Wastewater-based SARS-CoV-2 airport surveillance: key trends at the Cape Town International Airport.

Monitoring of SARS-CoV-2 RNA in wastewater has revealed the role of mobility in the transmission of coronavirus disease (COVID-19), and the surveillance of airport wastewater in cities across the world has demonstrated how travel entry points can give an indication of trends in transmission. This study undertook wastewater surveillance at the Cape Town International Airport (CTIA) to assess the use of a WBE approach to provide supplementary information on the presence of COVID-19 at a key air travel entry point in South Africa. Grab wastewater samples (n = 55) were collected from the CTIA wastewater pump station and analysed using quantitative real-time polymerase chain reaction (qRT-PCR) method. The study found a correlation between the wastewater data and clinical cases reported in the City of Cape Town during various time periods and during the peak of a COVID-19 wave. Highly elevated viral loads in the wastewater were observed at times there was increased mobility through the airport. The study also revealed elevated viral load levels at the airport despite the stricter restrictions and through the lower restrictions. The study findings indicate wastewater surveillance and airports can provide supplementary information to airport authorities to assess the impacts of imposed travel restrictions.

The benefit of using in vitro bioassays to screen agricultural samples for oxidative stress: South Africa's case.

Applied pesticides end up in non-target environments as complex mixtures. When bioavailable, these chemicals pose a threat to living organisms and can induce oxidative stress (OS). In this article, attention is paid to OS and the physiological role of the antioxidant defense system. South African and international literature was reviewed to provide extensive evidence of pesticide-induced OS in non-target organisms, in vivo and in vitro. Although in vitro approaches are used internationally, South African studies have only used in vivo methods. Considering ethical implications, the authors support the use of in vitro bioassays to screen environmental matrices for their OS potential. Since OS responses are initiated and measurable at lower cellular concentrations compared to other toxicity endpoints, in vitro OS bioassays could be used as an early warning sign for the presence of chemical mixtures in non-target environments. Areas of concern in the country could be identified and prioritized without using animal models. The authors conclude that it will be worthwhile for South Africa to include in vitro OS bioassays as part of a battery of tests to screen environmental matrices for biological effects. This will facilitate the development and implementation of biomonitoring programs to safeguard the South African environment.

Oxidative stress in the freshwater shrimp Caridina africana following exposure to atrazine.

The toxicity of pesticides to non-target organisms continues to be important in understanding the dynamic interactions between anthropogenic chemicals and ecosystem health. This study assesses biochemical markers to determine the effects that varying concentrations of atrazine (13.1-5557 µg/l) have on the freshwater shrimp, Caridina africana. Exposure and oxidative stress biomarkers were analysed and followed by univariate, integrated biomarker response v2 (IBRv2) and Kendall Tau correlation statistical analyses, to gain insight into the concentration-dependent responses. Oxidative stress biomarkers such as reduced glutathione content (GSH), glutathione-S-transferase activity (GST), superoxide dismutase activity (SOD) and catalase activity (CAT) were significantly correlated with increasing atrazine exposure concentration (p < 0.01). Bimodality has been seen when looking at both the univariate statistically significant differences as well as the IBRv2, with the first peak at 106.8 µg/l and the second peak at 5557 µg/l atrazine. The results indicate that while individual responses may indicate statistically significant differences, using correlation and integrated statistical analysis can shed light on trends in the adaptive response of these.

Pathogenic features of heterotrophic plate count bacteria from drinking-water boreholes.

Evidence suggests that heterotrophic plate count (HPC) bacteria may be hazardous to humans with weakened health. We investigated the pathogenic potential of HPC bacteria from untreated borehole water, consumed by humans, for: their haemolytic properties, the production of extracellular enzymes such as DNase, proteinase, lipase, lecithinase, hyaluronidase and chondroitinase, the effect simulated gastric fluid has on their survival, as well as the bacteria's antibiotic-susceptible profile. HuTu-80 cells acted as model for the human intestine and were exposed to the HPC isolates to determine their effects on the viability of the cells. Several HPC isolates were α- or ß-haemolytic, produced two or more extracellular enzymes, survived the SGF treatment, and showed resistance against selected antibiotics. The isolates were also harmful to the human intestinal cells to varying degrees. A novel pathogen score was calculated for each isolate. Bacillus cereus had the highest pathogen index: the pathogenicity of the other bacteria declined as follows: Aeromonas taiwanensis > Aeromonas hydrophila > Bacillus thuringiensis > Alcaligenes faecalis > Pseudomonas sp. > Bacillus pumilus > Brevibacillus sp. > Bacillus subtilis > Bacillus sp. These results demonstrated that the prevailing standards for HPCs in drinking water may expose humans with compromised immune systems to undue risk.

