Hexavalent chromium-containing waste from chromite ore processing is a major environmental health hazard due to its high toxicity. There have been instances of improper and unsafe disposal of this waste, leading to environmental health hazards.The objective of the present study was to identify the cause of yellow colored water discharge and reported health issues in nearby residents and cattle. In addition, it investigated the improper disposal of chromite ore processing residue (COPR), a hazardous waste, in an abandoned quarry in stromatolitic-limestone terrain in central-east India.42−), chlorine (Cl−), total hardness, calcium (Ca(II)), magnesium (Mg(II)), alkalinity and sodium (Na(I)) with proper sampling, quality assurance, and quality control protocols. Onsite Cr(VI) was analyzed using a chromium testing kit, and in the laboratory by atomic absorption spectrophotometry.Standard methods of analysis of water and wastewater were used for the analyses of variables, including hexavalent chromium (Cr(VI)), pH, sulfate  concentration observed was 1050 mg/L in leachate, 22 mg/L in surface water and 0.26 mg/L in the groundwater sample. Acute health effects were noted in cattle and by residents who consumed the highly contaminated water.A large volume of discharge of hexavalent chromium contamination from the COPR landfill was found, indicating the absence of containment features in the design . Disposal of COPR in an abandoned limestone mine is inadvisable. The highly fractured stromatolitic-limestone environment at the study site was found to offer almost no resistance to the mobilization of Cr(VI) due to the absence of organic or eukaryotic deposition in the stromatolitic environment. It was also noted that the drainage pattern of the area facilitates a possible translocation of contaminated discharge to the nearby river system. Nearby residents were unaware of the adverse impacts of the contaminated leachates and were using the contaminated water for bathing, washing, etc. Applicable Indian governmental regulations regarding the construction of hazardous waste landfills were found to be insufficient with respect to the use of inactive limestone mines as landfill sites.The authors declare no competing financial interests. Chromium (Cr) is an active oxidation-reduction element with two commonly encountered valence states, Cr(III) and Cr(VI), which are the most stable forms found in the environment.Chromium compounds play an essential role in any industrial economy and are widely used in metallurgy, the chemical industry, military, textiles, and machinery. Among the chromium salts, consumption of basic chromium sulfate and chromium oxide accounts for more than half of total consumption. Other widely used compounds include chromium chloride and the chromium salts of organic acids. Significant uses include chrome plating, manufacturing of dyes and pigments, leather tanning and wood preservation. These compounds are also used for drilling mud, as rust and corrosion inhibitors, textiles, and toner for copy machines.Chromium compounds include chromates, dichromates, and other derivatives like chromic oxides, chromium sulfate, etc. The process of mining chromium ore (mainly chromite) and processing to produce various compounds generate a considerable amount of chromium-rich solid waste, and liquid effluent and atmospheric emissions may contain Cr chiefly as Cr(III) or Cr(VI) species.Due to the toxicity of Cr(VI)-bearing substances, the pollution problem caused by the residue from metallurgical operations or manufacturing of chromium compounds has become a matter of worldwide concern.After the much publicized controversy of hexavalent chromium contamination of drinking water in the Hinkley community in California due to industrial operations, the issue of chromium contamination and its attendant health effects remain highly controversial.6COPRChromite ore processing residueTHTotal hardnessUSEPAUnited States Environmental Protection AgencyWater can be exposed to waste dumps, either in engineered secure landfills or ordinary municipal landfills, from rainwater percolation or groundwater flow. Depending on the protective strength of the landfill, water can lead to leaching of soluble inorganic and organic fractions of the waste. The liquid effluent generated is called leachate, which can undergo translocation from the landfill site.15There have been reported instances of hexavalent chromium transport due to large-scale groundwater flow in river basins, hence any large scale discharge of Cr(VI) has the potential to reach groundwater as well as rivers draining from the contaminated basin.22However, regulatory and public pressure on these industries is compelling them to dispose of wastes in landfills, instead of open dumping, as is prevalent in some areas of the world. The present study addresses Cr(VI) contamination of different environmental matrices including soil, surface water, and groundwater in the vicinity of a chromium waste disposal site in a landfill situated in an abandoned limestone quarry.. Limestone is the major mineral resource of the district, with an estimated 550 million tons of cement grade to ballast furnace grade limestone reserves in the area. The Bhilai steel plant mines a significant reserve of about 200 million tons of blast furnace grade limestone in the nearby town of Nandini. The area, comprised of villages such as Nadini-Khundini, Meresara, Semaria, Achholi, Ghotwani, Matargota, is being extensively mined for cement grade limestone for use in the cement plant and as paving material.The study location lies in the vicinity of a hazardous waste landfill constructed in a former limestone quarry in the Nadini-Khundini village of the Durg district in the Chhattisgarh state of India. The Durg district (20°23′-22°02′ N and 80°48′-81°57′ E) lies in the basin-plains of Chhattisgarh. The Shivnath River, a major tributary of the Mahanadi river system, drains from most areas in the district , and the details of the sampling locations are given in The sampling locations were distributed around the landfill site . Thus, this landfill quarry is a continuous source of leachate from the onset of the monsoon season in the month of May. Seepage from this quarry has been observed throughout most of the year with variation in the magnitude of the discharge, which is highest in the monsoon season and lower in the summer season.The landfill quarry is situated at a height of 280 feet above mean sea level and is the highest location in the sampled region Sampling was carried out as extensively as possible in the first circle (Sampling area 1) around the landfill site. Samples of soil, surface water, and groundwater were collected from the surrounding area in a grab sampling pattern while trying to maintain a sampling grid of appropriate distance. Around 55 samples were collected from sampling area 1, and 46 samples were collected from sampling area 2 and 29 samples were collected from sampling area 3. Sampling area 1 has an elevation of 285 to 280 m above sea level and is the highest region in the sampled area. The sampling points were mainly natural seepage points originating from the hanging walls of the landfill. Within a distance of about 500 m from the landfill, about 55 samples were collected in the periphery of the landfill. Sampling area 2 was an area approximately 1 km from the landfill with an elevation between 279 to 277 m. This area has many ponds and seepage discharge points, which were sampled to generate 46 samples. Sampling area 3 was an area 1 km to 5 km from the landfill. This area had many regular hand pumps and ponds, situated at an approximate elevation of 276 to 250 m. Around 29 samples were collected from this area.For quality assurance/quality control, water samples were collected in pre-washed plastic bottles, and soil samples were collected in dry polythene bags. No preservation was done, to maintain the prevailing oxidation state and the pH/redox potential conditions in the collected samples. The samples were refrigerated (<4°C) in the laboratory and analyzed as soon as possible.Quality control and quality check protocols consisted of the use of standard samples, analysis of blanks, duplicate analysis of every sample and inter-laboratory calibration checks. We adopted a laboratory independent quality control program by submitting duplicate field blanks and spiked samples.Both onsite and off-site analyses were conducted on the collected samples. The Cr(VI) was analyzed both onsite and in the laboratory. Onsite Cr(VI) was analyzed using AQUANAL-plus chromium . In the laboratory, total chromium was analyzed using an atomic absorption spectrophotometer . The analysis protocol used was as documented in a US Occupational Safety and Health Administration publication titled “Metal and Metalloid Particulates in Workplace Atmospheres (Atomic Absorption)” method number OSHA ID-121.23In the flame atomic absorption spectrophotometer analysis of chromium, a fuel-rich mixture of acetylene and nitrous oxide was used, and the measurement of absorption was carried out at 357.9 nm. Each sample was aspirated in the flame at least two times, and the average value was recorded. The minimum detection limit of the method was 0.04 μg/L. The stock solution of chromium was prepared by using analytical grade chromium trioxide (1.923 g) and was dissolved in water and acidified to pH 2 with pure and concentrated nitric acid. Calibration standards were prepared fresh every day by appropriate dilution of the stock solution. The calibration curve was cross-checked after every ten samples with appropriate use of calibration blanks.The concentration of Cr(VI) was determined colorimetrically by ultraviolet-visible spectrophotometry following the USEPA 7196A method as previously described., and the volume was made up to the volumetric mark. After about ten minutes, the absorbance was measured at 540 nm with a standard laboratory spectrophotometer following the usual calibration procedure.In the regular analysis method, an aliquot of the sample was taken in a volumetric flask and 2.0 mL diphenylcarbazide was added to it. The pH of the solution was brought to 2±0.5 by adding sulfuric acid (10% vol/vol)For quality control, the certified reference material “Chromium (VI) - Soil certified reference material” (CRM041) from Sigma-Aldrich  was used. The reported certified Cr(VI) content of this certified reference material was (86.5 ± 2.91 μg/g).Standard methods of analysis of water and wastewater by the American Public Health Association were used for the rest of the analyses.The landfill in the present study is located in a former limestone quarry in the Nadini-Khundini village of Dhamdha block. The landfill was created by a privately-owned chrome chemical company located in the light industrial area of Bhilai in the Durg district of Chhattisgarh state. State authorities