Urban mosquitoes and lamentous green algae: their biomonitoring role in heavy metal pollution in open wastewater channels in Nairobi, Kenya

Levels of Mercury (Hg), Lead (Pb), Chromium (Cr), Cadmium (Cd), Thallium (Tl), and Nickel (Ni) in samples of wastewater, lamentous green algae (spirogyra) and urban mosquitoes obtained from open wastewater channels in Nairobi industrial area, Kenya, was established. Industrial wastewater may contain hazardous heavy metals upon exposure. Aquatic organisms in wastewater may accumulate the toxic elements with time. Therefore, human population living in informal settlements in Nairobi industrial area risk exposure to such toxic elements. Biomonitoring using aquatic organisms can be key in metal exposure assessment.


Background
The wide application of heavy metals has raised concerns over their potential disadvantageous effects on human health and environmental modi cation [1]. Environmental pollution by heavy metals has been associated with mining, foundries, smelters, and other metal-based industrial operations [2].
Disadvantageous health effects associated with heavy metals in exposed humans and animals range from cancer, systems disorders, developmental anomalies, neurologic and neuro-behavioral disorders, hematologic disorders, DNA damage, cellular and tissue damage, and gastrointestinal toxicity [3,4,5,6,7]. According to Tchounwou and his colleagues [1], heavy metals toxicity depends on their dose, route of exposure, chemical property as well as age, gender, genetics, and nutritional status of the exposed individuals.
Biological monitoring of water quality involves use of aquatic organisms to detect the pollutants [8]. For instance, heavy metals have previously been reported in mosquito larvae [9]. Biomonitoring of aquatic pollutants using Culex mosquito larvae is advantageous because their larvae are common in urban areas where pollution level is likely to be high, and secondly, the Culex larvae proliferate fast and have a su cient developmental interval which gives time for heavy metal uptake [10,11,12].
According to Kitvatanachai and others [12], the routine collection of urban mosquitoes for medical research can also avail appropriate samples for monitoring environmental pollution by heavy metals. The uptake of pollutant metals by the mosquito larvae inhabiting contaminated water may occur through direct body absorption or indirectly through ingesting heavy metals contaminated materials. While adult mosquitoes suck nectar, honey and animal blood, their larvae lter algae and other plant materials from the water. However, the larvae of Toxorhynchites mosquitoes are predacious and feed on the larvae of other mosquito species, but in absence of a suitable prey they may feed on detritus or exhibit cannibalism [13]. According to Marten [14], abundance of algae usually provides favorable conditions for mosquito proliferation. Spirogyra lamentous algae which usually forms mats in the water serve as mosquito larvae food [15,16]. Some species of algae however, including those in the order Chlorococcales and the blue green algae (Cyanobacteria) have larvicidal effect because they are indigestible and toxic to mosquito larvae respectively [14]. Hexane and chloroform extracts from marine Phaephyta algae (Padina gymnospora) have been reported to display larvicidal activity against Aedes aegypti [17].
Certain species of algae have been reported to uptake heavy metals from contaminated water through biosorption and bioaccumulation [18]. Such species can therefore be used as indicators of the extent of water pollution and in removing pollutants from the wastewater, a process known as phytoremediation. Phytoremediation has emerged as a desirable technology which uses plants for removal of environmental pollution [19]. Both micro and macro algae have been shown to uptake heavy metals from contaminated water naturally and from experimental solutions in the laboratory [20,21]. Aquatic organisms in the lower trophic levels are better tools for natural biomonitoring of metal since they are among the rst in the food chain to be exposed to the pollutants [22]. The heavy metals taken up by the aquatic producers ow into the consumers in a food web through the various aquatic food chains.
The current study was therefore designed to establish the levels of heavy metals in samples of wastewater, lamentous algae (Order Zygnamatales: Genus Spirogyra) and mosquitoes (Order Diptera: Family Culicidae) both larvae and adults, that were obtained from open wastewater channels and the immediate vicinity in Nairobi industrial area, Kenya. The metallic elements studied were chromium (Cr), cadmium (Cd), mercury (Hg), lead (Pb), nickel (Ni), and thallium (Tl).

