Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro Biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia faba in the West region of Cameroon

Rodrigue Biguioh Mabvouna; Sali Ben Béchir Adogaye; Patrick Martial Pete Nkamedjie; Andrillene Laure Deutou Wondeu; Martin Sanou Sobze; Jean Blaise Kemogne; Vittorio Colizzi

doi:10.1371/journal.pone.0245379

. 2021 Feb 4;16(2):e0245379. doi: 10.1371/journal.pone.0245379

Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro Biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia faba in the West region of Cameroon

Rodrigue Biguioh Mabvouna ^1,^*, Sali Ben Béchir Adogaye ¹, Patrick Martial Pete Nkamedjie ², Andrillene Laure Deutou Wondeu ³, Martin Sanou Sobze ⁴, Jean Blaise Kemogne ³, Vittorio Colizzi ¹

Editor: Swatantra Pratap Singh⁵

PMCID: PMC7861400 PMID: 33539444

Abstract

At least 2.1 billion people around the world use contaminated drinking water, causing 485,000 diarrheal deaths each year, mostly among children under 5 years old. A study conducted 10 years ago in Bafoussam (West Cameroon) recorded concentrations of bacteria among surface and groundwater. High levels of bicarbonates, phosphates, chlorides and suspended matters were also found. The aim of this study was to assess the microbiological and chemical qualities of domestic water sources in 5 localities of the West region of Cameroon. Water samples from 22 water sources (wells, springs, water drilling and river) were aseptically collected in plastic bottles and transferred in 50 ml sterile tubes. For chlorinated water sources, 1 ml of Thiosulfate was added to the water sample; immediately placed in an ice box and transported to the laboratory for analysis. Water temperature and pH were measured on site. The microbiological quality of water was determined by testing Total Coliforms (TC) using the Micro Biological Survey method. 1 ml of each water sample was inoculated in the MBS vial initially rehydrated with 10 ml of sterile distilled water. The initial color of the vials is red. Color changes were monitored at three different time intervals (12h, 19h and 24h), corresponding to three levels of contamination. The chemical quality of water was assessed using micronucleus (MN) test in selected Vicia faba seeds secondary root tips permanently mounted in Dibutylphthalate Polystyrene Xylene mountant for histology after 72 hours of direct exposition in water samples and in dark. The mitotic indices and MN frequencies were evaluated in 10 root tips per site analysing 5000 cells per tip. Statistical analyses were done using Stata IC/15.0 software. The Student t-test was used for mean comparison and the significance level was set at 1%. The majority of samples were collected from wells (63.6%). The mean water pH ranged from 5.5 to 8.3 and the temperature varied from 23 to 26°C. A very high concentration of TC [>10³ CFU/ml] was found in 8 (36.4%) samples. 10 (45.5%) and 2 (9.1%) samples turned yellow at 19 and 24 hours respectively after incubation corresponding to TC concentration of [10<x<10³ CFU/ml] and [1<x< 10 CFU/ml]. The MN frequency was higher (P ≤ 0.01) compared to the negative control in 9 (40.9%) water samples indicating significant genotoxic effects of these water sources. This study highlighted the poor quality of domestic water sources in West region of Cameroon and the need to conduct regular monitoring of drinking water sources. Community capacity building on water treatment methods, including good wastes management should be implemented to help improve water quality.

Introduction

Adequate, accessible and safe drinking water source is often regarded as an important means of disease prevention [1]. Improving access to safe drinking-water can result in tangible benefits to health [2]. According to the World Health Organization (WHO) guidelines for drinking-water quality, safe water should not represent any significant risk to health over a lifetime of consumption, including different sensitivities that may occur between life stages [2]. In 2017, the WHO estimated that 785 million people lacked a basic drinking water service, including 144 million people who were dependent on surface water [3]. Infants and young children are the most exposed to waterborne diseases, especially those living in unsanitary conditions. At least 2.1 billion people around the world use a drinking water source contaminated with faeces; and according to forecasts, half of the world's population will be living in water-stressed areas by 2025 [3].

Water contaminants can be defined as any physical, chemical, biological, or radiological substance or material in water [2, 3]. Contaminants may be harmful if consumed at certain levels in drinking water [4]. Microorganism and chemical contaminants can transmit diseases such diarrhoea, cholera, dysentery, typhoid, cancers, infertility, immunological and neurological diseases [2–5]. Contaminated drinking water is estimated to cause 485,000 diarrheal deaths each year, mostly among children under 5 years old [3, 6]. There are many sources of contamination of drinking water resulting from domestic, agricultural or industrial activities [2–7]. Previously, attention was mainly focused on the microbiological quality of drinking water, but with the development of toxicology and the increase of knowledge on the risks of chemicals, studies of chemical quality of drinking water have increased. The microbiological quality of drinking water can be assessed by monitoring faecal indicator bacteria, such as Coliform bacteria [8–10].

Coliforms are a group of bacteria that are generally not harmful for human beings. They are present in the digestive tracts of humans and animals, thus in their faeces. Generally, the concentrations of faecal bacteria in water sample are low, and the quantity of possible bacteria present in the sample is high. Thus for water quality analyses, it is not practical to test the presence of each pathogen in the sample. As a result, the presence of other harmful biological organisms can be indirectly detected by testing Total Coliforms [11, 12]. Total Coliform bacteria are used as an indicator of other pathogenic bacteria mainly because their presence in water sample indicates environmental contamination and presence of water borne diseases bacteria [13]. Coliform bacteria also allow to assess the efficiency of water treatment (disinfection, chlorination or boiling), thus their presence indicates an insufficient, inadequate or absence of water treatment [14]. In addition to microbiological quality, chemical substances from human activities can also affect the quality of drinking water sources [15, 16].