permitted the landfill and operations began in an abandoned limestone quarry. The total quantity of disposed waste has not been specified, but it has been calculated that a significantly large quantity of waste generated from COPR has been dumped at the study location.Topographically, the Nandini-Khundini area is slightly undulating, with ground levels varying from 270 to 290 m above mean sea level. Red soil covers the mineralized limestone area with a clay overburden at a thickness varying from a fraction of meter to about 7 m, with an average thickness of soil of 4 m. The area is sub-arid in climate and receives a large amount of rainfall during the monsoon season . Due to the topographical features and high monsoonal rainfall, many indistinct streams run through the region following the slopes north and northwest.Geologically, a typical stromatolitic-limestone terrain topography is predominant. The study area contains large limestone mineralized beds which are horizontal to sub-horizontal (maximum dip ten degrees towards north). In the limestone deposit area of the mines, the bedding plane joints are found predominantly in the second to third bench along with numerous weak joints prevailing all over the area. The area is devoid of major geotectonic disturbances. The presence of columnar branching stromatolites indicates the studied area once had a shallow water marine environment with gentle slopes at the bottom. A protected tidal environment is best suited for such growth. The well-laminated nature of the limestone deposit rules out a deep-sea origin of rocks in the study area. Carbonate sedimentation in the study area can thus be expected to have been formed primarily from the chemical and biochemical process that has occurred in the clear, shallow, warm water marine environment.2CO3) to produce sodium chromate by reaction as in The study location is situated around an underground waste disposal site for COPR. Chromite ore processing residue was found to be produced in a factory situated in the industrial area of Bhilai in Chhattisgarh state where chromite ore is treated with lime (CaO) and soda ash  (maximum 22 mg/L) in the seepages were the first indication that the supposedly secure landfill had failed to keep wastes and rainwater separated. It was further noted that the landfill did not have an engineered system for collecting leachate and treating it before discharge.In the very first visit to the study location by the research team, yellow colored seepages were found to be coming from various pores and fissures. The chemical analysis of the yellow colored water confirmed a very high level of Cr(VI) contamination in the leachate from the landfill site. Out of the 130 sampled locations, 91 locations showed the presence of substantial amounts of hexavalent chromium . This can cause greater percolation of rainwater in the landfill, leading to inflow of water through the columns of waste and thus possible leaching of soluble components from the waste. As expected, the study site was found to be profusely discharging yellow colored liquid from numerous outlets, both from the veins and ore body along the dip and the stromatolite channels in the limestone bedrock in the rainy season. These discharges were found to coincide with the full onset of monsoon rains (July) and continued until the end of the post-monsoon season (December-January). Leachate discharges have led to the creation of a primary contamination zone , secondary contamination zone  and peripheral zone (extending up to ~ 5 km with a river boundary) as shown in The landfill is situated at a high point in the topography . This is expected, as limestone zones are generally rich in groundwater availability due to percolation of rainwater.In the study area, it was further observed that a considerable amount of groundwater seepage took place in limestone mines, even in the dry summer season . This is being done by using mine dewatering pumps to pump out the percolating water. The pumped-out water from nearby mines is discharged onto open fields. Many mine water discharges were found to be contaminated with high levels of hexavalent chromium. Thus, this mine dewatering water was found to be a major source of secondary contamination of nearby surface and groundwater sources.The studied landfill is situated at the highest elevation in the local topography. High rainfall and elevation have made this landfill an almost perennial source of contaminated seepage through the numerous discharge points in the laminar limestone strata. There are many adjoining limestone mines which are being actively mined. Thus, there are many exposed facies receiving contaminated seepage from the landfill as well as groundwater inflow. Therefore, for continuous mining operation the nearby mines need to pump out the accumulating water . The deposited yellow colored precipitate is a mixture of CaCrO4, calcium aluminochromate (3CaO•Al2O3•CrO4), tribasic calcium chromate (Ca3(CrO4)2) and sodium chromate (Na2CrO4).The concentrated landfill leachate, mine dewatering and contaminated groundwater plume are the principal causes of the groundwater and surface water contamination in the study location. The pond shown in It is well known that the presence of Cr(VI) in concentrations above 0.5 mg/L impart a characteristic yellow color to the impacted water.. In the trend analysis, surface water showed maximum contamination of Cr(VI) during the post-monsoon period  and a maximum of 2.53 mg/L during the pre-monsoon season. Thus, it can be concluded that Cr(VI) is being actively mobilized by percolating rainwater. The mine dewatering operation in the nearby limestone mines is contaminating the soil, surface and groundwater sources around the active mines.To identify the effect of seasonal changes in temperature and precipitation, sample collection and analysis were conducted in two different seasons, i.e., pre-monsoon (February–May) and post-monsoon (October–January). In these analyses, chromium levels showed a clear trend of seasonal variation  over pre-monsoon levels. The leachate showed a dominant presence of sulfate ions (SO42−) along with the sodium and chromium ions. This observation was consistent with the fact that the originating industry of the dumped wastes produced sodium chromate and dichromate from chromite ore.Two contaminants that showed significant differences in enrichment during the two seasons were Cr(VI) and sodium(I) ion (Na(I)). These two contaminants had a mean enrichment  of about 27 times for Cr(VI) and about 17 times in the case of Na(I)  revealed that changes in the concentration, or enrichment in case of the pH, total hardness (TH), alkalinity, calcium, and magnesium, were small compared to the results for chromium and sodium. High concentrations of Cr and Na during the monsoon season indicate that the leached ions originate from the landfill.Comparison of the mean results of two seasons in the impacted surface water  presents a relatively stable discharge condition prevailing at all the sampling locations. The low standard error, significant difference (SD) (except in case of Na(I)) and smaller values of kurtosis and skewness support this inference.The descriptive analysis of all samples collected during the post-monsoon season . This comparison showed very high enrichment for Cr(VI), SO42−, and Na(I) in the leachate impacted water. A significant increase in the TH, Ca(II), Mg(II) and a decrease in the alkalinity values was also noted, possibly due to dissolution of the bedrock limestone resulting in a mild increase in pH and a decrease in alkalinity (−38%). A very high enrichment in Cr(VI) (above 13999%), Na(I) (about 1531%) and SO42− (about 1411%) demonstrates the high degree of contamination in the Nadini-Khundini village in the Bhilai-Durg region.Enrichment of the chemical species in the impacted water was compared to background values (uncontaminated control sample) prevailing in the surface water As the waste dumped in the landfill is COPR, it is important to consider the impact of the pH of the percolating water. The solubility of Cr(VI) is controlled partially by Cr(VI)-hydrocalumite at pH >10.5 and by hydrotalcites at pH >8, in addition to adsorption of anionic chromate species onto inherently present metal oxides and hydroxides at pH <8. As pH decreases to <10, most of the Cr(VI)-bearing minerals will become unstable and their dissolution contributes to the increase in Cr(VI) concentration in a leachate solution.27In the present study, the pH noted in rain and surface water ranged between 6.75–7.80 with a SD of 0.35, which indicates a neutral pH which is likely to make the Cr(VI)-bearing minerals in COPR very susceptible to aggravated leaching, leading to heavy loading of Cr(VI).Even the groundwater used for drinking purposes, obtained from hand pumps in the affected village, showed the presence of Cr(VI) above the desired concentration of 0.05 mg/L according to the Indian standard for drinking water.30, as chromium levels start to rise with the onset of monsoon and peak around the post-monsoon season. The ingress of rainwater in the landfill is the apparent reason for the generation of the contaminated migrating groundwater plume. The dilution effect appears to operate under the subsurface due to a mixing of the percolating water and existing groundwater channels that exist in the limestone bedrocks. The reported annual total rainfall in the studied area was 1288 mm.Complete chemical analysis and statistical evaluation of the results were carried out. This analysis indicated that chromium was present in the form of chromium sulfate and sodium chromate. as evidenced by statistically insignificant (0.23) correlation of chromium with pH (suggesting a slightly acidic nature of the discharge) and a complete correlation (1 and 0.97) with sodium and sulphate, which underscores environmentally unaltered discharges. The correlation matrix in the post-monsoon season  retains the high correlation of chromium with sodium and sulfate (0.99 each) and in addition correlates significantly with pH, TH, calcium, magnesium, and alkalinity. Based on this, a limited neutralization of the attendant acidity and almost no attenuation of the chromium or sulfate was discernible in the environment around the landfill.The correlation analysis also showed extensive mobilization of chromium taking place during the monsoon season Groundwater near a landfill is mostly composed of percolated rainwater discharges from upgradient sources.Sampling area 3 was found to have a sizable number of inhabitants. It was also found that many people of the area work in the limestone quarry and are exposed to raw yellow colored leachates coming from the landfill area. The pond water, which was also contaminated with Cr(VI), was used for bathing and washing by the inhabitants. Questioning of local residents revealed that they were unaware