Results
Physico-chemical parameters of wastewater samples: The mean range for pH, temperature, total dissolved solids (TDS) and electrical conductivity (EC) of the wastewater samples were 7. 28  respectively. Both TDS and EC recorded were above the recommended limits by WHO ( Table 1). The physico-chemical parameters observed and recorded in the current study from the eight sampling sites differed signi cantly (F-test, P < 0.05).

Levels of heavy metals in samples of wastewater and tap water:
The Pb levels were highest ranging from 13.62 to 15.31 ppb, followed by Ni (4.96 to 6.91 ppb) and the lowest was Tl at 0.05 ppb. The mean concentrations of the heavy metals in acid digested wastewater samples followed an ascending order of Tl < Hg < Cd < Cr < Ni < Pb (Table 2). Mean concentration of Cr (7.49 ± 2.12 ppb) in wastewater samples that were not digested with acids was signi cantly higher than for the other elements studied ( Table 2). The mean concentrations of Pb and Cr in acidi ed wastewater samples were above the limits set by WHO, US EPA and Kenya. The levels of Hg, Cd, and Ni in acidi ed wastewater samples were below the limits set by WHO and Kenya. The level of Hg in wastewater samples was above the US EPA limit which is set at 0.00003 ppm (0.03 ppb). The mean concentration of thallium in wastewater was 0.04 ppb but standard limits for WHO, Kenya and US EPA were missing in the literature accessed. The mean concentrations of Hg, Pb, Cr, Cd, Tl and Ni in samples of tap water ranged between 0.01 to 0.2 ppb which were far below the standard limits set by WHO, US EPA, and Kenya ( Table 2).

Levels of the selected heavy metals in lamentous green algae
Filamentous green algae were sampled from 4 out of 8 (50 %) sampling sites ( Table 3). The mean concentration of heavy metals in the samples of wastewater and in lamentous green algae collected from the same site differed signi cantly (P > 0.05). The average heavy metal concentrations in lamentous green algae samples were between 500 to 5000 times more than the mean concentration of the same metals in wastewater samples in the same sampling site (Tables 2 and 3). The mean concentrations of heavy metal in lamentous green algae followed an ascending order of Hg < Tl < Cd < Ni < Cr < Pb and ranged from 0.057 to 110.62 ppm ( Table 3). The algae samples obtained from Railways Lower (D) and Davis & Shirtliff (E) sampling sites had signi cantly higher levels of heavy metals (P < 0.05) compared to those collected from Kartasi sampling sites (F1a & F1b) as shown in Table 3. Concentrations of Hg, Pb, Cr, Cd and Tl were 1.93 to 2.75 times higher in room temperature dried algae samples (that is before metal analysis) than in lyophilized algae samples ( Table 3). The Ni level was however higher in lyophilized algae samples than in the room temperature dried algae samples ( Table 3). The mean concentration of heavy metals in algae samples obtained from open wastewater channels were above the limits set for plants (vegetables) by WHO, Kenya and US-EPA except for thallium where the standard limits were missing in the literature accessed ( Table 3).

Levels of heavy metals in mosquito samples
There were no adult mosquitoes trapped at site C; and similarly, no mosquito larvae were available for sampling at sites B, F and H. However, the mean concentrations of Hg, Pb, Cr, Cd, and Ni in adult Culex mosquitoes' samples collected from Donholm site (H) was signi cantly higher than the means for the same elements at Kartasi site (F) as shown in Table 4. Similarly, the mean concentrations of Hg, Pb, Cr, Cd, Tl, and Ni in mosquito larvae samples collected from Sinai site (G) were signi cantly high ( Table 4).