In limited resources settings, the key issue in water safety policy is how to determine the microbiological and chemical qualities of drinking water with simple but valid methods. From a public health perspective, simple routine checking of drinking water should be designed to detect the presence or absence of microorganisms in water and the level of toxicity that can affect human health. In Cameroon, there are few studies assessing both microbiological and chemical qualities of drinking water sources in rural or urban settings. A study conducted 10 years ago in Bafoussam (West region) recorded higher concentrations of bacteria such as Escherichia coli, Salmonella, Shigella among surface and groundwater. Chemically, high levels of bicarbonates, phosphates, chlorides, and suspended materials were found in rivers and springs water samples [17]. To provide updated data and evidences for steering public health decisions, we performed a combined approach to assess the microbiological quality of domestic water sources by the detection of Total Coliforms (TC) using Micro Biological Survey (MBS) method and the chemical quality was assessed using the mutagenesis assay by the detection of genotoxic effects in root tips of Vicia faba.

Materials and methods

Study location

The study was conducted in the West region of Cameroon which has an estimated population of 1,921,590 inhabitants (from the 2008 population Census) [18]. Inhabitants are mostly farmers (coffee, potatoes, maize, beans, and vegetables production) and their farming activities require the use of fertilizers that could potentially pollute the environment including water sources.

Study design, period and water sampling

This was a cross-sectional study carried-out in five localities of west Cameroon (Bafoussam 1er, Bafoussam 2e, Foumbot, Galim and Kouoptamo) between October and November 2018. 22 samples from domestic water sources (wells, spring, drilling water and river) used as drinking water by local populations were aseptically collected in the morning before 12 o’clock. At each sampling site, 1 litre of water was directly collected in sterile plastic bottles. 50 ml of sample was taken in plastic tubes from collected water and 1 ml of Sodium Thiosulfate solution 10% (0.25g/50ml) was added to the sample; for chlorinated water sources. The tube containing the sample was immediately placed in an ice box and then transported to the laboratory for microbiological and genotoxic analysis. Temperature and pH of water were measured on site before placing the samples in the ice box. Water sources to be sampled were identified step by step according to their availability, and accessibility. Table 1 presents details on the water sources collected for analyses.

Table 1. Sampling locations, water sources categories, sample temperature (°C), geographic coordinates, and samples collection dates.

Sampling location	Water source category	Sample temperature (°C)	Geographic coordinates		Collection date
Sampling location	Water source category	Sample temperature (°C)	Latitude	Longitude	Collection date
Foumbot	Protected spring	24	5,5783333	10,6391667	23/09/2018
	River	24	5,5866667	10,6502778	23/09/2018
	Protected well	23	5,5558333	10,6125	24/09/2018
	Unprotected well	23	5,5722222	10,6158333	24/09/2018
	Unprotected well	23	5,4947222	10,6047222	24/09/2018
	Protected spring	24	5,4969444	10,5991667	24/09/2018
	Protected well	24	5,5422222	10,5930556	24/09/2018
Bafoussam 1er	Unprotected spring	24	5,4866667	10,5180556	23/09/2018
	Unprotected well	23	5,5116667	10,4933333	25/09/2018
	Drilling water	23	5,5008333	10,5216667	28/09/2018
Bafoussam 2e	Protected spring	24	5,5636111	10,5386111	24/09/2018
Bafoussam 2e	Protected well	23	5,5530556	10,5225	28/09/2018
Galim	Protected well	23	5,7280556	10,5225	23/09/2018
Kouoptamo	Unprotected well	26	5,6211111	10,4783333	23/09/2018
	Unprotected well	23	5,6186111	10,6252778	25/09/2018
	Unprotected well	22	5,645	10,5663889	25/09/2018
	Protected well	22	5,6344444	10,5447222	25/09/2018
	Protected spring	23	5,7394444	10,5788889	28/09/2018
	Protected well	23	5,7394444	10,6097222	28/09/2018
	Protected spring	23	5,6825	10,595	28/09/2018
	Unprotected well	23	5,6816667	10,5941667	28/09/2018
	Unprotected well	23	5,6486111	10,5611111	28/09/2018

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Total Coliforms detection

Quantitative detection of Coliforms in water samples was done using MBS-HACCP & water Easy test. This test is simple and can be performed by anyone, anywhere in an error-free way. The MBS method is a colorimetric reaction system based on the measurement of the catalytic activity of oxidoreductase enzymes of primary metabolism, which allows to determine a correspondence between microorganism and enzyme activity in the sample [19]. This method has been applied in similar context in Africa including Cameroon for the assessment of the microbiological safety of drinking water [20, 21], and had been found to be mostly adapted for developing countries as it requires less skills, time and can be done at low cost. The test comes with all the material necessary and the analysis doesn’t require other reagents or instruments. The MBS method is based on the visual observation of the color change in the suspension formed in the analysis vial used when the test sample is inoculated [19]. The color change (from red to yellow) occurs when the water sample added in the vial contains Coliforms bacteria. The changes in color according to time are proportional to the concentration of coliforms in the water samples, the greater the amount of microorganisms, the more rapid the change of color, thus, a positive result (contamination) [19–21]. The concentration of bacteria is expressed in Colony Forming Units per millilitres of water (CFU/ml) for the analysis water samples.

Before starting with the analysis, the Coliforms MBS (Coli MBS) vials were rehydrated with 10 ml of sterile distilled water and shaken to dissolve the reagent. 1 ml of each water sample was collected from plastic tubes using a sterile Pasteur pipette and inoculated in the Coli MBS vial. The vials were then closed and shaken for about 30 seconds for homogenization. Each analysis was performed twice and the vials were incubated at 37°C. The color changes of the Coli MBS reaction vials were monitored using the chromatic scale according to the standard protocol at three different time intervals (12 hours, 19 hours, and 24 hours), corresponding to three levels of contamination [19]. The initial color of the Coli MBS vials is red. A color change from red to yellow after 12 hours indicates a very high contamination (TC concentration > 10³ CFU/ml); a color change at 19 hours indicates a high contamination (TC concentration 10 < x < 10³ CFU/ml) and a color change at 24 hours corresponds to TC concentration of 1 < x < 10 CFU/ml. For the negative control, 1 ml of distilled water was inoculated in the Coli MBS vial [19].