of the possible ill-effects of the Cr(VI)-contaminated water, however, they reported adverse effects in exposed individuals and cattle.. Hence, it is evident that the surface water of the Shivnath River is at risk of contamination from the hazardous waste landfill at Nadini-Khundini village.The study region was found to be highly fractured and saturated with groundwater after a certain depth. In addition, the percolating rainwater migrates vertically downward from the landfill disposal area, which is located on the highest elevation point of the area. The maximum downward flows occur in the northern section of the landfill. The geographical flow of the water in the area is northward as evidenced by the regional topography, which culminates in the river Shivnath flowing in a north-easterly direction We noted the presence of stromatolites (a fossil relic from the marine environment) in the limestone deposits of the area. This feature lends credence to the layered depositional feature which is generally associated with a calm and shallow marine environment which might have prevailed in the region in the past geological history. This feature also indicates an absence of clay or eukaryotic organic depositions. This consideration is important for assessing the persistence of chromium in the contaminated groundwater plume.4), calcium aluminochromate (3CaO•Al2O3•CrO4), tribasic calcium chromate (Ca3(CrO4)2) and ferric chromate (Fe(OH)CrO4). This waste is considered to be highly hazardous due to high residual contamination of hexavalent chromium. This process of sodium chromate manufacturing has been abolished in western countries, but is still practiced in countries like China, India, Russia and Pakistan,In general, the chromite ore processing residue (COPR) waste are reported to be composed of calcium chromate (CaCrOTable 9). The results indicate favorable geochemical conditions for the mobilization and persistence of Cr(VI) and the absence of any inorganic or organic reductants in the landfill. The presence of Cr(VI) in the collected samples indicate that the study site an example of the hazardous waste mobilized contamination capable of causing potential adverse health effects noted at many other locations.36An analysis of the eleven contaminated groundwater samples and the ratio of Cr(VI) to total chromium showed that almost all samples contained more than 99% of chromium in the form of Cr(VI) , a topsoil analysis was performed. Ten samples were collected from the topsoil around the landfill, and average results are presented in As demonstrated in The question also arises whether the present location poses any other challenges compared to other limestone belts. Geologically, the limestone of the Durg district is pink limestone of the Raipur group, which is a part of the Chhattisgarh Supergroup formation. These rocks belong to the Proterozoic age.Hydrogeologically, the aquifers of the area belong to a fissured media type, which is discontinuous and unconfined to semiconfined. They are generally restricted to weathered mantle and fractures and are in contact with the limestone. The aquifers extend as much as 150 m below ground level. The compounding issue here is that the area around the landfill in question is being actively mined by two adjoining and nearby stone quarries. The slope geometry in such mines is very steep , and none have any provisions for berms or mine benches. This feature makes such quarries highly susceptible to instability, with tall face heights between 20 to 100 m. The percolating water seepage with high chromium content has caused problems for manual laborers working in the quarries, and to continue mining, chromium-laced seepage from all nearby quarries is pumped out by heavy duty pumps. This large discharge of water is a potential source of aquatic and environmental contamination.An issue of concern is the lack of knowledge in the literature about dilution and dispersion in fractured rock and limestone systems.38The present study can help to address the advisability of siting a hazardous waste landfill in a stromatolitic-limestone terrain. The results show that the landfill in Nadini-Khundini is causing considerable discharge of Cr(VI) due to the improper construction of the landfill and facilitated by the apparent lack of appropriate standards during its construction.The improperly designed hazardous waste landfill in Nadini-Khundini village in Bhilai and the siting of the landfill in limestone terrain and particularly in an abandoned limestone quarry is causing considerable discharge of Cr(VI) contaminated leachate. The landfill lacks any leachate collection or treatment mechanism. Furthermore, it has no surface water discharge mechanism after treatment and no monitoring of probable gaseous emissions. Other details of the waste type, volume, fill-area dimensions and related calculations are not available as the company is not willing to divulge this information. Entry to the landfill site is prohibited and secured by guards.As seen in The Siting Criteria in the Solid Waste Program (Ohio EPA) states that a sanitary landfill facility shall not be “located in a limestone quarry or sandstone quarry, unless deemed acceptable by the director”.The Canadian Council of Ministers of the Environment states that an engineered hazardous waste landfill facility needs to be in a location with appropriate environmental conditions.The Indian hazardous waste rules do not specifically discuss siting criteria, or address the suitability of siting in abandoned limestone quarries.47The industry which generated the landfilled waste is situated in the industrial estate at Nandini Road, Bhilai, Durg district. As per the records of the Chhattisgarh Environment Conservation Board, the industry generates category A-5 (concentration limit: ≥ 50 mg Cr(VI)/kg) hazardous waste. The annual production of the COPR sludge from this industry is 4500 million tons per annum with a waste generation factor of 552.15 (quantity of hazardous wastes generated per unit (ton of product)).The present case study indicates that implementation of the design principles was not taken into consideration at the landfill. The chromite ore processing waste dumped at the studied site is classified as Schedule II hazardous waste as per the Hazardous and Other Wastes (Management and Transboundary Movement) Rules, 2016.50The present study adds to the literature regarding issues of hazardous waste disposal in limestone-rich regions. Based on the results of the present study, it can be concluded that the landfill in question is poorly designed and improperly sited in an abandoned limestone quarry. The large amount of rainfall during the rainy season and its entry in the landfill has resulted in the generation of huge quantities of leachates from this landfill.Due to the surrounding alkaline medium, the condition at the site area favors the mobilization of Cr(VI). Hence the use of exhausted or abandoned quarries as hazardous waste landfill sites in India is likely to cause many further instances of ground and surface water pollution.Persistence makes it possible for any contaminant to retain its chemical, physical or biological properties for a prolonged period. Persistence depends on the chemical structure, conditions in the aquifer conducive to degradation such as microbial population, nutrients available to support microbial growth, and the type and quantity of ions in the background groundwater. As described above, the stromatolitic-limestone geographical area is almost devoid of any organic deposition or clay in the area. Thus the Cr(VI) being released from the landfill is not likely to be adsorbed or transform to any other ionic state in such conditions. Hence the Cr(VI) generated from the site in Nadini-Khundini is likely to be transported in a highly toxic state for long distances to the boundary of the hydrological region, i.e., the Shivnath River.The placement of a hazardous waste landfill in a limestone mine presents environmental and human health risks to the surrounding area. It is suggested that further delineation of the contaminated area and remediation and/or risk reduction actions to protect the public and natural resources from adverse health effects should be undertaken by the governmental authorities in this area. Remediation of the contaminates from this landfill is likely to be difficult and expensive. The appropriate authorities should consider the use of chemical reduction with sulfate-based materials as an immediate remediation step. This should be followed by bacterial bioremediation to ensure a safe and long-term solution.The present study aimed to identify and characterize the massive discharge of Cr(VI) from a hazardous waste landfill constructed in a former limestone quarry in the Nandini-Khundini area of Bhilai in central-east India. This landfill was officially permitted by the concerned authorities for disposal of wastes generated from a factory producing various chrome compounds. The raw material of the factory is chromite ore, and it is processed chemically to produce sodium chromate and dichromate. This process is known to produce considerable hazardous wastes containing a high amount of chromium and its intermediate compounds.The maximum Cr(VI) concentration measured in the leachate was 1050 mg/L and 22 mg/L in the impacted surface water with a mean value of 14 mg/L in the post-monsoon season. Groundwater also showed the presence of Cr(VI) with the highest value of 0.26 mg/L in the pre-monsoon season. The average rainfall in the area was greater than 1288 mm, indicating significant percolation of rainwater in the landfill, and a high level of discharge of Cr(VI) from the landfill has been reported. Correlation studies in different seasons showed almost no attenuation of chromium or sulfate is taking place in the plume path around the landfill.The present study also established that placing waste containing highly mobile compounds like Cr(VI) in a former limestone mine in a highly fractured stromatolitic-limestone environment is inappropriate. The geological environment in this area offers almost no resistance to the mobilization of Cr(VI). The absence of eukaryotic deposition has resulted in a minimal concentration of organic compounds in the soil or rocks of the study area. Therefore, highly toxic Cr(VI) is not being reduced to Cr(III) or other less harmful species. Furthermore, the drainage pattern in the study area indicates the possibility of translocation of contaminated discharge to the nearby river system on account of the favorable drainage pattern, non-reacting soil and prevailing lithology of the area. The lack of proper regulations, inadequate design, incomplete implementation of regulations, absence of monitoring and improper enforcement at the study site highlights the resulting adverse impacts on nearby residents and the environment.