The mean concentration of heavy metals in eld mosquitoes' samples followed an ascending order of Tl < Hg < Cd < Ni < Pb < Cr while that for the laboratory reared mosquito samples was Tl < Hg < Cd & Ni < Cr < Pb (Table 4). The mean concentration of Pb, Cr, Tl, and Ni in assorted eld mosquito samples were 1.3 to 2.4 times more than the mean concentration in the assorted laboratory reared mosquito samples. The mean concentrations for Hg (0.26 mg/L) and Cd (1.8 mg/L) in assorted laboratory-reared eld mosquitoes were 4.4 and 20 times more respectively than in assorted eld mosquitoes which were 0.059 mg/L (Hg) and 0.09 mg/L (Cd) as shown in Figure 1 and Table 4. The mean concentration of Pb, Cr, Cd, Tl, and Ni at Kartasi sampling site, in lamentous algae samples was 3 to 29 times higher than in assorted eld mosquito samples (Tables 3 and 4). The level of Tl was below the method detectable level which was set at 0.02 ppm in both the assorted eld and laboratory reared mosquito samples ( Figure 4 and Table 4).
The mean concentration of Pb, Cr, and Ni in both assorted eld and assorted laboratory-reared mosquitoes' samples ranged from 2.33 to 10.53 ppm, which was above the WHO permissible limits (0.5 to 2.0 ppm) for freshwater sh ( Table 4). The level of Hg in both assorted eld and assorted laboratoryreared mosquitoes' samples ranged from 0.06 to 0.26 ppm and was below the WHO permissible Hg levels (0.5 ppm) for freshwater sh. Similarly, the mean concentration of Cd in assorted laboratory-reared mosquito samples was 1.8 ppm and was above the WHO permissible Cd limits (1.0 ppm) for freshwater sh while the mean Cd level in assorted eld mosquito samples was 0.09 ppm (Table 4). WHO permissible limits for thallium in freshwater sh were missing in the literature accessed, and therefore no comparisons were made.
Correlation of the heavy metal levels in wastewater, algae, and mosquito samples Pairs of heavy metal concentrations such as Pb & Hg; Cd & Cr; Tl & Hg in algae samples correlated strongly, positively, and signi cantly (Table 5), where an increase in one element corresponded to an increase in the partner element in the pair of metals analyzed. Similarly, concentration of Tl & Cd in wastewater samples correlated positively (Table 5) (Table 5). Table 6 Table 6.

Discussion
According to Azam and others [39], insects are the dominant invertebrate faunal group that has been used in biomonitoring and bio assessment studies. This is because insects have a strong relationship with ecology [40]. Insects are abundant and they possess diverse morphologies and functions which enable them to display unique biochemical and genetical responses after their exposure to environmental changes including pollutions. In the current study, eld urban mosquitoes were evaluated for their possible role of bio-indication for heavy metal pollution in wastewater since they frequently encounter such water in open channels when accomplishing their life processes including feeding and reproducing. The results showed that the eld urban mosquito samples, majority of which belonged to Culex species as previously reported [41], had high levels of Pb and Cr compared to the laboratory-reared Anopheles and Aedes control mosquitoes that were reared in KEMRI -Nairobi, Kenya. This implied that the mosquitoes that breed in contaminated wastewater channels may have absorbed and accumulated Pb and Cr into their body tissues. This observation was supported further by establishing that the wastewater samples from the open channels from which the mosquito larvae were obtained had higher levels of the heavy metals when compared to tap water ( Table 2). Dechlorinated tap water is used for rearing of mosquitoes at KEMRI, Nairobi. Previous studies have shown that Culex mosquito larvae can be tools for natural biomonitoring of heavy metals since they are among the rst in the food chain to be exposed to the heavy metal pollutants [22,42]. It was also observed in the current study that the laboratory-reared mosquito samples had a slightly higher level of metals including Hg, Cd & Ni (Table 4). This could have been probably attributed to the rearing processes, equipment used, insectary, insect feed, and routine procedures in the rooms adjacent to the insectary where the rearing of mosquitoes took place. According to van der Fels-Klerx and colleagues [43], insects can become exposed to chemical hazards from the substrate used to grow them. Some of the heavy metals are known to escape into the air as tiny particulates [44] which would then easily contaminate the insectary and the mosquitoes being reared.