Micronucleus (MN) test

The MN test is considered to be one of the most suitable methods for identifying response to exposure to a complex mixture of contaminants [22, 23]. This test is used both as a short-term test in animals, humans and for the detection of the mutagenic potential of pollutants in air, water and soil, in environmental monitoring programs [24]. Its application in plant systems (Vicia faba, Allium cepa, Tradescantia sp. etc.) is particularly indicated for the detection of mutagens present in water and soil [25–28], allowing the analysis of raw environmental matrices without purification or concentration processes [22, 23]. For its sensitivity and reliability, MN test in Vicia faba roots is one of the most widely used methods in aquatic genotoxicity studies, also limiting the use of animal testing [29]. This tests is able to detect both clastogenic and aneugenic effects. Finally, its low cost and its simplicity of execution make it particularly suitable for monitoring polluted fresh water.

Mutagenesis assay in Vicia faba root tips

The MN test was performed in the secondary root tips of Vicia faba according to standard protocol [29, 30]. Micronuclei occur as small nuclei next to the main nucleus of interphase cells and their appearance is related to the formation of chromosome fragments or the loss of whole chromosomes during cell division (mitotic). They arise both from the loss of chromosome fragments, due to the absence of the centromere, and from the loss of entire chromosomes, due to induced functional damage to the mitotic spindle or the inhibition of the functions of other structures, including the interaction between the centromere-kinetochore and the fibers of the mitotic spindle involved in the process of segregation. Once induced, the lost fragment and chromosome, not binding to the fibers of the spindle, are excluded from the process of segregation.

Mutagenesis assay operating procedures

About 400 dry Vicia faba seeds were rehydrated for 24 hours in tap water. The seeds coats were removed and the seeds placed in moist cotton for germination at ambient temperature in the dark. Four to five days after seeding, the primary roots, about 3 cm in length were selected and their tips were cut off. For each sample, five seeds were selected. To perform the test, the primary roots of selected seeds were suspended in water samples contained in glass containers at ambient temperature and in the dark. Distilled water was used as negative control. No positive control was used for the experiment. After three days (72 hours), secondary roots of about 1–2 cm were obtained, cut off and fixed with a 3:1 solution of ethyl alcohol/acetic acid. After 30 minutes, the fixing solution was replaced by a new one and the roots were stored at + 4°C after covering each container with parafilm. Before staining, the roots were transferred from the fixing solution to tap water for 10 to 15 minutes at ambient temperature. The rehydrated roots were transferred to a preheated (60°C) 1N chloridic acid solution and placed in a water bath for 10 minutes. After complete removal of the chloridic acid solution, the roots were immersed in the Schiff reagent for 45 to 60 minutes at ambient temperature and in the dark. Stained roots were squashed on to slides in 45% acetic acid, then immerse slowly in liquid nitrogen and the coverslips were quickly removed using a scalpel blade. The slides were air-dried for 8 to 10 hours or overnight and permanently mounted in DPX mountant for histology. The mitotic indices and MN frequencies were evaluated in 10 root tips per site analysing 5000 cells per tip. The Student t-test was used to perform comparison between the average MN frequency at each site and the negative control. Statistical analyses were done using Stata IC/15.0 software (College Station, Texas 77845 USA, http://www.stata.com) and the significance level was set at 1% for the MN frequency and at 5% for proportions.

Ethical approval

This study does not report on or involve the use of any animal or human data or tissue, so ethical approval and consent were not required with reference to Order No. 079/A/MSP/DS of the Minister of Public Health of October 22, 1987 establishing and organizing an Ethics Committee on Research Involving Human Beings (article 2).

Results

The majority of the samples were collected from wells water (63.6%) and springs water (27.2%). The pH of the water samples collected ranged from 5.5 to 8.3 and the temperature varied from 23 to 26°C.

Microbiological quality

Concerning the microbiological quality of the samples, all the four types of water sources were contaminated with TC, except two wells where samples were found to be safe [TC counts <1 CFU/100 ml], (Table 2). A very high concentration of TC [>10³ CFU/ml] was found in 8 (36.4%) samples. 10 (45.5%) and 2 (9.1%) water samples turned yellow at 19 hours and 24 hours after incubation corresponding to TC concentration of [10<x<10³ CFU/ml] and [1<x < 10CFU/ml] respectively. The highest TC count was observed independently of the types of water sources analysed. Water sources form the sampling site of Kouoptamo appeared more contaminated. All the 9 samples of this locality were contaminated, 2 of them with very high TC concentration [>10³ CFU/ml].

Table 2. Total Coliforms concentration in water samples, level of contamination and time of analysis, West region, Cameroon, 2018.

Sampling location	Water source category	Total Coliform concentration (CFU/mL)	Level of contamination	Duration of analysis (hours)
Foumbot	Protected spring	1 < x < 10	Low	24
	River	> 10³	Very high	12
	Protected well	<1	Very low	No color change
	Unprotected well	10 < x < 10³	High	19
	Unprotected well	> 10³	Very high	12
	Protected spring	1 < x < 10	Low	24
	Protected well	10 < x < 10³	High	19
Bafoussam 1er	Unprotected spring	> 10³	Very high	12
	Unprotected well	10 < x < 10³	High	19
	Drilling water	10 < x < 10³	High	19
Bafoussam 2e	Protected spring	> 10³	Very high	12
Bafoussam 2e	Protected well	<1	Very low	No color change
Galim	Protected well	> 10³	Very high	12
Kouoptamo	Unprotected well	> 10³	Very high	12
	Unprotected well	10 < x < 10³	High	19
	Unprotected well	10 < x < 10³	High	19
	Protected well	> 10³	Very high	12
	Protected spring	10 < x < 10³	High	19
	Protected well	10 < x < 10³	High	19
	Protected spring	10 < x < 10³	High	19
	Unprotected well	10 < x < 10³	High	19
	Unprotected well	10 < x < 10³	High	19

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Chemical quality

Regarding the chemical quality of the water samples, the mutagenesis assay in root tips of Vicia faba revealed a significant genotoxicity in almost half of the samples (40.9%), Fig 1. Globally, the mean cells proliferation including mitotic cells of the examined root tips was similar to the negative control and the mitotic index values ranged from 5.4% and 8.8% (Table 3). The MN frequency was significantly higher (P ≤ 0.01) than the negative control value in 35.7% of wells water, 50% of springs and in the only river analysed. The Student t-test showed significant genotoxic effects in 9 water samples, of which 5 (55.6%) wells, 3 (33.3%) springs and a river (11.1%), (Table 4).