Negative marital quality is associated with poor health outcomes. Purpose in life can serve as a psychological resource to buffer the stressors experienced from a negative marital relationship. Yet, the associations among negative martial quality, a person’s level of purpose in life, and depressive symptoms have not been fully explored in a dyadic context. We examined the actor  and partner  effects of negative marital quality on depressive symptoms in couples and the potential mediating role of purpose in life. Structural equation modeling was used to analyze cross-sectional data on middle-aged and older married, heterosexual couples  who participated in the 2016 wave of the Health and Retirement Study. The final model had an acceptable fit to the data . At the actor level, negative relationship quality was positively associated with depressive symptom severity, and purpose in life mediated the relationship in wives and husbands. At the partner level, wives had more depressive symptoms when husbands reported higher negative marital quality. Comparatively, husbands had less depressive symptoms when their wives indicated a greater sense of purpose. Husbands also had a lower purpose in life when their wives had higher states of negative marital quality. This study highlights the psychological benefits of allaying negative perceptions of marital quality and enhancing the sense of purpose in middle-aged and older couples. The results support a focus on dyadic approaches to improve the psychological health, and potentially, the physical health status of middle-aged and older couples.


Conservative treatments such as exercise are recommended for the management of people with neurogenic claudication from spinal stenosis. However, the effectiveness and mechanisms of effect are unknown. This protocol outlines an a priori plan for a secondary analysis of a multicentre randomised controlled trial of a physiotherapist-delivered, combined physical and psychological intervention  programme).We will use causal mediation analysis to estimate the mechanistic effects of the BOOST programme on the primary outcome of disability (measured by the Oswestry Disability Index). The primary mechanism of interest is walking capacity, and secondary mediators include fear-avoidance behaviour, walking self-efficacy, physical function, physical activity and/or symptom severity. All mediators will be measured at 6 months and the outcome will be measured at 12 months from randomisation. Patient characteristics and possible confounders of the mediator-outcome effect will be measured at baseline. Sensitivity analyses will be conducted to evaluate the robustness of the estimated effects to varying levels of residual confounding.Ethical approval was given on 3 March 2016 . The results of this analysis will be disseminated in peer-reviewed journals and at relevant scientific conferences.ISRCTN12698674. Causal mediation analysis of clinical trials can explain how an effective intervention works, or why an ineffective intervention does not work.This protocol describes a planned analysis of the underlying causal mechanisms of the Better Outcomes for Older people with Spinal Trouble rehabilitation programme; a combined physical and psychological intervention for people with neurogenic claudication symptoms.We propose an exploratory analysis of several potential causal pathways based on theoretical mechanisms of action, acknowledging the risks associated with multiple testing.The proposed models will estimate the extent to which candidate mediators  mediate effects on disability measured by the Oswestry Disability index.Neurogenic claudication (NC) is a leading cause of disability and lost independence in older adults.15LSS has become the most common reason for spinal surgery in people over 65 years of age.17The Better Outcomes for Older people with Spinal Trouble (BOOST) trial is a large multicentre randomised controlled trial (RCT) testing the effectiveness of a 12-week combined physical and psychological intervention delivered by physiotherapists in a group setting. The primary focus of this randomised trial is to estimate the effect of the BOOST intervention on patient-reported disability. There is, however, growing recognition of the importance of evaluating hypothesised treatment mechanisms in randomised trials.We present an a priori protocol for a secondary analysis of the BOOST trial to evaluate the effects on the primary mediator of a change in walking capacity, and to explore alternative mediators of changes in fear-avoidance behaviour, walking self-efficacy, physical function, physical activity and symptom severity. The aim of this planned analysis is to contribute to our understanding of treatment mechanisms and provide valuable information to guide future intervention development and implementation.This study is a causal mediation analysis of the National Institute of Health Research (NIHR) funded BOOST trial. The BOOST trial is a high-quality multicentred RCT. It aims to evaluate the clinical-effectiveness and cost-effectiveness of a physiotherapist-delivered, combined physical and psychological intervention for community-dwelling older adults with symptoms of NC, compared with a control treatment of best practice (standardised advice and education). BOOST trial details are published separately in the trial protocol.25Patient and public involvement has been embedded in the BOOST trial and is described fully in the trial protocol.25In total, 438 participants with symptoms of NC from across 15 primary-care and secondary-care centres in the UK have been randomised to the trial intervention or control treatment at a 2:1 ratio. We recruited participants over the age of 65 years from spinal clinics within primary and secondary care centres (n=394) and via a primary care survey of community-dwelling older adults (n=44). They were eligible for the study if they presented with symptoms consistent with NC  and were able to participate safely in the group rehabilitation programme.A detailed description of the BOOST intervention and its development have been published elsewhere.We developed the intervention collaboratively with clinical experts and patients to address some of the unique challenges of managing back problems in older people. These include age-related physical changes of reduced muscle strength and fitness, in addition to psychological factors such as depression28–31The control intervention consists of a 1 hour individual session with a physiotherapist for assessment, advice and education about NC and being physically active, advice on exercises including flexion-based exercises, use of medications and when to seek more advice. The advice and education are reinforced in a leaflet and a further two review appointments are available if required.The Oswestry Disability Index (ODI)33The ODI comprises a series of questions about how pain impacts on the patient’s ability to carry out a range of daily functions. For each question, possible answers are scored on a scale of 0–5. These scores are summed and expressed as a percentage of the maximum possible score for disability, with higher scores representing worse disability. In the BOOST trial, ODI scores are measured for each participant at baseline, and at 6 and 12 months after randomisation.Important secondary outcome measures for the BOOST trial focus on walking capacity, and on fear-avoidance behaviour, walking self-efficacy, physical function, physical activity and symptom severity: the 6-minute walk test provides an objective measure of walking capacity and entails a timed measure of walking distance which is easily performed in the clinical setting.We will test the mechanisms of the BOOST intervention for patients with NC by estimating the extent to which different potential mediators explain the effect of the BOOST intervention on participants’ ODI outcome scores. This will be achieved by deconstructing the total effect of the BOOST treatment into indirect and direct effects. The indirect effect is the path through which the intervention affects the outcome via a selected mediator. The direct effect is the remaining total effect that is not encompassed by the indirect effect.Walking capacity has been selected as the primary mediator for our causal mediation analysis model. Walking limitation is a hallmark of disability in LSSThe BOOST intervention is underpinned by a strong theoretical premise for addressing age-related decline in mobility. Exercise components of the intervention aim to improve physical function by targeting reduced muscle strength, balance and sensory impairments.Our final alternative mediator is symptom severity (SSS). Little is known about how exercise affects NC symptom severity.4 70To understand the mechanisms of the BOOST intervention, we plan to test our primary hypothesis that changes in disability will be mediated primarily by changes in walking capacity, and that various mechanisms might explain these changes in walking capacity: We hypothesise that the progressive exercise component of the BOOST intervention will exert its effect on walking capacity via initial changes in physical function and physical activity levels during treatment. We further hypothesise that the cognitive behavioural components of the intervention will exert effects on walking capacity through reductions in fear-avoidance behaviour and increased self-efficacy. There is also the potential for both the exercise and the cognitive behavioural components of the intervention to reduce symptom severity, resulting in increased walking capacity.Our secondary hypothesis is that the causal effects of the BOOST intervention on disability will be explained through changes in fear-avoidance behaviour, self-efficacy, physical function, physical activity and/or symptom severity (SSS) without exerting any effects on walking capacity.If the BOOST intervention is found to be effective, causal mediation analysis will explain how the intervention works. Conversely, if the intervention is not found to be effective, causal mediation analysis will identify where the hypothesised mechanism broke down.71The decision-making process that will guide our analyses is laid out in If the intervention is effective, our primary objective is to estimate the extent to which the joint effects of walking capacity, fear-avoidance behaviour, walking self-efficacy, physical function, physical activity and symptom severity mediate this effect.Our secondary objective will be to further refine this model through the candidate primary mediator, walking capacity; and via five secondary mediators . If the indirect effect through the primary mediator is significant, we will consider sequentially ordered multiple mediator models through secondary mediators. The serial multiple mediator models accounting for potential confounders are presented in the directed acyclic graphics in If the intervention is not effective, our primary objective is to determine where the causal paths break down. All potential mediators  will be evaluated.