Laboratories have been associated with increased concentration of speci c pollutants depending on the nature of experiments that are being conducted [45]. In a study on indoor air quality in research laboratories, Valavanidis and Vatista [46] established that respirable suspended particulates (RSP) reached 700 µg/m 3 in spring and summer period. Similarly, Rumchev and colleagues [47] in their study on indoor air quality in 15 university laboratories established that the particulate matter (PM 2.5 and PM 10 ) were signi cantly high in Chemistry, Engineering and Biology laboratories. Suspended particulates in the air may include black carbon, heavy metals, spores, dust, pollen grains, liquid aerosols among others, and they tend to be in large quantities in heavily polluted areas and premises.
The mean concentration of heavy metals was highest in lamentous green algae, followed by eld mosquito larvae or adult mosquito samples and lowest in wastewater, giving an ascending sequence of wastewater < mosquitoes < lamentous algae. At Kartasi sampling site, the mean concentration of Pb, Cr, Cd, Tl, and Ni in lamentous algae samples was 3 to 29 times higher than in assorted eld mosquito samples. This observation was in line with a previous study carried out by Kitvatanachai and others [12] which showed that the levels of Pb was higher in Cx quinquefasciatus than in wastewater from the factories and the areas close to the factories. Aquatic insects accumulate heavy metals in their bodies from contaminated aquatic ecosystems because they become exposed during their vital developmental stages and processes including embryogenesis, larval development, and pupation [48,49]. The emerging and surviving imagoes of aquatic insects are therefore likely to have elevated levels of heavy metal in their bodies as well. In our current studies, eld assorted mosquitoes had high concentration of Pb and Cr when compared to the assorted laboratory-reared mosquitoes. Previous studies though, have shown that the process of metamorphosis can be a survival challenge for aquatic insects in metal contaminated aquatic ecosystems because the larvae become exposed to extra stress that enhance the mortality of the imagoes [50]. The urban mosquitoes, majority of which are Culex pipiens [41] can breed in wastewater, although when exposed to increased speci c heavy metal concentration, their breeding potential is reduced [51]. According to Dom and colleagues [52], the Aedes mosquitoes, the key dengue vectors appear to develop adaptations to cope with increased heavy metal concentration in polluted waters. The urban mosquitoes that can breed and survive in polluted waters especially in crowded areas are therefore a health hazard because they can serve as vectors of infectious diseases as well as pollutants contaminated blood sucking insects. It was also shown in our study that the mean levels of Pb, Cr, and Ni in assorted eld mosquito samples, which comprised of adults and their larvae, were above the WHO permissible levels for freshwater sh. This agreed with a previous study that reported an increased Pb levels in Cx. quinquefasciatus mosquito larvae that were obtained from Pb-contaminated wastewater [12]. Both the assorted eld and assorted laboratory-reared mosquito samples had mean Hg levels that were below the WHO permissible levels for freshwater sh. This was in line with a study carried out in North America in which the methylmercury in mosquitoes was a little less than that typically found in sh [53].
From previous studies, algae belonging to the genus spirogyra have a signi cant potential absorbent for heavy metal from contaminated water [54,55]. Our current study established that the mean concentration of heavy metals in lamentous green algae was 500 to 5000 times more than the mean concentration of the same metals in the wastewater samples collected from the same site. According to Sunish and Reuben [56], lamentous algae in the mosquito breeding water have nutritive value necessary for mosquito development and adult emergence. Therefore, when the mosquito larvae feed on heavy metal contaminated lamentous algae, the heavy metals may get transferred into their tissues. Feeding process is one of the main pathways through which aquatic invertebrates obtain metals from their surroundings [53,57]. Our study clearly illustrates occurrence of bioaccumulation of heavy metals in the mosquitoes and aquatic lamentous algae inhabiting contaminated open wastewater channels in Nairobi industrial area, Kenya. This was in line with previous studies which established that contaminated wastewater could lead to a build-up of heavy metals in soils, food crops and macrophytes [58,59]. According to Gokce [60], use of algae for environmental biomonitoring can be advantageous and suitable because algae is spatially dense, easy to sample where available and store. Similarly, mosquitoes can breed rapidly in stagnant water and are easy to sample, especially the larvae.