A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2',7'-dichlorodihydrofluorescein diacetate assay.

Exposure to xenobiotics can increase the production of reactive oxygen species (ROS). When detoxification organs such as the intestines and liver cannot neutralise these xenobiotics, it can induce oxidative stress and cause damage to tissues. Therefore, cell-based bioassays that indicate intracellular ROS production are a useful screening tool to evaluate the effect of these chemicals. Although flow cytometry is commonly used to measure ROS in cells, many research laboratories in the Global South do not always have access to such specialised instrumentation. Therefore, we describe a sensitive but low-cost method that can easily be used to determine ROS production in vitro. This method employs the fluorogenic dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), which emits fluorescence after being oxidised to a fluorescent derivative. Since the H2DCF-DA bioassay indicates non-specific ROS production it can be used as a marker of overall oxidative stress. This method was validated by exposing human duodenum epithelial adenocarcinoma (HuTu-80) and rat liver epithelial hepatoma (H4IIE-luc) cells to agricultural soil samples.•Production of ROS can be determined in vitro in intestinal and liver cells.•This method is inexpensive and can be easily performed in standard laboratories.•The method provides a tool for the high-throughput screening of environmental samples.

Distribution of antibiotic resistance genes and antibiotic residues in drinking water production facilities: Links to bacterial community.

There is a rapid spread of antibiotic resistance in the environment. However, the impact of antibiotic resistance in drinking water is relatively underexplored. Thus, this study aimed to quantify antibiotic resistance genes (ARGs) and antibiotic residues in two drinking water production facilities (NW-E and NW-C) in North West Province, South Africa and link these parameters to bacterial communities. Physicochemical and ARG levels were determined using standard procedures. Residues (antibiotics and fluconazole) and ARGs were quantified using ultra-high performance liquid chromatography (UHPLC) chemical analysis and real-time PCR, respectively. Bacterial community compositions were determined by high-throughput 16S rRNA sequencing. Data were analysed using redundancy analysis and pairwise correlation. Although some physicochemical levels were higher in treated than in raw water, drinking water in NW-E and NW-C was safe for human consumption using the South African Water Quality Guideline (SAWQG). ARGs were detected in raw and treated water. In NW-E, the concentrations of ARGs (sul1, intl1, EBC, FOX, ACC and DHA) were higher in treated water than in raw water. Regarding antimicrobial agents, antibiotic and fluconazole concentrations were higher in raw than in treated water. However, in NW-C, trimethoprim concentrations were higher in raw than in treated water. Redundancy analysis showed that bacterial communities were not significantly correlated (Monte Carlo simulations, p-value >0.05) with environmental factors. However, pairwise correlation showed significant differences (p-value <0.05) for Armatimonas, CL500-29 marine group, Clade III, Dickeya and Zymomonas genera with environmental factors. The presence of ARGs and antibiotic residues in the current study indicated that antibiotic resistance is not only a clinical phenomenon but also in environmental settings, particularly in drinking water niches. Consumption of NW-E and NW-C treated water may facilitate the spread of antibiotic resistance among consumers. Thus, regulating and monitoring ARGs and antibiotic residues in drinking water production facilities should be regarded as paramount.

Determining superoxide dismutase content and catalase activity in mammalian cell lines.

Traditional methods for determining superoxide dismutase (SOD) content and catalase (CAT) activity rely on measuring the absorbance of individual tissue (biological) samples using a cuvette and spectrophotometer, rather than cell cultures. Although there are kits available for SOD and CAT assays, these allow for high-throughput analysis of samples and might be too expensive for research laboratories in countries from the Global South, such as South Africa. This paper describes a simple and cost-effective method to determine SOD content and CAT activity in mammalian cell cultures following exposure to environmental chemical mixtures by measuring absorbance in 96-well microplates. Moreover, the equipment used for this method is considered standard for cell culture laboratories, while the reagents and consumables are easily obtainable.•Antioxidant enzyme levels can be measured in vitro in cell cultures.•The supernatant obtained can be used to determine protein concentration, SOD content, and CAT activity.•This method is simple and affordable, allowing for the analysis of multiple samples (up to 32 samples per microplate).