‘medflex’ and ‘mediation’ packages.73 74We will use a model-based inference approach for causal mediation analysis.To estimate a joint indirect effect, we will consider a natural indirect effect of the intervention on the outcome that is exerted through a vector of all six mediators . The advantage of this approach is that we can consider all possible mechanisms simultaneously and relax the assumption of omitting confounders of the mediator-outcome effect that is affected by randomisation. We will use the imputation-based approach outlined by SteenTo consider the mediators independently, we will construct single mediator models for the ODI outcome. For each single mediator model , we will estimate the intervention-mediator effect, the mediator-outcome effect, the average causal mediation effect (ACME), average direct effect (ADE) and the average total effect (ATE). We will also estimate the proportion mediated in each single mediator model. The ACME is the average intervention effect through the mediator; ADE is the average intervention effect that works through all other mechanisms excluding the selected mediator; and the ATE is the average effect of the intervention on the outcome. The ATE is the sum of the ACME and ADE on the additive scale. The proportion mediated is the fraction of the ATE that is explained by the ACME.We will fit two regression models: the mediator model and the outcome model. The mediator model will use linear regression with treatment allocation as the independent variable, and the mediator as the dependent variable, with the baseline level of the mediator as a covariate. The outcome model for disability will also use linear regression. Each outcome model will be constructed with the mediator as the independent variable, the outcome as the dependent variable and treatment allocation, baseline values of the mediator and outcome variable in addition to the set of observed pre-treatment confounders as covariates. Because the mediators are not randomised, it is possible for the mediator-outcome effects to be confounded. To address this problem, we will adjust for the effects of pre-treatment confounders based on theorised causal relationships with the mediators and outcome variables from expert clinical knowledge and available evidence. These potential confounders include age, body mass index, sex, number of comorbidities, in addition to baseline measures of disability, physical function, walking capacity, self-efficacy, physical activity, symptom severity and fear-avoidance behaviour, and also baseline frailty (frailty index/grip strength); use of walking aids; general pain (Nordic pain Q); exercise self-efficacy; and attitudes to ageing. We will also conduct a sensitivity analysis to determine the robustness of the estimated ACME and ADE to possible unmeasured confounding.78Using mediator and outcome regression models, we will simulate potential values of the mediator for each participant under each level of the intervention; then simulate potential outcome values for each participant under all combinations of the intervention and simulated mediator values. From these observed and simulated potential values of the mediator and outcome, we will calculate point estimates for the ACME, ADE and ATE and their 95% CIs using 1000 bootstrapped simulations with the percentile method.multimed function from the mediation package to estimate the ACME and ADE, and the sensitivity parameters.For multiple mediator models, we will use an expanded framework.We will assess the proportion of missing mediator and outcome data. We will conduct all analyses on complete cases if the proportion of missing data is <5%. If missing data exceeds 5% we will use multiple imputations by chain equations to impute 10 datasets using the ‘mice’ package.80To facilitate clinical interpretation, we will calculate how much change in a given mediator would equate to a minimally clinically important difference (MCID) of disability (5 point change in ODI scoreWe do not currently know whether non-surgical interventions are effective in reducing disability reported by older people with NC symptoms. The BOOST trial will provide important high-quality evidence about the effectiveness of an intervention incorporating components to address both physical and psychological impairments. The targets of this comprehensive intervention include walking capacity, physical function, physical activity, self-efficacy, unhelpful beliefs about activity and symptom severity. We cannot assume, however, that if the intervention improves any of these variables, the participants will experience benefits in patient-reported outcomes, such as the ODI.Mediation analysis conducted in parallel to an RCT can address specific questions about the underlying mechanisms of the intervention being tested. This protocol describes the planned causal mediation analysis that we will undertake to evaluate hypothetical mechanisms that underlie the effect of the BOOST rehabilitation programme. This will help us to understand how the BOOST intervention works, or doesn’t work.If the BOOST intervention is found to be effective and this mediation analysis indicates that improvements in ODI scores are mediated by walking capacity, which in turn is mediated by improved self-efficacy, then this provides valuable information for refining the intervention with a greater focus on self-efficacy to optimise treatment effectiveness. If, on the other hand, the BOOST trial is not found to produce a significant improvement in patient-reported disability, then the analysis will help us to understand where the hypothesised causal mechanisms of treatment break down. For example, we may find that although the BOOST intervention improves walking capacity, this does not translate into an improvement in ODI score and may therefore not be an appropriate treatment target. The causal analysis will, therefore, provide valuable information for designing, refining and implementing an effective intervention.Because this is an exploratory study, we have selected several mediators based on our current clinical understanding and evidence for exercise and behavioural treatments. Several potential causal pathways will, therefore, be explored, and we acknowledge the risks associated with this multiple testing. If alternative pathways of mediation emerge during the analysis of the trial and associated qualitative data, we will investigate these further. The publication of this protocol does not preclude other analyses if indicated. Future studies may be needed to further explore the inter-relationship of the mediators explored in our analyses.how an intervention affects outcomes, and therefore may not identify the most effective treatment targets for a particular patient population. Mediation analysis of clinical trials can estimate how much the total effect of the treatment can be attributed to pre-specified treatment targets. Improving our understanding of these treatment mechanisms can generate evidence that can be used to tailor and refine treatments and optimise clinical effectiveness. In this study, we will estimate the causal mediation effects of a rehabilitation programme incorporating exercise and behavioural components for patients with NC.Clinical trials do not address questions around


The aims of this study were to examine the prevalence of orofacial pain symptoms in Chinese older adults at the end of life, and to investigate risk factors related to orofacial pain. The sample derived from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), a national respresentative sample of the oldest-old. The results showed that the 6-month prevalence of pain when chewing or biting at the end of life was 11.1%, and the rate was 5% for jaw joint pain/facial pain. Lower SES, smokers, and having chronic diseases were associated with having orofacial symptoms. Unexpectedly, the results revealed that dentate older adults  who brushed their teeth more often were more likely to have orofacial symptoms. Older adults have poor oral health, particularly at the end of their life. This study highlights the importance of improving oral health for vulnerable older adults.


Solid evidence has shown financial resources play important roles in housing decisions among older adults. Despite the growing research on the joint assessment of income and assets as valid economic well-being, little attention is paid to its role in relocation in old age. Drawing from the Behavioral Model of Elderly Migration, this study examined to what extent financial resources are associated with the likelihood of moving in later years. The data came from the 2017 Panel Study of Income Dynamic (PSID). A sample of 1354 people, 65 years and older, was used in the analyses. We used the annuitized approach, which is different from conventional approaches that assume people draw down all available assets to satisfy daily needs and leave no assets for use in later years. We (1) assessed annuitized assets based on the 2019 IRS Mortality Table, (2) assessed yearly income using supplementary income (i.e. income plus non-discretionary expense). A final indicator of the summed score was used in a logistic regression to predict the likelihood of moving. A set of covariates known to affect later- year relocation at an individual level , environmental level  are controlled for. In clear conflict with previous studies, we found annual financial resources did not significantly influence relocation among older adults. The notable absence of the well-known role of the economic factor provides critical initial evidence about the importance of simultaneous assessment of financial resources for the literature on later year relocation.


