampling sites with high bacterial counts. This indicates that this pathogen was not treated efficiently with chlorine in KSWWTP, which exposes the public to high risk. Fayyad & Al-Sheikh (2001) reported that most chlorine residual that applied at KSWWTP was inorganic monochloramine (NH2Cl) that has lower disinfection efficiency and stability than free chlorine. Therefore, bacterial contamination such as pathogenic Shigella sp. will grow and reproduce and the disinfection process will be unreliable. This pathogen is transmitted by direct contact with an infected individual, who may excrete up to 109Shigellae per gram of feces (Faruque et al. 2002). Although person-to-person contact is the main mode of transmission of this pathogen, food-borne transmission via raw vegetables and water-borne transmission have also been documented (Faruque et al. 2002). Shigella sp. was the highest bacteria isolated from site 1 (Table 1). Our results are in agreement with Qublan's (2001) study, which investigated the distribution of Shigella and other pathogens in Zarqa River and showed that the river water had high densities of coliforms, Salmonella, and Shigella. Fandi et al. (2009) reported that King Talal Dam water total coliform and fecal coliform extensively exceeded the permissible limit, where all sampling sites showed the contaminated status of E. coli. Therefore, since the water of King Talal Dam reservoir is used to irrigate lands of the middle and southern regions of the Jordan Valley, it is highly alarming that all irrigation water channels have bacterial contamination. A study done by Goldstein et al. (2012) found that S. aureus was detected at four USA wastewater treatment plants in the influents and in some of their effluents. Such finding is in agreement with our results for the presence of this pathogen in the effluents of the KSWWTP and even in the water of the Zarqa River itself.

Results from this study indicated that differences in temperature between seasons affect the bacterial count number and the total coliform count. In some systems, E. coli survival is promoted by warmer temperatures (Chandran & Hatha 2005). However, an inverse relationship between E. coli and temperature was observed, in which E. coli survival was increased with a decrease in water temperature, possibly through temperature-prompt changes in cell membrane fluidity (McLain & Williams 2008). Furthermore, McLain & Williams (2012) measured the impact of treated wastewater irrigation on the levels of fecal indicator bacteria in irrigated soils, levels of E. coli and Enterococcus in Arizona, USA. They found higher levels of E. coli in winter than in summer and the levels of fecal indicator bacteria decreased from summer to winter, possibly related to low winter water use and corresponding death of residual bacteria within the system. Taken together, reduction in bacterial contamination in treated wastewater effluent must be performed by improving the treatment system in the wastewater treatment plant itself in order to overcome the overload that comes from municipal and industrial waste.

Potential mutagenicity

Thousands of chemicals are released into ground and surface water from factories and other industrial activities and it is important to identify the most relevant sources of these hazardous compounds. These sources include effluent from petrochemical industries and agricultural runoff, which contain both pesticide and fertilizer residues. Municipal wastewaters, industrial and agriculture effluents, and natural agents (De Flora et al. 1991; Bünger et al. 2007; Krishnamurthi et al. 2008)) can account for mutagenicity of environmental matrices air, water, and soil (Minissi et al. 1998). The positive mutagenic activity in all water samples from all sampling sites found in this study indicated that the effluents of KSWWTP had mutagenic substances. The high positive mutagenicity results from both of Kherbet al-Samra (K1 W and K2 W) sampling sites might be due to high concentration of mutagenic pollutants and the disinfectant chlorine in the effluent-treated water before it was diluted with surface water. However, as water flow passed to the other sampling sites ending with the King Talal Dam-2 water reservoir, the mutagenicity decreased because of the increase in water dilution with surface water and precipitation that collected in the King Talal Dam-2, especially during the winter season. Additionally, since the King Talal Dam-2 reservoir is controlled by a natural resource watching station and periodically patrolled by environmental police, the illegal dumping of wastewater from trucks and nearby factories is highly regulated.

There are many reasons behind the high mutagenicity results obtained in this study. The treated wastewater was not treated efficiently, the industrial wastewater reaching the KSWWTP by the sewage system was not properly treated at the factories before releasing it to the municipal sewers, and the disinfection by chlorine in the wastewater treatment process might be the source of potential mutagenic activity. Trace quantities of chlorinated hydrocarbon compounds in water may also be attributed to the chlorination of organic residues by chlorine added as a disinfectant in the wastewater treatment process (Weiner & Matthews 2003). Khan & Roser (2007) using Ames test studied chlorination of treated wastewater. Their results showed that the majority of the mutagenic activity appeared to be derived from the chlorination process itself.