Using Wastewater Surveillance to Compare COVID-19 Outbreaks during the Easter Holidays over a 2-Year Period in Cape Town, South Africa.

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown to be an important approach to determine early outbreaks of infections. Wastewater-based epidemiology (WBE) is regarded as a complementary tool for monitoring SARS-CoV-2 trends in communities. In this study, the changes in the SARS-CoV-2 RNA levels in wastewater during Easter holidays in 2021 and 2022 in the City of Cape Town were monitored over nine weeks. Our findings showed a statistically significant difference in the SARS-CoV-2 RNA viral load between the study weeks over the Easter period in 2021 and 2022, except for study week 1 and 4. During the Easter week, 52% of the wastewater treatment plants moved from the lower (low viral RNA) category in 2021 to the higher (medium to very high viral RNA) categories in 2022. As a result, the median SARS-CoV-2 viral loads where higher during the Easter week in 2022 than Easter week in 2021 (p = 0.0052). Mixed-effects model showed an association between the SARS-CoV-2 RNA viral loads and Easter week over the Easter period in 2021 only (p < 0.01). The study highlights the potential of WBE to track outbreaks during the holiday period.

The role of effect-based methods to address water quality monitoring in South Africa: a developing country's struggle.

Water is an important resource, and it is a worldwide struggle to provide water of good quality to the whole population. Despite good governing laws and guidelines set in place to help protect the water resources and ensure it is of good quality for various consumers, the water quality in South Africa is worsening due to lack of management. The deteriorating infrastructure is becoming progressively worse, due to corruption and insufficient funds. The ever-increasing number of toxicants, as well as the identification of emerging chemicals of concern, are also challenges South Africa is facing. Chemical analysis cannot determine the total biological effect of a mixture of chemical compounds, but this shortcoming can be addressed by adding effect-based methods (EBMs) to water quality monitoring programmes. In this paper, the current status of water quality monitoring in South Africa is discussed, as well as the capacity of the country to add EBMs to its water quality monitoring programmes to protect and improve human and animal life. Created in Biorender.com.

HIV-antiretrovirals in river water from Gauteng, South Africa: Mixed messages of wastewater inflows as source.

South Africa has the highest number of people living with the human immunodeficiency virus (HIV). High usage of HIV-antiretroviral drugs (ARVs) for the treatment of the acquired immunodeficiency syndrome (AIDS) leads to the presence of ARVs in the environment. Wastewater is a major contributor of pharmaceuticals in surface and drinking water as wastewater treatment plants (WWTPs) are not designed to remove these compounds. Pharmaceuticals in the environment pose risks and the effects of ARVs on non-target organisms are largely unknown. The concentrations of ARVs in surface water upstream and downstream from WWTPs in rivers were determined. The samples were extracted by solid-phase extraction and analysed by using liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Five ARVs were quantified, mostly in downstream samples of the WWTPs, indicating wastewater as a source of ARVs, but this was not apparent in all cases. Nevirapine, lopinavir, and efavirenz were frequently detected; the highest concentrations being lopinavir and efavirenz at 38 µg/L and 24 µg/L, respectively. Aquatic ecosystems are at risk due to the constant input of pharmaceuticals that include large amounts of everyday use and the release of ARVs. This study highlights the potential of increased water pollution worldwide should more people consume increased quantities of pharmaceuticals.

Agrochemicals in freshwater systems and their potential as endocrine disrupting chemicals: A South African context.

South Africa is the largest agrochemical user in sub-Saharan Africa, with over 3000 registered pesticide products. Although they reduce crop losses, these chemicals reach non-target aquatic environments via leaching, spray drift or run-off. In this review, attention is paid to legacy and current-use pesticides reported in literature for the freshwater environment of South Africa and to the extent these are linked to endocrine disruption. Although banned, residues of many legacy organochlorine pesticides (endosulfan and dichlorodiphenyltrichloroethane (DDT)) are still detected in South African watercourses and wildlife. Several current-use pesticides (triazine herbicides, glyphosate-based herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and chlorpyrifos) have also been reported. Agrochemicals can interfere with normal hormone function of non-target organism leading to various endocrine disrupting (ED) effects: intersex, reduced spermatogenesis, asymmetric urogenital papillae, testicular lesions and infertile eggs. Although studies investigating the occurrence of agrochemicals and/or ED effects in freshwater aquatic environments in South Africa have increased, few studies determined both the levels of agricultural pesticides present and associated ED effects. The majority of studies conducted are either laboratory-based employing in vitro or in vivo bioassays to determine ED effects of agrochemicals or studies that investigate environmental concentrations of pesticides. However, a combined approach of bioassays and chemical screening will provide a more comprehensive overview of agrochemical pollution of water systems in South Africa and the risks associated with long-term chronic exposure.