Inter-elemental analysis of the metals in the different samples collected from the channels revealed several strong, positive, and signi cant correlations. Such pairs included Pb & Hg; Cd & Cr; Tl & Hg in algae samples. These correlations suggested that the pairs of the metals may have had a common source, most likely the industries whose wastes were draining into the open channels in the study area. Such industries probably were releasing speci c wastes that were rich in certain elements, hence a positive correlation of such elements. This explanation was in line with previous studies carried out in Nigeria and Pakistan [61,62]. The signi cant correlation coe cients between pairs of metals in samples of wastewater, lamentous algae and mosquito larvae strongly suggested that the sources of the heavy metal pollution in the study area was mainly anthropogenic.
Our current study raises a few public health implications such as, people can easily become exposed to heavy metal pollutants when clearing and unblocking the wastewater channels when they clog. Prolonged heavy metal exposure can lead to serious toxicity and exposure to potential carcinogenic agents in humans [63]. The heavy metal contaminated wastewater pollutes the surface runoffs after the rains, which then spread the pollutants into the residential areas, soils, crops and public places.
Contaminated wastewater may over ow from the channels onto the highways during the heavy rains hence exposing the road users to the pollutants. The mosquitoes that breed successfully from contaminated wastewater channels may accumulate heavy metals in their bodies with time through direct diffusion of such metals into their bodies or by ingesting heavy metal contaminated plant materials that includes algae. Such mosquitoes may therefore serve as both disease vectors as well as pollutants contaminated piercing and blood sucking insects. Studies to verify whether mosquitoes with elevated heavy metals in their tissues can spread such elements through their bites are however lacking. Such a study can involve comparing the levels of heavy metals in salivary glands of mosquitoes exposed and those not exposed. In India, wastewater has been used for microalgae cultivation for biofuel production [64]. The current study has shown that algae present in contaminated wastewater absorbs and accumulates the pollutants, in this case heavy metals. The levels of heavy metals in the algae were higher than in wastewater due to bioaccumulation in the current study. Therefore, harvesting microalgae grown in contaminated wastewater can be a health hazard especially where the level of ignorance and poverty levels are high and inadequate use of safety measures when handling such algae. In Kenya, commercial cultivation of microalgae for biofuel production is still facing many challenges [65] and even when this economic activity picks, use of untreated wastewater to cultivate the microalgae should be highly discouraged.

Conclusion And Recommendation
The heavy metal concentration in the samples analysed followed an ascending sequence of wastewater < mosquitoes < lamentous algae. The mean concentration of Pb, Cr, and Ni were relatively higher than those of Tl, Hg, and Cd in wastewater, lamentous algae, and eld mosquito samples. The mean concentration of the heavy metals in eld mosquito samples followed an ascending order of Tl < Hg < Cd < Ni < Pb < Cr. The levels of Pb, Cr, Tl, and Ni in assorted eld mosquito samples was signi cantly higher than in assorted laboratory-reared mosquito samples. The assorted laboratory-reared mosquito however had slightly higher levels of Hg and Cd when compared to the assorted eld mosquitoes. The levels of Pb, Cr, and Ni in both the eld and laboratory reared mosquitoes were above the WHO permissible limits for freshwater sh. The concentration of Pb, Cr, Ni, and Cd in wastewater were above the limits set by WHO, Collection and preparation of wastewater samples: A standard 350 mL dipper was used to collect the wastewater samples from the open channels and placed into clean reagent plastic bottles. The samples were collected in triplicates in equal portions. Two separate portions were separately digested with concentrated hydrochloric (HCL) acid and concentrated nitric (HNO 3 ) acid respectively by adding three drops of the respective acid per 100ml of wastewater sample. Acidi cation was meant to inhibit adsorption of dissolved elements onto the interior walls of the plastic bottles as well as preventing microbial reactions [66]. A third portion of wastewater was not acidi ed to act as a control. Similarly control triplicate samples of tap water were also collected from selected sites in the study area, two portions were acidi ed while one portion was not. All the samples were labeled appropriately, packaged, and stored in low temperature.
