Lysine L-lactylation is the dominant lactylation isomer induced by glycolysis.

Lysine L-lactylation (Kl-la) is a novel protein posttranslational modification (PTM) driven by L-lactate. This PTM has three isomers: Kl-la, N-ε-(carboxyethyl)-lysine (Kce) and D-lactyl-lysine (Kd-la), which are often confused in the context of the Warburg effect and nuclear presence. Here we introduce two methods to differentiate these isomers: a chemical derivatization and high-performance liquid chromatography analysis for efficient separation, and isomer-specific antibodies for high-selectivity identification. We demonstrated that Kl-la is the primary lactylation isomer on histones and dynamically regulated by glycolysis, not Kd-la or Kce, which are observed when the glyoxalase system was incomplete. The study also reveals that lactyl-coenzyme A, a precursor in L-lactylation, correlates positively with Kl-la levels. This work not only provides a methodology for distinguishing other PTM isomers, but also highlights Kl-la as the primary responder to glycolysis and the Warburg effect.

Survey of bioavailable PCDDs, PCDFs, dioxin-like PCBs, and PBBs in air, water, and sediment media using semipermeable membrane devices (SPMDs) deployed in the Hartbeespoort Dam area, South Africa.

A survey of bioavailable polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), dioxin-like PCBs (dl-PCBs), and polybrominated biphenyls (PBBs) from ambient air, water and sediment was performed in the Hartbeespoort Dam area in South Africa, a region where data on highly toxic Stockholm Convention persistent organic pollutants (POPs) is scanty. The sampling was designed to simulate POP bioaccumulation in benthic and aquatic dwelling organisms as well as ambient air for estimation of ecological risk. The objective was to survey the spatiotemporal distribution and fate of bioavailable priority persistent organic compounds in the Hartbeespoort Dam in summer, autumn and winter seasons and to validate the utility of a comprehensive two-dimensional gas chromatography-time of flight mass spectrometry (GCxGC-TOF) method for PCDD/F, PCB, and PBB analysis. The highest detection rates for bioavailable priority POPs were for PCB 77 and PCB 126 which were detected in 15 and 16 of the 22 samples, though the majority of the detections were < LOQ for PCB 77. Overall, PCB 126, PBB 10 and PBB 49 recorded the highest quantified bioavailable concentrations per site in SPMDs deployed in the Hartbeespoort Dam. The SPMDs deployed in air at the Magalies River site in winter recorded the highest toxic equivalency quotient (TEQ) of 29.77 pg TEQ SPMD-1. The highest TEQs recorded for SPMDs deployed in the sediment phase were 10.2, 3.3, and 3.2 pg TEQ SPMD-1, recorded at the Harbour site in summer, Dam wall in summer and Harbour in winter respectively. In water, SPMDs deployed at the Crocodile River site recorded the highest TEQ of 0.81 pg TEQ SPMD-1 in summer. TEQ data shows that air carries significant bioavailable dl-toxicity compared to the water phase, and sediment generally carries the highest dl-toxicity. Detection rates for bioavailable PBBs were generally very low, with < 3 detections being quantified above the LOQ for the majority of the sites. Statistical analysis of TEQs computed at all sites, using AVOVA shows that the dispersion of TEQs in the Hartbeespoort Dam is largely homogenous as the differences between the TEQs were insignificant (p > 0.05).

Organ-specific bioaccumulation of PCBs and PAHs in African sharptooth catfish (Clarias gariepinus) and common carp (Cyprinus carpio) from the Hartbeespoort Dam, South Africa.