Currently, a global analysis of the information available on the relative composition of the floral scents of a very diverse variety of plant species is missing. Such analysis may reveal general patterns on the distribution and dominance of the volatile compounds that form these mixtures, and may also allow measuring the effects of factors such as the phylogeny, pollination vectors, and climatic conditions on the floral scents of the species. To fill this gap, we compiled published data on the relative compositions and emission rates of volatile organic compounds (VOCs) in the floral scents of 305 plant species from 66 families. We also gathered information on the groups of pollinators that visited the flowers and the climatic conditions in the areas of distribution of these species. This information allowed us to characterize the occurrence and relative abundances of individual volatiles in floral scents and the effects of biotic and climatic factors on floral scent. The monoterpenes trans-β-ocimene and linalool and the benzenoid benzaldehyde were the most abundant floral VOCs, in both ubiquity and predominance in the floral blends. Floral VOC richness and relative composition were moderately preserved traits across the phylogeny. The reliance on different pollinator groups and the climate also had important effects on floral VOC richness, composition, and emission rates of the species. Our results support the hypothesis that key compounds or compounds originating from specific biosynthetic pathways mediate the attraction of the main pollinators. Our results also indicate a prevalence of monoterpenes in the floral blends of plants that grow in drier conditions, which could link with the fact that monoterpene emissions protect plants against oxidative stresses throughout drought periods and their emissions are enhanced under moderate drought stress. Sesquiterpenes, in turn, were positively correlated with mean annual temperature, supporting that sesquiterpene emissions are dominated mainly by ambient temperature. This study is the first to quantitatively summarise data on floral-scent emissions and provides new insights into the biotic and climatic factors that influence floral scents. We further determined whether phylogeny, reliance on different types of pollinators, and climate were correlated with floral VOC richness, scent composition, and rate of emission. We hypothesized that the compositions and rates of emission of floral scents have been preserved throughout evolutionary history, and we aimed to differentiate between the effects of phylogeny on floral scents and the effects of biotic and climatic factors. We expected that the pollination syndrome would be correlated with VOC richness, composition, and emission rate of floral scents. Finally, we hypothesized that climate would exert some selective pressures on the production and emission of floral scents, thus positively or negatively stimulating the emissions of all or some compounds under the environmental conditions where each species grew and flowered.We exhaustively searched the Web of Knowledge and Google Scholar for studies of floral scent using combinations of the keywords “floral”/“flower” and “volatiles”/”VOCs”/“scent”. We chose studies that provided complete data on the emission rates and/or relative percentages of all floral VOCs emitted. We discarded studies that did not report the complete bouquet of VOCs of the floral scents but focused only on particular compounds, thus omitting other compounds that were emitted but were not the focus of the study. We finally selected 58 studies that provided information on the complete compositions of floral scents of one or more species. The references for all the studies from which we used data to make our database can be found in WorldClim database: Mean Annual Temperature, Max Temperature of Warmest Month and Min Temperature of Coldest Month (K), Mean Annual Precipitation, Precipitation of Wettest Month, and Precipitation of Driest Month (L/m2). We calculated the Gaussen index of aridity using the climatic data as: Gaussen index = annual precipitation/. We further obtained information on the pollinators that visited the flowers of each plant species as described by the source studies.Each case in our database corresponded to a description of the floral scent of one plant species in one study. The name of the species (and subspecies when appropriate) was entered as provided by the source study, and equivalent synonyms following the Angiosperm Phylogeny Group III classification system, the genera, and the families were also recorded in our database. We obtained the longitudes and latitudes of the populations from which individuals of each species were sampled according to the methods described in the papers, and several climatic variables were obtained from the -1 flower-1μg h, -1 inflorescence-1μg h, -1 g DW-1μg h, and -1 g FW-1μg h), depending on the methods used in each study. Some studies specified the isomer(s) of various isomeric compounds in the floral scent of the species, but others did not. Several compounds thus appear in our database as repeated variables with and without a specified isomer.We classified floral VOCs based on their biosynthetic pathways, which was the predominant classification in all our data sources and enabled comparisons with phylogenetic hypotheses. We thus divided floral VOCs into the nine major classes: fatty acid derivatives, amino acid derivatives, benzenoids, monoterpenes, sesquiterpenes, irregular terpenes, nitrogen-containing compounds, sulphur-containing compounds, and miscellaneous compounds. We entered the data on the floral scent of each species as “presence/absence” (1/0), “relative percentages of the total blend” (%), and “emission rates” for all individual compounds and for the nine major groups of floral volatiles identified above. The source studies did not always provide emission rates, but all studies provided percentages of the total floral VOC blend. Emission rates were provided in various units . The description of pollinators differed across classification levels amongst the studies, so we created different binary variables to characterize the spectrum of pollinators, some of which were included in other groups  and provided information that was redundant to some extent. Both higher and lower classification levels, however, were useful for conducting different comparisons to answer different questions.We obtained information from the source studies of the pollinators that visited each plant species. Several species were pollinated or visited by different pollinator groups, so we created several binary variables in our database to indicate whether a particular plant species was or was not pollinated by a particular pollinator group  to important degrees could not be classified into these groups and were therefore not included in the classification figures or analyses that required this classification.We further classified as many plant species as possible  as predominantly pollinated by phylosig function from the R package phytools  and P values based on the variance of phylogenetically independent contrasts relative to tip shuffling randomisation  and the pollination syndromes .We also used the phylogenetic tree to reconstruct the ancestral states of floral VOC emissions and pollination syndromes. We used stochastic character mapping  to reconphytools , simulatkruskal function of the agricolae package (number of comparisons).We used the Kruskal-Wallis (K-W) test for non-parametric data to test for differences in floral VOC richness and in the percentages of the classes of volatiles between plants pollinated by different pollinator groups. We further used the K-W test to compare the percentages of the most common floral volatiles, i.e. benzaldehyde, limonene, linalool, trans-β-ocimene, and benzyl alcohol, in floral blends amongst plants pollinated by different pollinators. K-W tests were conducted with R software using the  package . We perfphylolm function of the phylolm package, which fits phylogenetic linear models, allowing us to exclude the effect of phylogenetic distance  based on two distance measures and then fitted the pollination system onto the ordination using the ge vegan . We usedre, dA,B , that coWe compiled 851 VOCs in the floral scents of 305 plant species belonging to 66 families  as a group than in anemophilous species (pollinated by wind), although the differences were not significant  are almost scentless , likely P < 0.001, P < 0.001, The higher richness , because it is a more standardized unit for describing emissions from flowers or any other plant organs/tissues.Some studies of the composition of floral scents provided emission rates for all floral compounds, but many studies provided only the relative percentages. The relative composition of floral scents is very relevant information, but actual emission rates could contribute more to our understanding of floral scents. We therefore encourage authors to quantify and describe the emission rates for each compound when possible. We also encourage authors to use the same reference units when providing emission rates, which would simplify the inclusion of their results in combined analyses. We noticed that μg hThe responses of floral scents to different environmental climatic factors such as temperature or drought are highly plastic , as are Phyllospheric microorganisms have important effects on the composition of floral volatile emissions . MicroorFloral scents are subject to many evolutionary pressures exerted by biotic and abiotic environmental factors. The need to attract pollinators is a major reason why animal-pollinated angiosperms have evolved complex and diverse floral scents. Plants have thus evolved various sets and mixtures of floral volatiles that help promote flower constancy, which in some cases stimulate the attraction of specific groups of pollinators. We identified some patterns indicating that particular VOCs were associated with particular pollination syndromes. Our results support the concept that key compounds or compounds originating from specific biosynthetic pathways play a significant role in mediating the interactions of flowers with their pollinators. Other floral visitors (e.g. herbivores and larcenists) and floral inhabitants  also exert important selection pressures on plant secondary metabolism and floral scents. All these selection pressures act in different ways on floral phenotypes and they may difficult the appearance of patterns established across the entire phylogeny of flowering plants (or even within major plant clades) of shared emissions of complex VOC mixtures associated with the attraction of a particular pollinator group. We identified several significant relationships between climatic variables and the richness, relative composition, and emission rate of floral VOCs. Our results suggest that climate is a relevant factor determining the composition and emission rate of floral scents, in addition to the strong and well-known selective pressures exerted by biotic agents such as pollinators and other floral visitors. We therefore hypothesize that climate can also select for floral scents that contain compounds with different physicochemical properties, increasing the suitability of floral scents to the environmental conditions that plants experience during flowering.All datasets presented in this study are included in the article/GF-A and JP conceived and designed the study. GF-A compiled all the information contained in the database used in the study. MF-M and RJ significantly contributed to the statistical analysis of data by conducting some of the analyses and advising GF-A on the suitability of all the analyses employed. GF-A wrote the manuscript, and JP, RJ, MF-M, and IF supervised and contributed to the writing. All authors contributed to the article and approved the submitted version.This research was supported by the Austrian Science Fund , the Spanish Government grant CGL2016-79835, the Catalan Government grant SGR 2017-1005, the European Research Council Synergy grant ERC-2013-SyG-610028 IMBALANCE-P and the Catalan Government grant FI-2013. MF-M is a postdoctoral fellow of the Research Foundation – Flanders (FWO). The authors declare no conflicts of interest.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer [JM] declared a past co-authorship with one of the authors [RJ] to the handling Editor.