Fig 1 — *Vicia faba* cells in proliferation: (A) non genotoxic water source slide, mitotic cells are circled in blue; (B) genotoxic water source slide, mitotic cells are circled in black and micronuclei are circled in red.

Table 3. Mean per 5000 cells ± Std. Err. of mitotic cells and mitotic index in root tips of Vicia faba exposed to water samples, West region, Cameroon, 2018.

		Mitotic cells		Mitotic index (%)
Sampling location	Sources	Mean	SE	Mean	SE
Foumbot	Spring	365	±16.98	7.3	±0.3
	River	349	± 12.3	6.9	±0.2
	Well	277	±13.7	5.5	±0.2
	Well	278	±51.2	5.5	±1
	Well	316	±52.4	6.3	±1
	Spring	293	±25.8	5.8	±0.5
	Well	273	±25.1	5.4	±0.5
Bafoussam 1er	Spring	321	±12.38	6.4	±0.2
	Well	339	±41.5	6.7	±0.8
	Water drilling	385	±43.8	7.7	±0.8
Bafoussam 2e	Spring	284	±38.9	5.6	±0.7
Bafoussam 2e	Well	374	±41.1	7.4	±0.8
Galim	Well	293	±18.9	5.8	±0.3
Kouoptamo	Well	314	±22.7	6.2	±0.4
	Well	317	±17.1	6.3	±0.3
	Well	348	±24.9	6.9	±0.4
	Well	343	±35.1	6.8	±0.7
	Spring	367	±22.7	7.3	±0.4
	Well	279	± 23.3	5.5	±0.4
	Spring	441	± 39.3	8.8	±0.7
	Well	378	±45.8	7.5	±0.9
	Well	349	±45.9	6.9	±0.9
Total	-	331	±9.3	6.6	±0.1
Negative control	Tap water	332	±16.5	6.6	±0.3

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Table 4. Mean per 5000 cells ± Std. Err. of MN frequency in root tips of Vicia faba exposed to water samples, West region, Cameroon, 2018.

Sampling location	Sources	Mean	SE	[99% CI]
Foumbot	Spring	4.1	±0.52	[2.3–5.8]
	River	11.8^*	±0.6	[10.2–13.3]
	Well	3.9	±0.3	[2.6–5.1]
	Well	4.3	±0.4	[2.7–5.8]
	Well	3.6	±0.4	[2.2–4.9]
	Spring	4.1	±0.2	[3.2–4.9]
	Well	3.6	±0.3	[2.6–4.5]
Bafoussam 1er	Spring	13.6^*	±0.8	[11.7–16.4]
	Well	4.1	±0.5	[2.4–5.7]
	Water drilling	4	±0.4	[2.5–5.4]
Bafoussam 2e	Spring	13.8^*	±0.8	[11.1–16.4]
Bafoussam 2e	Well	4.2	±0.3	[2.9–5.4]
Galim	Well	4	±0.3	[2.7–5.2]
Kouoptamo	Well	4	±0.2	[3.1–4.8]
	Well	3.8	±0.2	[2.8–4.7]
	Well	10.5^*	± 1.4	[5.8–15.1]
	Well	9.6^*	±0.7	[7–12.1]
	Spring	10.8^*	±1.2	[6.6–14.9]
	Well	9.7^*	±0.9	[6.6–12.7]
	Spring	4	±0.4	[2.4–5.5]
	Well	12.1^*	±1.3	[7.5–16.6]
	Well	8.9^*	±1.2	[4.9–12.8]
Negative control	Tap water	3.4	±0.26	[2.7–4]

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*The MN frequency is significantly higher (P ≤ 0.01) than the negative control value.

Discussion

Water from wells, drilling, spring and river are the major domestic water sources in sub-Saharan Africa and therefore are essential resource for human activities and for consumption. The control of their quality remains a concern in many countries. Water represents one of the principal sources of diseases transmission and the use of unsafe water can cause serious health problems due to the presence of potential contaminants. The use of simple and rapid methods for both microbiological and chemical water qualities check that can be performed anywhere even by unskilled personnel is one of the key components of effective water management policy, especially in resource-limited countries. In this study, we used a combined approach for water microbiological and chemical qualities check. MBS method results showed that almost all types of water sources contained TC and higher concentrations of TC was found among wells. Our findings are consistent with the results of previous study in Bafoussam (West Cameroon), which reported high concentration of TC in groundwater (well water and spring water) mostly during rainy season with TC concentration higher than 10³ CFU/ml [17]. Our study was also conducted during rainy season suggesting possible high bacteria growth in groundwater than in surface water (running water) in this period. This could be favoured by the combined effect of high temperatures (from 23 to 26°C) and pH (from 5.5 to 8.3). It is also important to note that most of wells were closed to latrines and/or human habitation and hence phreatic slicks of wells could communicate with latrines during rainy season due to additional water flow. External contamination by runoff water might also explain high TC concentration observed as bacteria could be transported by water from latrines and/or dumps to wells. Additional investigations are required to further assess these relations. In 20 (90.9%) water samples, TC counts were above the recommended levels (<1 CFU/100ml) set by WHO [2, 31], including treated sources (chlorinated sources) indicating an insufficient or inadequate treatment of the sources. However, the TC concentration of untreated source water was found to be higher than treated water sources with no statistically significant difference. Coliform bacteria and other harmful biological organisms have the same origin. The detection of coliforms is simple and rapid. Their quantity in water sample is higher than other pathogenic bacteria. As other dangerous organisms, coliforms are sensitive to water treatment such as disinfection, boiling, thus assessing coliforms as indicator of microbiological quality of drinking water can be a reasonable approach of testing the presence or absence of other pathogenic bacteria in water.