The distribution of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in the muscle, liver, spleen and kidney tissue of two fish species was studied using an optimised diatomaceous earth assisted modified QuEChERS extraction method. Five-year-old free-ranging male African sharptooth catfish (Clarias gariepinus) and 5-year-old male common carp (Cyprinus carpio) sampled from the Hartbeespoort Dam in South Africa were used for method development. Acetonitrile extraction produced more precise recoveries than hexane extraction. Fluorene and naphthalene were the most abundant PAHs detected in the majority of fish tissues analysed. PAH bioaccumulation in 5-year-old carp and 5-year-old catfish was in the order muscle > kidney > liver > spleen and liver > muscle > kidney > spleen, respectively. PCBs were mostly detected in carp spleen and kidney. Two-year-old carp were analysed to determine PCB and PAH bioaccumulation trends. The differences in ∑16PAH concentrations between the four organs tested were all statistically insignificant for the 3 fish tested (p > 0.05). All other organs with the exception of 5-year-old carp spleen and 5-year-old carp kidney recorded total 31 PCB concentrations (∑31PCB) < 25 ng g-1. Only 5-year-old carp spleen (∑31PCB of 592 ng g-1) and 5-year-old carp kidney (∑31PCB of 561 ng g-1) had significant differences (p < 0.05) from the spleen and kidney in 5-year-old catfish and 2-year-old carp. Whilst the carp and catfish sampled can be considered low PCB risk foods, 5-year-old carp muscle can be considered to be a high PAH risk food, with a benzo(a)pyrene concentration of 7 µg g-1, based on the EU Commission Regulation 2005/208/EC pertaining to the maximum permissible benzo(a)pyrene level in fresh fish muscle.

Use of the Chemcatcher® passive sampler and time-of-flight mass spectrometry to screen for emerging pollutants in rivers in Gauteng Province of South Africa.

Many rivers in urbanised catchments in South Africa are polluted by raw sewage and effluent to an extent that their ecological function has been severely impaired. The Hennops and Jukskei Rivers lying in the Hartbeespoort Dam catchment are two of the worst impacted rivers in South Africa and are in need of rehabilitation. Passive sampling (Chemcatcher® with a HLB receiving phase) together with high-resolution tandem mass spectrometry-targeted screening was used to provide high sensitivity and selectivity for the identification of a wide range of emerging pollutants in these urban waters. Over 200 compounds, including pesticides, pharmaceuticals and personal care products, drugs of abuse and their metabolites were identified. Many substances (~ 180) being detected for the first time in surface water in South Africa. General medicines and psychotropic drugs were the two most frequently detected groups in the catchment. These accounted for 49% of the emerging pollutants found. Of the general medicines, antihypertensive agents, beta-blocking and cardiac drugs were the most abundant (28%) classes detected. The Hennops site, downstream of a dysfunctional wastewater treatment plant, was the most polluted with 123 substances detected. From the compounds detected, peak intensity-based prioritisation was used to identify the five most abundant pollutants, being in the order caffeine > lopinavir > sulfamethoxazole > cotinine > trimethoprim. This work provides the largest available high-quality dataset of emerging pollutants detected in South African urban waters. The data generated in this study provides a solid foundation for subsequent work to further characterise (suspect screening) and quantify (target analysis) these substances.

Source characterisation and distribution of selected PCBs, PAHs and alkyl PAHs in sediments from the Klip and Jukskei Rivers, South Africa.

A study of the distribution of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) utilising 16 priority PAHs, benzo(e)pyrene, perylene, 19 alkylated PAHs and 31 ortho substituted PCBs in South Africa is presented. It was aimed to (a) deduce characteristic contamination patterns for both PCBs and PAHs and (b) provide the first comprehensive dataset for establishment of source characterisation of PCBs and PAHs. This is in line with new South African legislation on mandatory monitoring of PCB and PAH emissions. Bar charts, principal component analysis (PCA) and biplots were utilised to identify signature contamination patterns and distribution of PCBs and PAHs within the Jukskei and Klip Rivers. Sediments from the Jukskei and Klip River catchments both showed distinct contamination signatures for hexa to nonachlorinated PCBs, characteristic of contamination by Aroclor 1254 and 1260 technical mixtures. PCB signature patterns in order of abundance were 138 > 180 > 206 > 153 > 187 > 149 and 138 > 153 > 180 > 149 > 187 > 110 > 170 for the Jukskei and Klip River sediments, respectively. The upstream Alberton point had the highest Σ31 PCB and Σ (parent+alkyl) PAH concentrations in the Klip River of 61 and 6000 µg kg-1 dry weight (dw), respectively. In the Jukskei River, the upstream Marlboro point had the highest Σ31 PCB concentration of 19 µg kg-1 dw and the N14 site recorded the highest Σ (parent+alkyl) PAH concentration of 2750 µg kg-1 dw. PAH concentrations in both the Jukskei and Klip Rivers were significantly higher than the PCB concentrations. Fluoranthene, phenanthrene and pyrene were found in the highest concentrations in both the Jukskei and Klip River sediments. Both the Jukskei and Klip River sediments showed trends of a mixed pyrogenic-petrogenic PAH source contamination.