In this study, the compounds that contributed to the observed mutagenic activity were not identified. Batarseh (2003) studied the toxic, mutagenic, and carcinogenic substances in Zarqa River and reported the presence of many organic pollutants in the Jordanian environment either in reclaimed water or in sewage sludge. Some of these substances were polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs: like DDT, aldrin, and dieldrin), chlorinated benzenes (CBz) and organochlorine pesticides (OCP). All these substances were mutagenic and carcinogenic and can reach the food chain easily (Batarseh 2003). Furthermore, Qublan (2001) studied the distribution of heavy metals in Zarqa River water. His results showed that chromium and cadmium were above the recommended levels according to Jordanian Standards. Chromium and cadmium are cytotoxic and mutagenic substances to humans (Bagchi et al. 2000). Ansari & Malik (2009) study showed the presence of genotoxic agents in some farms in India that used wastewater for irrigation. All tested samples exhibited significant mutagenicity with Ames test, suggesting the possible role of contaminating pesticides in the wastewater. On the other hand, Durgo et al. (2009) investigated wastewater polluted by fertilizers from a nearby factory in Croatia. Using the alkaline comet assay, they found significant DNA damaging potential of wastewater on human leukocytes; however, no significant result was found using Ames test. This indicated that the Ames test could not detect some mutagens and genotoxic substances. El-Asslouj et al. (2009) studied the genotoxic potential of Settat River in Morocco, which contained wastewater using the micronucleus test and cellular proliferation index. The tested water samples induced a clear increase in the frequency of micronucleated cells and a lower cell proliferation in human peripheral blood lymphocytes in vitro. Furthermore, Miadoková et al. (2000) investigated the mutagenic/carcinogenic potential of acid-mine water from the Slovak mining area Rudňany that contained a high load of toxic metals. Their study results obtained from the modified pre-incubation Ames assay proved that 1,000-fold diluted wastewater exhibited mutagenic effect on all S. typhimurium strains. Similarly, Joliboisa & Guerbeta (2005) evaluated the genotoxic potential of different wastewaters (industrial, hospital, and domestic) samples collected in the Rouen area on S. typhimurium mutagenicity assay with or without metabolic activation. The study revealed that 65% of tested samples showed positive mutagenic results.

The use of contaminated wastewater containing industrial wastes in soil and crop irrigation has serious public health consequences due to the entry of toxic pollutants into the human food chain (Hanjra et al. 2012). The high levels of heavy metals in wastewater could have carcinogenic and fatal effects when ingested in high amounts (Amlan et al. 2012).

CONCLUSION

The following conclusions are drawn:

  • There was high contamination with different types of pathogens and high levels of coliforms in all sampling sites without specific prevalence pattern.

  • The isolated pathogenic bacteria were Salmonella sp., Shigella sp., B. cereus, and S. aureus.

  • The isolated opportunistic pathogens were A. lwoffii, E. meningosepticum, P. fluorescens, and B. licheniformis.

  • Differences in temperature between seasons affect the bacterial count number and the total coliform count.

  • All water samples collected from all sites during the study period showed positive mutagenic activity except the samples that were collected from King Tallal Dam-2 site in February and May.

  • The highest positive mutagenicity percentage among all water samples was recorded in May from Kherbet al-Samra-2 (58%) followed by Kherbet al-Samra-1 (56%) sampling site.

  • The positive mutagenic activity and high bacterial pollution exposes the public to high risk of bacterial infection.

  • Improving the disinfection efficiency in the wastewater treatment plant is important to reduce water resources contamination and environmental pollution and minimize potential toxicity and exposure of the public to pathogenic bacteria.

  • Increasing the number of samplings throughout the year is necessary to monitor the bacterial contamination and collect additional information concerning the amount of toxicity/mutagenicity of the treated wastewater effluents.

ACKNOWLEDGEMENTS

This research was funded by Jordan University of Science and Technology-Deanship of Research, grant No.63/2011.

DATA AVAILABILITY STATEMENT

All relevant data are included in the paper or its Supplementary Information.

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