We also applied MN test in Vicia faba roots tips as a first alarm system for mutagenic effects of domestic waters sources. The genotoxicity was measured by direct exposition of Vicia faba roots to water samples. Other methods using plants such as Tradescantia [32–34]; aquatic animals e.g. amphibian, fishes, mussels [24, 35, 36] exist and can be used with the difference that they are more complex to perform compared to the MN test in Vicia faba roots. The mitotic index found in this study ranged from 5.4% to 8.8% corresponding to range found in optimal conditions of roots growth and cell proliferation [37]. Our results show a significant genotoxicity in almost half of water samples. Well water, springs and river showed high genotoxic effects compared to the negative control. In line with findings from a study that reported genotoxic activity in ground and surface water for drinking, samples collected from spring and river were the most genotoxic [38, 39]. To our knowledge, there is no study on the water genotoxicity in Cameroon and our study is the first one investigating water genotoxicity using Vicia faba seeds in Cameroon including the Central African sub region. The rare genotoxic studies carried-out in Africa were almost done on plants used for traditional medicine using other methods such as comet essay, Salmonella microsome assay [40–42]. Our approach has the merit of being simple and reliable. It is especially adapted to study environment quality in areas like West Cameroon where there are intensive agricultural activities. The results have highlighted a potential health risk for the populations who live around this area.

Conclusion

Water quality monitoring is essential to attest whether water sources are safe for human consumption or not. The use of valid methods for water analysis is also important. Results showed that almost all water samples were found to be contaminated by TC including treated water sources, highlighting the poor quality of domestic water sources in West region of Cameroon and lack of appropriate knowledge regarding water treatment techniques. Some water sources indicated genotoxic effects on Vicia faba root tips. Results demonstrated the need to conduct regular monitoring of drinking water sources. Community empowerment on water treatment methods and wastes management need to be implemented to help improve water quality. As previous studies have shown that disinfectants can induce an increase in the water genotoxicity, water treatment should be done following recommended protocols. There is also a need to design adequate faeces disposal systems, mostly around water drinking sources.

Supporting information

S1 Dataset

(XLS)

Click here for additional data file.^{(33KB, xls)}

Acknowledgments

The authors express their gratitude to the administrative staff of the Evangelic University of Cameroon.

Abbreviations

CFU: Colony Forming Units
HACCP: Hazard Analysis Critical Control Point
MBS: Micro Biological Survey
MN: micronucleus
TC: Total Coliforms
WHO: World Health Organization

Data Availability

All relevant data are within the paper and supporting information files.

Funding Statement

This study was supported by Bread for the World (Brot für die Welt). https://www.brot-fuer-die-welt.de/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0245379.r001

Decision Letter 0

Swatantra Pratap Singh

6 Jul 2020

PONE-D-20-17828

Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia Faba in the West Region of Cameroon

PLOS ONE

Dear Dr. Mabvouna,

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Additional Editor Comments (if provided):

The article titled “Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia Faba in the West Region of Cameroon” is interesting, however, it has very high similarity with a previous article on "Microbiological quality of water sources in the West region of Cameroon: quantitative detection of total coliforms using Micro Biological Survey method". This article cannot be accepted in the present form and need Major revision in terms of presentation, methodology, and discussion. The manuscript lacks many technical details that require clarification and/or improvement. Authors are suggested to address the reviews comments in order to make the manuscript suitable for publication and provide point-by-point justification or rebuttal against the comments:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The work has been found to be very interesting, however there are some major suggestions with the respect to the design of the experiment and also discussions in the writeup:

1) Details about the sampling to be given in more depth

2) As the microbial load of the water is to be tested, the time of the day or even the temperature at the time of collection is crucial, please provide the data

3)Why E.coli, please explain the need for ennumeration of the indicator, depth in literature lacking especially in the introduction and discussion

3) Why an enzymatic assay like MBS, why not membrane filtration? Explicitly mention the advantages and the underlying principle and the rationale behind the choice of the method

4)Micronucleus test is a standard method, please cite where it has been adapted from.

5)Flowchart on the protocol for micronucleus test is recommended

6)22 samples may be too few to come up with a valid conclusion

Reviewer #2: Manuscript Number: PONE-D-20-17828

Title: Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia Faba in the West Region of Cameroon

Authors: Rodrigue Biguioh Mabvouna, Sali Ben Béchir Adogaye, Patrick Martial Pete Nkamedjie, Adrillene Laure Wondeu Deutou, Martin Sanou Sobze, Jean Blaise Kemogne and Vittorio Colizzi

Article Type: Research Article

Journal: PLOS ONE

Comments: The manuscript highlights the poor quality of domestic water sources in the Central African sub-region. The study deals with the microbial and genetic toxicity induced factors responsible for deteriorating water quality of different natural/domestic sources of drinking water. While this is worthy of investigation, the manuscript lacks in clarity of presentation, especially the methodology and discussion section. The manuscript provides a good read on detection of total coliforms (TC) which was done using the Micro Biological Survey-HACCP & water Easy test along with micronucleus test to assess the genetic toxicity caused due to the poor water quality. However, the approach of the study lacks many technical details that need clarification and/or improvement. Authors should improve the usage of the English language while revising the manuscript. Authors are suggested to address following comments in order to make the manuscript suitable for publication and provide point-by-point justification or rebuttal against each comment:

1. The content of abstract requires modification. Authors are suggested to make the abstract more clear and informative. Provide concise and factual abstract briefly and sequentially stating the aims (objectives), major results, and principal conclusions of the work. Derive the conclusion at last. Provide the data (values) for significant findings of the present study.

2. Abstract, line 29, and Study design and period, line 123, At each site,…genotoxic analysis. The word ‘litter’ should be replaced by ‘litre’ which is a unit of volume.

3. Introduction, lines 78-80, According to World Health Organization…between life stages. Please rephrase the sentence.

4. Total coliforms detection, Lines 130-131, what does it mean by rapid color change? Specify the changes in color corresponding to a definite range for the determination of increasing or decreasing coliform count.

5. Total coliforms detection, Lines 130-131, which reagent is the author talking about? Please mention.

6. Total coliforms detection, Lines 141-145, please mention the specific color change and concentration range (very high, high and low) for coliform count after 24 hours. Also clarify the reason for the reducing coliform counts with the passage of time.