Utility of an alternative method (to USEPA Method 1613) for analysis of priority persistent organic pollutants in soil from mixed industrial-suburban areas of Durban, South Africa.

This study evaluates the adequacy of a USEPA Method 1613 alternative analytical method for analysis of persistent organic pollutants (POPs) in soil from the immediate vicinity of industrialized areas in the eThekwini municipal area in South Africa. The objective of this study is in line with the Stockholm Convention Article 11 on research, development, and monitoring. Furthermore, it became imperative to find an alternative analytical procedure to USEPA Method 1613 that could cater to studies conducted in Africa where recent reviews have indicated that most African countries lack the technical and instrumental capacity for performing analysis of dioxin-like compounds according to USEPA Method 1613, which entails the use of high-resolution chromatography and high-resolution mass spectrometry instrumentation. The study aimed to ascertain the utility of an alternative two-dimensional gas chromatography-time of flight mass spectrometry method for analysis of trace-level priority POPs in soil, along with a fast single quadrupole gas chromatography-mass spectrometry method. The analytical methods were applied to the analysis of POPs on soil samples from industrial areas with oil refineries and a pulp and paper manufacturing company, while other samples were collected near the electricity substations and a landfill site. Analytical results showed BDE 209 as the dominant contaminating polybrominated diphenyl ether (concentration ranges from 0.006 to 5.71 ng g-1 ). Polybrominated biphenyls (PBBs) 9, 10, and 49 were the dominant PBBs detected in 78% of the sites tested, although their concentrations were below the limit of quantification (LOQ). Polychlorinated dibenzo-p-dioxins and furans and dioxin-like polychlorinated biphenyls detected could not be quantified above their respective LOQs, indicating that the Durban area has low priority pollutant contamination levels compared to other regions around the world. The methods developed are a starting point that will inform considerations for routine evaluation and management of soil contamination, which plays a vital role in environmental management. Integr Environ Assess Manag 2023;19:749-762. © 2022 SETAC.

Adjustment of Matrix Effects in Analysis of 36 Secondary Metabolites of Microbial and Plant Origin in Indoor Floor Dust Using Liquid Chromatography-Tandem Mass Spectrometry.

Exposure to microbial agents in water-damaged buildings is a major public health concern. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a primary tool for testing environmental samples for microbial secondary metabolites (SMs); however, matrix effects can lead to inaccurate results in exposure assessment. Applying a universal internal standard (ISTD) and a matrix-matched calibration can adjust for matrix effects, as shown by our previous study. However, there are only few isotope-labeled internal standards for SMs available on the market. In this study, we determined the best-performing ISTDs among ten candidates (nine 13C-labeled isotopes and one unlabeled analogue) for each of 36 SMs. We analyzed school floor dust spiked with the 36 SMs to identify the best-performing ISTDs (initial experiment) and examined reproducibility with the selected ISTDs and the same spiked dust (validation 1). We also tested applicability for the selected ISTDs using spiked dust collected from different schools (validation 2). The three experiments showed that 26, 17, and 19 SMs had recoveries within the range 100 ± 40%. 13C-ochratoxin A and 13C-citrinin were most frequently selected as the best ISTDs for the 36 SMs, followed by deepoxy-deoxynivalenol, 13C-sterigmatocystin, and 13C-deoxynivalenol. Our study shows that using the identified, best-performing analogous ISTDs for those metabolites may improve testing accuracy for indoor dust and help better estimate exposure effects on potential health.

Seasonal variation of chloro-s-triazines in the Hartbeespoort Dam catchment, South Africa.