Antifungal agents, yeast abundance and diversity in surface water: Potential risks to water users.

South African surface waters are subject to various forms of pollution. Recent findings in aquatic systems suggest an association exists between yeast diversity, chemical pollutants and land coverage, which are important water quality determinants. Yeast abundance and diversity, as well as antifungal agents in two river systems in South Africa, were investigated and related to the existing land coverage. Yeast abundance and diversity were determined from environmental DNA by quantitative polymerase chain reaction and next-generation sequencing, respectively, of the 26S ribosomal ribonucleic acid (rRNA) gene. Antifungal agents were qualitatively and/or quantitatively detected by ultra-high-pressure liquid chromatography-mass spectrometry. Analyses of 2 031 714 high-quality 26S rRNA sequences yielded 5554 amplicon sequence variants (ASVs)/species. ASV richness and Shannon-Wiener index of diversity reflected the southward flow of the river with higher values observed downstream compared to the upstream. Fluconazole concentrations were quantifiable in only two samples; 178 and 271 ng L-1. Taxonomically, at least 20 yeast species were detected, including the dominant Candida tropicalis, Cryptococcus spp. as well as the lesser dominant Bensingtonia bomiensis, Fereydounia khargensis, Hericium erinaceus, Kondoa changbaiensi, Pseudozyma spp. and Sphacelotheca pamparum. The two dominant species are known opportunistic pathogens which had antifungal resistant traits in previous studies from the same rivers and therefore is a public health threat. The present study provides further evidence that yeasts should be included as part of water quality parameters, especially in developing countries where much of the population are economically disadvantaged, and also immunocompromised due to age and disease.

May agricultural water sources containing mixtures of agrochemicals cause hormonal disturbances?

Agricultural chemicals end up in the environment as complex mixtures and it is their combinatorial effects that need to be evaluated, rather than the traditional single effect of the active ingredients. This study emphasises effects-directed analyses (androgen receptor (AR) activity) of such environmentally relevant mixtures. Soil, where glyphosate and 2,4-dichloro-phenoxyacetic acid (2,4-D) were sprayed on Bt maize, were extracted with rainwater. This allowed to test the bio-available fraction. AR effects were measured with an in vitro reporter-gene assay using MDA-kb2 cells. The cells were exposed to: single active ingredients; formulations; environmentally relevant concentrations of the active ingredients and formulations; as well as rainwater extracts. The AR was activated by rainwater extracts from soil that received a pre-and post-emergent Roundup application. The testosterone equivalents (TTEQs) derived from AR activation exceeded international drinking water trigger values. We conclude that (i) rainwater run-off from maize sprayed with Roundup and 2,4-D contained androgen active substances and (ii) the chronic exposure to this water may cause endocrine disrupting effects in humans and aquatic life which emphasise the need for intensified monitoring of environmental water resources.

Cytotoxicity of Ag, Au and Ag-Au bimetallic nanoparticles prepared using golden rod (Solidago canadensis) plant extract.

Production and use of metallic nanoparticles have increased dramatically over the past few years and design of nanomaterials has been developed to minimize their toxic potencies. Traditional chemical methods of production are potentially harmful to the environment and greener methods for synthesis are being developed in order to address this. Thus far phytosynthesis have been found to yield nanomaterials of lesser toxicities, compared to materials synthesized by use of chemical methods. In this study nanoparticles were synthesized from an extract of leaves of golden rod (Solidago canadensis). Silver (Ag), gold (Au) and Ag-Au bimetallic nanoparticles (BNPs), synthesized by use of this "green" method, were evaluated for cytotoxic potency. Cytotoxicity of nanomaterials to H4IIE-luc (rat hepatoma) cells and HuTu-80 (human intestinal) cells were determined by use of the xCELLigence real time cell analyzer. Greatest concentrations (50 µg/mL) of Ag and Ag-Au bimetallic were toxic to both H4IIE-luc and HuTu-80 cells but Au nanoparticles were not toxic. BNPs exhibited the greatest toxic potency to these two types of cells and since AuNPs caused no toxicity; the Au functional portion of the bimetallic material could be assisting in uptake of particles across the cell membrane thereby increasing the toxicity.