7. Total coliforms detection, lines 141-145, A color change…103 CFU/ml. Please rephrase the sentence.

8. Of the 4 water sources sampled (as mentioned in study design and discussion), results of drilling water is not mentioned in Table 1. Further, results lack the complete details on TC concentration range along all the sources. Also, provide the coordinates for the sampling locations.

9. The reason for opting/choosing Vicia faba as a study plant is not mentioned in the methodology. Authors need to explain with proper rationale or any background study.

10. MN (micronucleus) test abbreviation must be given in the sub headings rather than providing details within the text body for better understanding of readers. Also brief introduction about MN test is not provided as given for Micro Biological Survey-HACCP & water Easy test for coliforms. Please include.

11. The major criticism of the manuscript lies in the lack of technical details provided for the methodology adopted. Authors are suggested to make this section clearer and explanatory. How well water is supposed to be more contaminated with higher coliform count during rainy reason and not in the surface water source? Explanation provided is not satisfactory and subject to contradiction. More meaningful and concrete justification is required.

12. Discussion, TC concentration detected highest among wells but later genotoxicity effects has been reported in spring water, river water as well as well water. What could be the possible reason for such differences? Authors need to address the same.

13. What specific genotoxicity effects do author referring to?? Whatever genetic changes/ mutation detected in the experiments is nowhere mentioned in the manuscript. Please clarify.

14. Discussion, line 215, “However, the TC concentration of untreated source water was found to be higher than treated water sources with no significant difference” – the sentence itself showing significant differences in TC concentration. Please rephrase suitably.

15. Discussion, lines 217 – 219, (Other standardized……..environmental pollutions) – past studies and future need for more such studies needs to be differentiated well. Rephrase the sentence carefully.

16. Discussion, line 232, intensive agricultural activities instead of “intensives”. Please check the whole manuscript for other grammatical errors.

17. The conclusion section must state the key findings of the study. Authors are suggested to draw major inferences/primary conclusions first from the results followed by the secondary conclusions reached through the critical analysis.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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PLoS One. 2021 Feb 4;16(2):e0245379. doi: 10.1371/journal.pone.0245379.r002

Author response to Decision Letter 0

13 Oct 2020

Responses to general comments

We are not the first authors of the laboratory protocols used in this study. We have added the references of these protocols in the main manuscript to allow reproducibility of the results.

A marked-up copy of the manuscript highlighting changes made to the original version was prepared and uploaded separately. An unmarked version of the revised paper without tracked changes was also submitted.

Our financing statement remains unchanged.

Academic Editor

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Author’s responses: We revised the manuscript according to PLOS ONE style requirements, including file naming.

Author’s responses: A table with sampling location names, water source categories, geographic coordinates and date of sample collection was added in the methods section.

3. We note that [Figure(s) 1] in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth).

Author’s responses: We are the authors of the figure 1. We have added details of the software used to generate the figure in the revised version of the manuscript.

4. Please ensure that you refer to Figure 3 in your text as, if accepted, production will need this reference to link the reader to the figure.

Author’s responses: The correction has been done. There was confusion between tables and figures.

5. Please include a copy of Table 2 and 3 which you refer to in your text on page 17 and 18 (there are two tables uploaded named as Figure 2 and Figure 3).

Author’s responses: The correction was done.

Author’s responses: The ethics statement was added in the Methods section and included in the manuscript.

7. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Author’s responses: We change our declaration; the data sets used and/or analysed during the current study are available without restrictions.

8. In your revised cover letter, please address the following prompts:

Author’s responses: There are no ethical or legal restrictions on sharing data set. Data can be viewed without restrictions.

Author’s responses: We changed our Data Availability statement and we will upload the study data set as Supporting Information files.

Additional Editor Comments

Author’s responses: Here, we provide a point-by-point response to the comments made by reviewers.

In the first study "Microbiological quality of water sources in the West region of Cameroon: quantitative detection of total coliforms using Micro Biological Survey method" we applied the MBS method as simple and rapid test to check water potability. In this second study, we combined the MBS and micronucleus test using Vicia faba seeds to propose an approach for bacteriological and chemical routine quality check of drinking water which can be easily implemented in developing countries.

Comments to the Author

1. Is the manuscript technically sounds, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Partly

Author’s responses: All experiments conducted in this study were done according the standard's protocols (citations added in the main manuscript). We have further commented on our results to support our conclusion.

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Author’s responses: Statistical analyses were done using Stata IC/15.0 software (College Station, Texas 77845 USA, http://www.stata.com), and the main results were presented and interpreted.

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Authors responses: Nothing to add

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: No

Author’s responses: The manuscript was submitted to an English-speaking person for language revision and improves its quality.

5. Review Comments to the Author

Authors responses:

Reviewer reports

Reviewer #1

The work has been found to be very interesting; however, there are some major suggestions with the respect to the design of the experiment and also discussions in the write-up:

1) Details about the sampling to be given in more depth

Author’s responses: More details in the sampling procedure of water sources were added in the manuscript. Water was collected (in the morning before 12 o’clock), we didn’t apply a statistical sampling method to identify water source to be collected. The sources of water were identified step by step according to their availability, accessibility, and their use as drinking water by the local populations.

2) As the microbial load of the water is to be tested, the time of the day or even the temperature at the time of collection is crucial, please provide the data

Author’s responses: The date and time of water sampling were provided in the manuscript. Temperature and pH of water were measured before leaved the site. The temperatures and pH didn’t significatively vary between water sources. The averages values of both measurements were given in the results section.

3) Why E. coli, please explain the need for enumeration of the indicator, depth in literature lacking especially in the introduction and discussion

Author’s responses: We didn’t enumerate E. coli but Total Coliforms bacteria which include Escherichia coli (major species in the faecal coliform group). The MBS test doesn’t permit to clearly identify the bacteria present in sample water. We provided the justification of using TC as water pollution indicator in the manuscript.

4) Why an enzymatic assay like MBS, why not membrane filtration? Explicitly mention the advantages and the underlying principle and the rationale behind the choice of the method

Author’s responses: We mentioned in the main manuscript the advantages of MBS and cited previous studies which applied this test for water analysis in Africa and in Cameroon.