Seasonal variation of eight chloro-s-triazine herbicides and seven major atrazine and terbuthylazine degradation products was monitored in the Hartbeespoort Dam catchment using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS/MS). Lake, river and groundwater were sampled from the Hartbeespoort Dam catchment over four seasons and the downstream Jukskei River was monitored during the winter season. Triazine herbicide concentrations in the Hartbeespoort Dam were in the order atrazine>simazine>propazine>ametryn>prometryn throughout the four seasons sampled. Triazine herbicide concentrations in the Hartbeespoort Dam surface water were highest in summer and gradually decreased in successive seasons of autumn, winter and spring. Terbuthylazine was the only triazine herbicide detected at all sampling sites in the Jukskei River, though atrazine recorded much higher concentrations for the N14 and Kyalami sites, with concentrations of 923 and 210ngL-1 respectively, compared to 134 and 74ngL-1 for terbuthylazine. Analytical results in conjunction with river flow data indicate that the Jukskei and Crocodile Rivers contribute the greatest triazine herbicide loads into the Hartbeespoort Dam. No triazine herbicides were detected in the fish muscle tested, showing that bioaccumulation of triazine herbicides is negligible. Atrazine and terbuthylazine metabolites were detected in the fish muscle with deethylatrazine (DEA) being detected in both catfish and carp muscle at low concentrations of 0.2 and 0.3ngg-1, respectively. Desethylterbuthylazine (DET) was detected only in catfish at a concentration of 0.3ngg-1. With atrazine herbicide groundwater concentrations being >130ngL-1 for all seasons and groundwater ∑triazine herbicide concentrations ranging between 527 and 367ngL-1, triazine compounds in the Hartbeespoort Dam catchment may pose a risk to humans and wildlife in light findings of endocrine and immune disrupting atrazine effects by various researchers.

Contaminants of emerging concern in the Hartbeespoort Dam catchment and the uMngeni River estuary 2016 pollution incident, South Africa.

A quantitative assessment of pollutants of emerging concern in the Hartbeespoort Dam catchment area was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to establish the occurrence, source and distribution of 15 environmental pollutants, including 10 pharmaceuticals, 1 pesticide and 4 steroid hormones. Seasonal sampling was conducted in the Hartbeespoort Lake using sub-surface grab sampling to determine the lake's ecological status and obtain data for establishment of progressive operational monitoring. The Jukskei River, which lies upstream of the Hartbeespoort Dam, was sampled in the winter season. Five year old carp (Cyprinus carpio) and catfish (Clarias gariepinus) were also sampled from the Hartbeespoort Dam to study bioaccumulation in biota as well as to estimate risk associated with fish consumption. In the Jukskei River, the main source of 11 emerging pollutants (EPs) was identified as raw sewage overflow, with the highest ∑11 EP concentration of 593ngL-1 being recorded at the Midrand point and the lowest ∑11 EP concentration of 164ngL-1 at the N14 site located 1km downstream of a large wastewater treatment plant. The Jukskei River was found to be the largest contributor of the emerging contaminants detected in the Hartbeespoort Dam. In the Hartbeespoort Dam EP concentrations were generally in the order efavirenz>nevirapine>carbamazepine>methocarbamol>bromacil>venlafaxine. Water and sediment were sampled from the uMngeni River estuary within 24h after large volumes of an assortment of pharmaceutical waste had been discovered to be washed into the river estuary after flash rainfall on 18 May 2016. Analytical results revealed high levels of some emerging pollutants in sediment samples, up to 81ngg-1 for nevirapine and 4ngg-1 for etilefrine HCL. This study shows that efavirenz, nevirapine, carbamazepine, methocarbamol, bromacil and venlafaxine are contaminants that require operational monitoring in South African urban waters.

Effects of environmentally relevant sub-chronic atrazine concentrations on African clawed frog (Xenopus laevis) survival, growth and male gonad development.