The Coronavirus pandemic is recording unprecedented deaths worldwide. The temporal distribution and burden of the disease varies from setting to setting based on economic status, demography and geographic location. A rapid increase in the number of COVID-19 cases is being reported in Africa as of June 2020. Ethiopia reported the first COVID-19 case on 13 March 2020. Limited molecular laboratory capacity in resource constrained settings is a challenge in the diagnosis of the ever-increasing cases and the overall management of the disease. In this article, the Ethiopian Public Health Institute (EPHI) shares the experience, challenges and prospects in the rapid establishment of one of its COVID-19 testing laboratories from available resources. The first steps in establishing the COVID-19 molecular testing laboratory were i) identifying a suitable space ii) renovating it and iii) mobilizing materials including consumables, mainly from the Malaria and Neglected Tropical Diseases (NTDs) research team at the EPHI. A chain of experimental design was set up with distinct laboratories to standardize the extraction of samples, preparation of the master mix and detection. At the commencement of sample reception and testing, laboratory contamination was among the primary challenges faced. The source of the contamination was identified in the master mix room and resolved. In summary, the established COVID-19 testing lab has tested more than 40,000 samples (August 2020) and is the preferred setting for research and training. The lessons learned may benefit the further establishment of emergency testing laboratories for COVID-19 and/or other epidemic/pandemic diseases in resource-limited settings. Coronaviruses (CoV) are a large family of viruses that cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV). The novel coronavirus SARS-CoV-2) is a new strain which had not previously been identified in humans . The nov is a newenv), nucleocapsid (N), spike (S), RNA-dependent RNA polymerase (RdRp), and ORF1 genes. The sensitivities of the tests to individual genes are comparable [The most common laboratory testing method for SARS CoV-2 is by detecting the virus nucleic material (RNA) using RT-PCR. The test uses nasopharyngeal/oropharyngeal swabs or other upper respiratory tract specimens, including sputum and saliva samples . A variemparable .As of 12 July 2020, the COVID-19 pandemic has affected more than 12.9 million people worldwide resulting in more than 567,914 deaths [When COVID-19 was spreading out of Wuhan, China in January 2020, Ethiopia had limited capacity in the molecular diagnosis of coronaviruses. With unprecedented effort and as the lead national referral laboratory, the EPHI started managing testing for COVID-19 by early March 2020. However, the limited testing capacity at EPHI could not keep pace with the increasing demand for testing. Initially, a COVID-19 testing laboratory was initiated by the Virology/Influenza team at the EPHI. Following the quest for expansion of testing at the EPHI, the Malaria and Neglected Tropical diseases (NTDs) research team at EPHI also took the initiative to establish a complementary COVID-19 testing laboratory with the available resources initially allotted for Malaria and NTD research activities.In addition, several public universities, research institutes and hospitals in the country joined the effort by establishing their own COVID-19 testing laboratories. Herein, we have documented the processes followed and challenges encountered during the successful establishment of the COVID-19 laboratory by the Malaria and NTDs research team at the EPHI.The Malaria and NTDs team at EPHI had limited capacity in molecular testing. The existing resource focus was on Chelate extraction capacity and molecular testing of drug resistance using conventional thermocycler and gel electrophoresis. The laboratory was mainly inactive due to weak procurement systems for molecular reagents. However, the key equipment for molecular work including thermocycler, centrifuges, vortex, hotplate and pipettes was available in the laboratory.To develop clear structures of accountability and leadership, a steering committee was formed by voluntary team members from the Malaria and NTDs research team to lead the laboratory establishment. The nucleus team comprised of the laboratory manager, quality officer, safety officer and the team leader. The group started identifying space in parallel with mobilizing materials and consumables. Available equipment and consumables in the Malaria and NTD research laboratory and additional consumables provided by the national Emergency Operation Centre (EOC) targeting COVID-19 testing were taped and utilized for establishment.COVID-19 molecular laboratory testing has its own set of safety requirements with the need to have at least level two safety cabinets or equivalents . The staSimultaneously with room renovation, the team started locating and collecting standard materials required for the new laboratory. The EPHI is a historic institute (more than 100 years since establishment) and large institute with significant human potential and equipment . The firIn parallel, part of the team was mobilizing molecular laboratory instruments such as pipettes, tips, vortex, centrifuges, spines, hotplates, biohazard bags and other consumables to start the actual testing. Most of them were collected from the Malaria and NTDs laboratories at EPHI.In the process of infrastructure and equipment organization, the team benefited from and was capacitated in leadership, team work, critical thinking in problem solving and leveraging human and material resources at the institute.The establishment team generally agreed to have a separate setup for dirty rooms (sample reception and extraction) and clean rooms (master mix). Sample flow was organized in such a way that the dirty rooms do not contaminate the clean rooms. Maximum efforts were exerted to prevent contamination and infection from the dirty rooms .Figure PCR reagents and Personal Protection Equipment (PPE) were made available through the national COVID-19 response team and aid from the Chinese philanthropist Jack Ma ,8. The tThe collected samples were extracted using Da An nucleic acid extraction kit ; a master mix was prepared using Da An detection kit for Novel Coronavirus (2019-nCoV) RNA  and amplified. The results were twofold; the system worked but the result was undetermined and non-interpretable. Similar tests and results were repeated two to three times. Potential contamination from unknown source was suspected. In the meantime, since the setup of the laboratory was convenient, experts from EPHI’s virology unit requested to extract their samples using the extraction facility, this was in part to avoid the overcrowding at the virology laboratory. The extracts were all contaminated when tested in the newly established laboratory. The nucleus team started searching for the source of the contamination one by one.The entire extraction room, the safety cabinet and all materials including freezers were cleaned using bleach and 70% alcohol to control contamination from nucleic acid and other cellular contaminants that may interfere with assay detection. The room was fumigated with formaldehyde and neutralized. The safety cabinet was exposed to UV light and the laminar fan was kept turned on and running for a couple of days.In the amplification room, all the pipettes were calibrated per the national standards and exposed to UV light; the door to the extraction room was closed and human movement was restricted, except for authorized persons. All the rooms were thoroughly cleaned and fumigated.The team ran the experiment again; however, the result was further frustrating as it showed that the contamination had not yet been removed. The situation was considered as a total laboratory contamination and a corrective action plan was agreed upon. Given the inability to turnaround the samples in the expected timeframe, the COVID-19 national coordinating team notified the Malaria and NDTs team to return the samples. Repeated attempts at extraction and detection failed following which the samples were returned by keeping aliquots. The search for the source of contamination continued. At this stage, most colleagues were frustrated and little to no support remained.To mitigate the challenges, an experiment was designed to extract the samples and master mix in different COVID-19 testing laboratories at the EPHI and the detection was run using different instruments .Table 1.The results indicated that the source of the contamination was the master mix room and potentially the master mix cabinet, particularly the ill working internal airflow. The team decided to remove the safety cabinet from the laboratory and kept on searching for laminar flow from different laboratories. At the same time, a master mix was prepared without the cabinet and limited contamination was found. Whereas samples were requested again for processing by the Malaria and NTDs team, the national COVID-19 testing team was hesitant to accommodate the request. The team received a few samples, ran experiments, and observed encouraging results. Before releasing the results, the team found a new laminar flow fitted with a UV light from one of the sister laboratories at EPHI. The experiment was repeated, and the results were confirmed. The results were confirmed in another laboratory and finally the new laboratory under the Malaria and NTDs team joined the chain of national COVID-19 testing laboratories with an initial testing capacity of 50–100 samples per day, with turnaround time of 24 hours. The new laboratory adopted a sample pooling strategy with four samples pooled together and only pools that have tested positive are individually extracted while the negative pools being reported as negative samples [The laboratory design was revised , it is rThe Emergency Operation Centre (EOC) of the centre for Public Health Emergency Management (cPHEM) at EPHI is the coordinating body for COVID-19 surveillance, diagnosis, and response. The laboratory section at the EOC coordinates the sample collection from health facilities, communities, congregate settings and vulnerable groups. The sample management unit at the EOC receives samples collected by trained health professionals using standard viral transporting medium (VTM) reinforced by triple packaging, following receipt the samples get registered and distributed to COVID-19 testing laboratories in Addis Ababa. After sample processing, using the Demographic and Health Information System-2 (DHIS-2) platform negative results dispatched to patients via short message service (SMS) and by email to institutions whereas the case management unit of the EOC communicate positive results to the relevant entities for appropriate action .Figure The new laboratory is named Parasitology Covid-19 testing Laboratory at EPHI (PCL-EPHI). With a strong commitment, teamwork, leadership and strong management support, an effective COVID-19 testing laboratory was established in a resource-limited setting, at a point in time when laboratory capacity expansion was most critical. Some of the lessons learned include: 1) Repurpose existing laboratory spaces using national and WHO guidelines 2) Mobilize underutilized resources such as equipment and human resources for COVID-19 laboratory setup. Particularly equipment available for health research and diagnostics 3) Collaborate with local and international health experts and equipment manufacturers and agents to solve laboratory issues 4) Ensure that space and equipment identified for COVID-19 testing is sterile and contamination free from the outset. Universities (there are 40 public universities in Ethiopia), hospitals and research institutes may follow a similar approach to establish COVID-19 testing and/or other emergency laboratories using available resources.Currently, COVID-19 is increasingly spreading in Ethiopia as part of the global pandemic. The Ethiopian population is estimated to be 114 million  and the