5) Micronucleus test is a standard method, please cite where it has been adapted from

Author’s responses: We provided the citation in the text.

6) Flowchart on the protocol for micronucleus test is recommended

Author’s responses: We described all the steps of the MN analysis in the Methods section and we cited the source of the protocol. We think that these are sufficient for the results reproducibility.

7) 22 samples may be too few to come up with a valid conclusion

Author’s responses: As we mentioned above, the sources of water to be sampled were identified step by step according to their availability, accessibility. It is also important to note that we are in context of water scarcity.

Reviewer #2

Author’s responses: We did our best to address the comments and improve the quality of the manuscript.

Author’s responses: The abstract was revised as recommended.

2. Abstract, line 29, and Study design and period, line 123, At each site,…genotoxic analysis. The word ‘litter’ should be replaced by ‘litre’ which is a unit of volume

Author’s responses: Done

3. Introduction, lines 78-80, According to World Health Organization…between life stages. Please rephrase the sentence

Author’s responses: Done

Author’s responses: A rapid color change (from red to yellow) indicates a very high concentration of coliforms in the water sample. We rephrased the sentence. The change in color according to the time of analyses and the corresponding concentrations of coliforms are given in the main manuscript.

5. Total coliforms detection, Lines 130-131, which reagent is the author talking about? Please mention

Author’s responses: We are talking about test, not a reagent. The MBS kit comes in a pack containing all the material necessary for the analysis without need additional reagent. More details on MBS-HACCP & water Easy test are given in the main manuscript.

6. Total coliforms detection, Lines 141-145, please mention the specific color change and concentration range (very high, high, and low) for coliform count after 24 hours. Also clarify the reason for the reducing coliform counts with the passage of time.

Author’s responses: There is one change in color from red to yellow (contamination). The results were interpreted according to the standard protocol. We specified the level of Coliforms concentration according to the time required for the color change. 12h = very high contamination; 19h = high contamination and 24h = low contamination. The color change is proportional to the concentration of coliforms in the water samples, the greater the number of microorganisms, the more rapid the change of color, thus, a positive result (contamination).

7. Total coliforms detection, lines 141-145, A color change…103 CFU/ml. Please rephrase the sentence.

Author’s responses: Done

Author’s responses: Results of drilling water were mentioned; we used a synonym, now we cleared mentioned drilling water in all tables. We provide all result details available when using MBS method. The most important result is the indication of whether TC bacteria are present or not and the level of concentration. The coordinates of sampling locations were provided in the manuscript.

9. The reason for opting/choosing Vicia faba as a study plant is not mentioned in the methodology. Authors need to explain with proper rationale or any background study.

Author’s responses: The reasons for opting/choosing Vicia faba were given in the Methods section.

Author’s responses: Done

Author’s responses: Additional details on the methods used were given, and we specified (and cited) that we used the standard protocols. The analyses used in this study are simple to execute and don’t require sophisticated instruments or additional reagent (MBS method). We provide all the steps of our methodology necessary for the reproducibility of the results.

We are talking about concentration (quantity) of bacteria in well water. A previous study conducted in the same area (Bafoussam), during the same season found similar results. We think that bacteria growth is most high in groundwater than surface water (running water), and the combined effect of temperatures (from 22 to 26 °C) and pH (5.5 to 8.3) could favour bacteria growth. The proximity between wells and latrines could also favour contamination (by runoff water or slick phreatic communication). We added other justifications in the manuscript.

12. Discussion, TC concentration detected highest among wells, but later genotoxicity effects has been reported in spring water, river water as well as well water. What could be the possible reason for such differences? Authors need to address the same.

Author’s responses: There is no relation between genotoxicity and TC concentration, and results are coherent. If genotoxic effect is detected in water this indicates the presence of chemicals (disinfectant, heavy metal, etc.), and generally there are toxic for bacteria or limit bacteria growth (low concentration). Finally we noted in the study that some wells were chlorinated (that’s why 1 ml of Thiosulphate was added to water sample to neutralize the chlorine), so it’s consistent if genotoxic effects are also detected in well water.

13. What specific genotoxicity effects do author referring to?? Whatever genetic changes/ mutation detected in the experiments is nowhere mentioned in the manuscript. Please clarify.

Author’s responses: Micronucleus as mutagenesis endpoint (chromosome fragments). We clarified in the manuscript.

Author’s responses: No statistically significant difference at the significance level set. We rephrased.

Author’s responses: Done

16. Discussion, line 232, intensive agricultural activities instead of “intensives”. Please check the whole manuscript for other grammatical errors

Author’s responses: Done

Authors responses: Done

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(55KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0245379.r003

Decision Letter 1

Swatantra Pratap Singh

25 Nov 2020

PONE-D-20-17828R1

PLOS ONE

Dear Dr. Mabvouna,

Please submit your revised manuscript by 15th Dec 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Swatantra Pratap Singh, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The article has been improved, however few miner corrections are required before publication as suggested by the reviewers.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The work done is interesting however some suggestions on improving the clarity of the writeup is recommended:

Line 130: 1 mL of what concentration of thiosulphate? Is it sodium thiosulphate, if yes please specify, if not please mention the salt name explicitly

Line 133: Mention explicitly the temperature and pH were taken before or after placing the samples in the ice box

Table 1: The temperature mentioned is for the water or ambient air temperature? Also, mentioning the time of collection is suggested

Line 160-164: Add the reference

For the section of total coliform detection: The test details insufficient, which enzymes play a crucial role in the color change? More details on the mechanism of color change needs to be explained to understand how the kit works?

Line 219: Why so much variation in the pH, was it because the samples were collected at different time frames of the day, was the time of collection not kept constant? Or was there appearance or disappearance of algal blooms?

Reviewer #2: The manuscript has been revised in accordance with the comments provided. Authors have also satisfactorily answered and clarified the doubts raised. The revised manuscript is now suitable for acceptance and publication in the Journal.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2021 Feb 4;16(2):e0245379. doi: 10.1371/journal.pone.0245379.r004

Author response to Decision Letter 1

4 Dec 2020

Additional Editor Comments (if provided):

The article has been improved; however few miner corrections are required before publication as suggested by the reviewers.