Measuring the physico-chemical parameters of water samples: The physico-chemical parameters of the wastewater samples including temperature, pH, electrical conductivity (EC), and total dissolved solids (TDS) were measured immediately after collection of wastewater samples at the site using a digital electronic device (HANNA Instruments, H1991300, Romania) and recorded appropriately.
Collecting samples of lamentous green algae: Filamentous green algae ( Figure 3) were collected in triplicates from the open wastewater channels using a large plastic strainer and packaged in well labeled brown paper bags. The strainer was then rinsed in deionized water before being used again. All the samples collected were transferred to Kenyatta University Biochemistry laboratory for further processing.
Preparation of green algae samples for heavy metal analysis: The lamentous green algae samples were divided into two parts. One part was air dried at room temperature for several days while the remaining part was lyophilized (freeze dried). Both air dried and lyophilized algae samples were ground and sieved to obtain a ne powder as described by Ngure & Kinuthia [67]. The powder was then weighed and packaged in well labeled brown small envelops to await metal analysis. Brie y, lyophilization involved extracting the algae samples using de-ionized water for 36 hours on an electrical shaker, followed by ltering the extract obtained using clean muslin cloth on a water pump. About 200 ml of the ltrate was then put on clean stainless-steel tray and placed in the deep freezer for 24 hours at negative 45 o C. The samples were then retrieved and placed in a freeze-drier for a further 24 hours at negative 50 o C to complete lyophilization.
Outdoor trapping of adult mosquitoes: Adult mosquitoes were trapped using surveillance standard Centers for Disease Control and prevention (CDC) light traps as described by Mweya and colleagues [68] using carbonated dry ice as the bait. The traps were set in potential breeding sites and amidst the vegetation where applicable (Figure 4) within the factory premise. The trapping commenced from 6:00 PM to 6:00 AM each day. The average number of CDC traps set per sampling site per night was seven depending on the size of the compound. The mosquito trapping activity was carried out daily for two weeks. The eld mosquitoes were trapped near the sites where wastewater and algae samples had been collected from.
Collection of mosquito larvae from wastewater: Mosquito larvae were collected during the day preferably midmorning, from open wastewater channels. Three dips (triplicate) were taken to obtain the larvae from the wastewater, using the standard 350 mL dipper. If less than ten mosquito larvae were captured in the rst three attempts, additional two dips were done to obtain a sizable number. The dipper contents were then transferred onto a white plastic tray. The mosquito larvae were sorted, counted and their number per dip per site recorded. The larvae were then placed in plastic Whirl-Pak ® bags (Bio Quip, Rancho Dominguez, CA) which were approximately half full of the same wastewater from which the larvae were collected. The Whirl-Pak bags containing the larvae were then tightly closed to retain air before transporting to the laboratory as described by Rueda and others [69], where they were identi ed and preserved.
Preservation of adult mosquitoes and mosquito larvae in the eld: The trapped mosquitoes were processed as described by Tchouassi and colleagues [70]. The trapped mosquitoes were anaesthetized and killed using triethylamine while still in the trap. The mosquitoes were then sorted, counted, and put in Nunc tubes. The adult mosquitoes were then preserved in liquid nitrogen until when they were required for identi cation at Kenya Medical Research Institute (KEMRI), before processing them further for metal analysis. Similarly, the mosquito larvae were preserved as described by James-Pirri and others [71]. Brie y, the mosquito larvae were retrieved from the Whirl-Pak bags and placed in hot water at a temperature of 87 o C for 50 seconds after which they were removed using a strainer. The larvae were then preserved in Dietrich's solution and later transferred into 75% ethanol for further preservation until when they were required for identi cation and processing for metal analysis.
Morphological identi cation of the trapped eld mosquitoes: Both mosquito larvae and adults were identi ed using morphological features up to species level under a stereomicroscope. Appropriate mosquito taxonomic keys for the Sub-Sahara Africa and the East African region [72,73,74] were used.