Lymphoedema is a well-known concern for cancer survivors. A crucial issue in lymphoedema is that we cannot predict who will be affected, and onset can occur many years after initial cancer treatment. The variability of time between cancer treatment and lymphoedema onset is an unexplained mystery. Retrospective cohort studies have investigated the risk factors for lymphoedema development, with extensive surgery and the combination of radiation and surgery identified as common high-risk factors. However, these studies could not predict lymphoedema risk in each individual patient in the early stages, nor could they explain the timing of onset. The study of anatomy is one promising tool to help shed light on the pathophysiology of lymphoedema. While the lymphatic system is the area least investigated in the field of anatomical science, some studies have described anatomical changes in the lymphatic system after lymph node dissection. Clinical imaging studies in lymphangiography, lymphoscintigraphy and indocyanine green (ICG) fluorescent lymphography have reported post-operative anatomical changes in the lymphatic system, including dermal backflow, lymphangiogenesis and creation of alternative pathways via the deep and torso lymphatics, demonstrating that such dynamic anatomical changes contribute to the maintenance of lymphatic drainage pathways. This article presents a descriptive review of the anatomical and imaging studies of the lymphatic system in the normal and post-operative conditions and attempts to answer the questions of why some people develop lymphoedema after cancer and some do not, and what causes the variability in lymphoedema onset timing. Lymphoedema is a well-known side-effect of post-cancer treatment. The standard treatment for lymphoedema includes the daily wearing of a compression garment on the affected body part to prevent the progression of the disease . ManagemOne of the crucial issues in lymphoedema is that oncologists cannot predict which patients will suffer from the disease in the future and which will be spared. Lymphoedema can develop at any time from six months to many years after completion of cancer treatment, and, thus, the onset is variable . AlthougThe study of lymphatic anatomy is considered to be one of the promising tools to help explain the pathophysiology of lymphoedema. The lymphatic system is a part of the vascular system, but it has been far less studied than the blood circulatory system. The role of the lymphatic system is to collect and transport interstitial tissue fluid and protein substances that have escaped from the blood capillaries. The dysfunction of the lymphatic system causes oedema in the affected regions. Cancer cells also travel via the lymphatic system, and the process of cancer cells reaching the regional lymph nodes and taking root there is known as lymphogenous metastasis. Hence, the lymphatic system is commonly targeted in cancer treatment to prevent the disease from spreading. In this way, cancer treatment is a direct cause of lymphoedema.Previous anatomical studies have focused on the development of a lymphoedema model in animals and patients affected by lymphoedema, but less attention has been paid to patients who survived cancer and did not develop lymphoedema afterwards ,10,11,12This article presents a review of studies into the anatomical changes that occur in the lymphatic system post-lymph node dissection and discusses anatomical theories as to why some patients develop lymphoedema and others do not.Precise knowledge of normal lymphatic anatomy provided the baseline information needed to identify the changes to lymphatic structures resulting from lymph node dissection. Lymphatic vessels originate as avalvular lymphatic capillaries in the superficial dermis and connect to valvular pre-collectors in the deep dermis. Pre-collectors merge with each other and change course to run vertically from the skin to the deep layer. These vertical vessels connect to the superficial lymph-collecting vessels running horizontally in the subcutaneous layer. The deep lymph-collecting vessels run alongside the major arteries below the deep fascia  18,19].,19.18,19In lymphoedema, excess lymph fluid and proliferation of adipose tissue occur predominantly in the subcutaneous fat layer above the deep fascia ,21. ThesThe skin can be divided into superficial lymphatic territories called “lymphosomes”  22,23].,23.22,23There are two types of lymph nodes, known as regional and interval nodes. The axillary and inguinal nodes are regional nodes, and the popliteal and epitrochlear nodes are interval nodes . The difThe focus in lymphoedema is generally the lymphatics of the upper or lower limb, and the torso is often overlooked. The lymphatics in the torso are divided by the sagittal midline and horizontal line at the umbilical level . Each quThe presence of dermal backflow is a specific imaging criterion for the diagnosis of lymphoedema. This indicator was originally used in lymphangiography . When raDermal backflow is composed of dilated dermal lymphatic capillaries and pre-collectors . It deveDermal backflow has two functions in regulating lymph transport in lymphoedema . Firstly, if patent, superficial lymph-collecting vessels remain in the affected limb, it bridges the gap between the non-patent and patent vessels. Secondly, if all the superficial lymph-collecting vessels have deteriorated, the dermal backflow becomes the only pathway to drain lymph fluid from the affected area. The lymph capillaries and pre-collectors in the dermis are several times smaller than the lymph-collecting vessels , so if dThe lymphatic system can regenerate after lymph node dissection. Animal studies found that although lymph nodes themselves did not regenerate, the lymphatic vessels could regenerate to develop new lymph drainage pathways  36,37].,37.36,37Clinical imaging studies using lymphangiography, lymphoscintigraphy and ICG lymphography also found that the lymphatic system in the affected limb could reconnect to the remaining lymph nodes through lymphangiogenesis ,40,41,42When a patient undergoes lymph node dissection and does not develop lymphoedema later, the common assumption is that a part of the original lymphatic pathway is intact. For example, breast surgeons do not skeletonize the axillary vein during axillary node dissection, because anecdotal experience suggests an increasing chance of lymphoedema development. However, anatomical studies indicate that both the breast and arm sentinel nodes were located in level-one nodes in the lateral axillary region . AlthougWe have previously reported that the superficial and deep lymph-collecting vessels are independent of each other in normal anatomy ,19. HoweThe lymphatic system in the torso can be affected by lymph node dissection, with either breast or abdominal oedema identified. Newer techniques have been developed to reduce the extent of breast surgery, and breast-conserving therapy is becoming a more popular option in early breast cancer . Breast-Breast and abdominal oedema can be independent of, or associated with, lymphoedema in the limbs. In some cases of advanced breast cancer-related lymphoedema, dermal backflow covered the whole arm and extended to the front chest. The tracer drained to the internal mammary lymph nodes via the perforating lymphatic vessels or crossed the front sagittal midline and drained to the contralateral axillary nodes via normal lymphatics in the unaffected breast  17]..17].In these cases, the lymphatic system in the torso worked as a collateral lymphatic drainage pathway from the affected arm. Similar anatomical changes were observed in leg lymphoedema. Advanced unilateral leg lymphoedema sometimes drained to the contralateral inguinal nodes via the lymphatics in the lower abdomen. The possible interaction of the lymphatic system between the limbs and torso should be considered in post-lymph node dissection because limb lymphoedema can cause breast and abdominal oedema.Knowledge of normal lymphatic anatomy and an understanding of how it changes post-operatively may shed light on the pathophysiology of lymphoedema. Olszewski conducted lymphangiography studies to investigate post-operative lymphoedema in animal models . His resWhen axillary lymph node dissection is performed, the superficial lymphatic drainage pathway from the arm is interrupted and a surgical break is created because arm sentinel nodes are located in level-one nodes in the lateral axilla . The deeWhen the break occurs, lymph fluid leaks from the stump of the lymph-collecting vessels, and the surgical drains remove the pooled fluid in the defective tissue. Lymphoedema does not develop immediately because excess fluid is removed from the wound.Lymphangiogenesis occurs during the wound-healing process to restore the break caused by surgery. New lymphatics are regenerated from the distal stumps of the arm lymph-collecting vessels and connect to the remaining lymph nodes . The patThe regenerated lymphatics develop from immature vessels into stable, mature vessels. If this process is successful, lymphoedema may not develop at all. Scar formation, delayed wound-healing with seroma, post-operative irradiation, and cellulitis are the factors that are considered to adversely impact the maturation process and may cause regression of the immature vessels.If the temporarily restored lymphatics lose function, lymphoedema will develop. Obstruction of the superficial lymph-collecting vessels causes incompetent valves to form in the pre-collectors, resulting in dermal backflow, forming a connection between patent and non-patent lymph-collecting vessels ,42. If tOlszewski’s study found that the gap in time between lymph node dissection and lymphoedema development may be explained by the length of time taken for the initial bridging process through lymphangiogenesis to occur, followed by the regression of the regenerated lymphatics . Akita’sSeveral ideas have been proposed to prevent the development of lymphoedema. One is the vascular endothelial growth factor C (VGEF-C) drug that has been developed to the clinical trial stage . AnotherThe exact factors that determine which patients will develop lymphoedema after cancer treatment have not yet been identified. However, medical imaging plays an important role in screening patients before lymphoedema develops, and techniques to do this are advancing all the time. Anatomical theories are also vital in promoting understanding about what structural changes occur post-surgery and how these changes correlate with lymphoedema development.Lymphoedema is a debilitating condition that affects many people who have survived cancer. However, we are still unable at present to identify who among them will develop lymphoedema and who will not. This paper has reported on the anatomical changes that occur after surgery and how they contribute to the maintenance of lymphatic drainage pathways. We have also described how the mechanism of lymphangiogenesis works to bridge the gap in the lymphatics caused by surgery and demonstrated that regression of the immature lymphatic vessels may explain the delayed onset of cancer-related lymphoedema.