Author’s responses: Corrections as suggested by the reviewers have been done (see below).

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author:

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Author’s responses: nothing to report

________________________________________

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Author’s responses: We added more information in the revised manuscript as requested by Reviewer #1

________________________________________

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Author’s responses: nothing to report

________________________________________

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: No

Reviewer #2: Yes

Author’s responses: We already change our data availability statement: Data used for this study are fully available without restriction and the data set was uploaded as supporting information during the revision process.

________________________________________

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Author’s responses: nothing to report

________________________________________

6. Review Comments to the Author

Author’s comments:

It’s not a dual publication. As we already responded, in the study "Microbiological quality of water sources in the West region of Cameroon: quantitative detection of total coliforms using Micro Biological Survey method" we applied the MBS method as simple and rapid test to check water potability. In this second study, we combined the MBS and micronucleus test using Vicia faba seeds to propose an approach for bacteriological and chemical routine quality check of drinking water which can be easily implemented in developing countries.

Regarding the research ethics, or publication ethics, our manuscript does not report on or involve the use of any animal or human data or tissue, so an ethics approval and consent are not required with reference to Order No. 079/A/MSP/DS of the Minister of Public Health of October 22, 1987 establishing and organizing an Ethics Committee on Research Involving Human Beings (article 2).

Reviewer #1:

The work done is interesting however some suggestions on improving the clarity of the writeup is recommended:

1. Line 130: 1 mL of what concentration of thiosulphate? Is it sodium thiosulphate, if yes please specify, if not please mention the salt name explicitly

Author’s responses: It is 1 ml of Sodium Thiosulfate solution 10% (0.25g/50ml). We specified in the revised manuscript.

2. Line 133: Mention explicitly the temperature and pH were taken before or after placing the samples in the ice box

Author’s responses: Done: Temperature and pH of water were measured on site before placing the samples in the ice box. We mentioned explicitly it the revised manuscript.

3. Table 1: The temperature mentioned is for the water or ambient air temperature? Also, mentioning the time of collection is suggested

Author’s responses: The temperature mentioned is for the water samples, we specified it in the revised manuscript. We did not accurately record the time of sample collection, but as mentioned in the revised manuscript, sample collection was done in the morning before 12 o’clock.

4. Line 160-164: Add the reference

Author’s responses: Done!

5. For the section of total coliform detection: The test details insufficient, which enzymes play a crucial role in the color change? More details on the mechanism of color change needs to be explained to understand how the kit works?

Author’s responses: MBS method is a colorimetric reaction system based on the measurement of the catalytic activity of oxidoreductase enzymes of primary metabolism, which allows you to determine a correspondence between microorganism and enzyme activity in the sample. This precision was added in the revised manuscript.

6. Line 219: Why so much variation in the pH, was it because the samples were collected at different time frames of the day, was the time of collection not kept constant? Or was there appearance or disappearance of algal blooms?

Author’s responses: Some factors can explain the large variation of the water pH, such as time of samples collection. But think that we have minimized this influence since all the samples were collected in the same period (in the morning before 12 o'clock). Rather, we think that this variation of the pH may be due to the difference in depth of the water sources (wells, water drilling) or to the composition of the underground rock. We will take these factors into account in future studies.

Author’s responses: nothing to report

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Author’s responses: nothing to report

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(43KB, doc)}

PLoS One. doi: 10.1371/journal.pone.0245379.r005

Decision Letter 2

Swatantra Pratap Singh

30 Dec 2020

PONE-D-20-17828R2

Dear Dr. Mabvouna,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Swatantra Pratap Singh, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

As per reviewers comments and we please to accept the article for the publication.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The paper has covered a very important topic and the authors have tried and included all the revisions suggested by the reviewers

Reviewer #2: The manuscript has been revised suitably. The revised manuscript is now suitable for acceptance and publication in the Journal.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

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PLoS One. doi: 10.1371/journal.pone.0245379.r006

Acceptance letter

Swatantra Pratap Singh

12 Jan 2021

PONE-D-20-17828R2

Dear Dr. Mabvouna:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Swatantra Pratap Singh

Academic Editor

PLOS ONE

Associated Data

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Supplementary Materials

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Data Availability Statement

All relevant data are within the paper and supporting information files.

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PERMALINK

Microbiological quality and genotoxicity of domestic water sources: A combined approach using Micro Biological Survey method and mutagenesis assay (micronucleus test) in root tips of Vicia faba in the West region of Cameroon

Rodrigue Biguioh Mabvouna

Sali Ben Béchir Adogaye

Patrick Martial Pete Nkamedjie

Andrillene Laure Deutou Wondeu

Martin Sanou Sobze

Jean Blaise Kemogne

Vittorio Colizzi

Roles

Abstract

Introduction

Materials and methods

Study location

Study design, period and water sampling

Table 1. Sampling locations, water sources categories, sample temperature (°C), geographic coordinates, and samples collection dates.

Total Coliforms detection

Micronucleus (MN) test

Mutagenesis assay in Vicia faba root tips

Mutagenesis assay operating procedures

Ethical approval

Results

Microbiological quality

Table 2. Total Coliforms concentration in water samples, level of contamination and time of analysis, West region, Cameroon, 2018.

Chemical quality

Fig 1.

Table 3. Mean per 5000 cells ± Std. Err. of mitotic cells and mitotic index in root tips of Vicia faba exposed to water samples, West region, Cameroon, 2018.

Table 4. Mean per 5000 cells ± Std. Err. of MN frequency in root tips of Vicia faba exposed to water samples, West region, Cameroon, 2018.

Discussion

Conclusion

Supporting information

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Decision Letter 0

Swatantra Pratap Singh

Roles

Author response to Decision Letter 0

Decision Letter 1

Swatantra Pratap Singh

Roles

Author response to Decision Letter 1

Decision Letter 2

Swatantra Pratap Singh

Roles

Acceptance letter

Swatantra Pratap Singh

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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