Sub-chronic toxicity of environmentally relevant atrazine concentrations on exposed tadpoles and adult male African clawed frogs (Xenopus laevis) was evaluated in a quality controlled laboratory for 90 days. The aim of this study was to determine the effects of atrazine on the survival, growth and gonad development of African clawed frogs. After exposure of tadpoles to atrazine concentrations of 0 (control), 0.01, 200 and 500 µg L-1 in water, mortality rates of 0, 0, 3.3 and 70% respectively were recorded for the 90 day exposure period. Morphometry showed significantly reduced tadpole mass in the 500 µg L-1 atrazine exposed tadpoles (p < 0.05). Light microscopy on testes of adult frogs exposed to the same atrazine concentrations using hematoxylin and eosin (H&E) and Van Gieson staining techniques revealed gonadal atrophy, disruption of germ cell lines, seminiferous tubule structure damage and formation of extensive connective tissue around seminiferous tubules of frogs exposed to 200 µg L-1 and 500 µg L-1 atrazine concentrations. Ultrastructural analysis of the cellular organelles using transmission electron microscopy (TEM) revealed significant amounts of damaged mitochondria in testosterone producing Leydig cells as well as Sertoli cells. Biochemical analysis revealed reduced serum testosterone levels in adult frogs at all exposure levels as well as presence of six atrazine metabolites in frog serum and liver. The results indicate that atrazine concentrations greater than the calculated LC50 of 343.7 µg L-1 cause significant mortality in tadpoles, while concentrations ≥200 µg L-1 adversely affect reproductive health of adult frogs and development of tadpoles sub-chronically exposed to atrazine.

Distribution of 2,3,7,8-substituted polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofurans in the Jukskei and Klip/Vaal catchment areas in South Africa.

Comprehensive two dimensional gas chromatography (GCxGC)-µECD analysis was used to determine 2,3,7,8-substituted dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) distribution in the Jukskei and Klip/Vaal catchment areas from ten sites previously identified as persistent organic pollutant hotspots in major rivers in the Gauteng province of South Africa. Five sediment samples from the Jukskei River catchment area and five sediment samples from the Kilp/Vaal River catchment area were collected for analysis. The extracts were screened for dioxin-like activity using the DR-Luc bioassay prior to GCxGC-µECD analysis. All sediment samples tested positive for dioxin-like activity with total activity ranging from 16 to 37 pg toxic equivalents (TEQ) g(-1) dry weight (dw) for the Jukskei River catchment and 1.5-22 pg TEQ g(-1) dw for the Klip/Vaal River catchment, indicating that the Jukskei River catchment area had higher concentrations of total dioxin-like compounds. Confirmatory tests for the presence of the most potent seven PCDDs and ten PCDFs conducted using GCxGC-µECD revealed presence of 11 PCDD/Fs and 6 PCDD/Fs in the Jukskei and Klip/Vaal River catchments respectively. Total organic carbon (TOC) and particle size distribution analysis were conducted to understand the distribution of PCDD/Fs within the Jukskei and Klip/Vaal catchments.

Alternative calibration techniques for counteracting the matrix effects in GC-MS-SPE pesticide residue analysis - a statistical approach.

This paper investigates the efficiency of application of four different multivariate calibration techniques, namely matrix-matched internal standard (MMIS), matrix-matched external standard (MMES), solvent-only internal standard (SOIS) and solvent-only external standard (SOES) on the detection and quantification of 20 organochlorine compounds from high, low and blank matrix water sample matrices by Gas Chromatography-Mass Spectrometry (GC-MS) coupled to solid phase extraction (SPE). Further statistical testing, using Statistical Package for the Social Science (SPSS) by applying MANOVA, T-tests and Levene's F tests indicates that matrix composition has a more significant effect on the efficiency of the analytical method than the calibration method of choice. Matrix effects are widely described as one of the major sources of errors in GC-MS multiresidue analysis. Descriptive and inferential statistics proved that the matrix-matched internal standard calibration was the best approach to use for samples of varying matrix composition as it produced the most precise average mean recovery of 87% across all matrices tested. The use of an internal standard calibration overall produced more precise total recoveries than external standard calibration, with mean values of 77% and 64% respectively. The internal standard calibration technique produced a particularly high overall standard deviation of 38% at 95% confidence level indicating that it is less robust than the external standard calibration method which had an overall standard error of 32% at 95% confidence level. Overall, the matrix-matched external standard calibration proved to be the best calibration approach for analysis of low matrix samples which consisted of the real sample matrix as it had the most precise recovery of 98% compared to other calibration approaches for the low-matrix samples.