Laboratory rearing of mosquitoes: Anopheles gambiae s.s., Kisumu strain and Aedes aegypti, Mombasa strain laboratory colonised mosquitoes were reared in the laboratory at KEMRI, following the protocol described by Das and colleagues [75]. Mosquito rearing was carried out in the insectary that was maintained at a temperature ranging from 27 to 28 o C and approximately 80 % humidity on a 12h/12h light and darkness cycle. Optimal larval concentrations were maintained to avoid possible effects of competition. Mosquito larvae were fed on nely ground Sera Vipan staple diet TM (Sera, Germany) while adults were offered a fresh 10% (w/v) glucose solution meal daily and fed on hamster (Mesocricetus auratus) as a source of blood meals for egg production. Mosquito larvae were reared in de-chlorinated tap water. De-chlorination of the tap water was achieved by allowing the tap water in a bucket to stand in the insectary chamber for at least 24 hours. These laboratory-reared mosquitoes obtained served as a control in the current study to enable us to compare the levels of heavy metals in eld trapped and laboratory mosquitoes.
Preparation of the mosquito samples for metal analysis: Both the eld and laboratory-reared mosquitoes were separately dried from an open room on brown papers, ground and then sieved to obtain a ne powder. The mosquito powder was then weighed, packaged in small new brown envelops and labeled appropriately for metal analysis.
Analysis of heavy metals for the different samples: The analysis of heavy metals was carried out at Mineral Laboratories, Bureau Veritas Commodities Ltd, Vancouver, Canada. The protocols included aqua regia digestion ultra-trace inductively coupled plasma mass spectroscopy (ICP-MS) for algae and mosquito samples; and ICP-MS (solutions > 0.1% total dissolved solids (TDS)) for water samples as described by the American Herbal Products Association [76]. The digest solution was nebulized, and sample aerosols transferred to argon plasma. The high temperature plasma then produced ions, which were then introduced into the mass spectrometer. The mass spectrometer then sorted out the ions according to their mass-to-charge ration and nally, the ions were quanti ed with an electron multiplier detector. Certi cates of analysis and quality control reports for all the samples analyzed were awarded by the Bureau Veritas, Canada.
Data Analysis: The statistical package for the social sciences (SPSS) version 20 for Windows at 5% level of signi cance was used for data analysis. Descriptive statistics involved computing mean, standard error (SE), and standard deviation (SD) for the different variables measured in wastewater, algae, and mosquito samples. One-way analysis of variance (ANOVA) was used to establish whether the differences within and between groups were signi cant or not. Tukeys and Games-Howell Post hoc tests were carried out to establish the pairs of variables that were signi cantly different. Correlation analysis was carried out to establish the nature of relationship, level of signi cance between concentrations of heavy metals in different samples. Pairwise correlation coe cients for the levels of selected heavy metals in wastewater, algae, and mosquito samples were also computed.

Limitations
We acknowledge the limitations of the current study which included: limited samples of lamentous green algae from the sampling sites and the challenge faced in obtaining adequate powdered mosquito samples for adults and larvae separately for metal analysis, hence forcing us to prepare assorted (mixed) mosquito samples for metal analysis. All the datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Con ict of interests
The authors declare that there are no con icts of interests, both nancial and non-nancial competing interests, associated with this manuscript.

Authors Contributions
GK -designed the proposal, sourced for funding, collected samples from the eld and was involved in data analysis; VN -oversaw heavy metals analysis of the samples, collection of samples from the eld and their preparation for metal analysis; and assisted in data analysis; LK -reviewed the proposal; oversaw rearing of the Anopheles and Aedes mosquitoes in the laboratory for control experiments; and advised on data analysis. All the authors reviewed and corrected the manuscript before it was submitted for publication.
Due to technical limitations, table 1, 2, 3, 4, 5, 6 is only available as a download in the Supplemental Files section. Figure 1 Showing comparison of heavy metal concentration (ppm) in assorted eld and assorted laboratoryreared mosquito samples.   Showing CDC mosquito trap on a tree branch in the premise of Kartasi Industries, Nairobi.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TablesEBOV2021.docx