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The development of an analytical system for β-nmethylamino- l-alanine and investigation of distribution of producing organisms and extent of freshwater contamination
Expanded Title:Beta-N-methylamino-L-alanine (BMAA) is produced by free-living cyanobacteria, with one of these being marine. BMAA has also been identified as a potential risk to human health as it is implicated in Alzheimer’s disease, Parkinsonism and Amyotrophic Lateral Sclerosis (ALS). The possibility of sustained 2. PROJECT ABSTRACT the extent of raw water contamination and treatment efficacy. Since biotoxins are often present in low concentrations, large volumes of water needed to be concentrated to be able to quantify BMAA. In order to assess the extent of free BMAA contamination of water, a concentration method needed to be developed. OBJECTIVES The stated aims of the proposal relating to BMAA research were: • To establish and validate the method to test for free and bound Beta-N-methylamino-L-alanine (BMAA) in water and cyanobacterial bloom samples • To establish a method to concentrate environmental BMAA from large volumes of water for the purpose of analysis • To initiate the formation of an axenic culture collection of South African cyanobacterial isolates and establish the ubiquity of BMAA production by cyanobacteria • To determine the prevalence of cyanobacterially produced BMAA in South African freshwater impoundments RESULTS AND CONCLUSIONS A culture collection of South African cyanobacterial isolates was established, expanded and maintained as part of this project. Over 100 impoundments were sampled over a period of two years and all culturable cyanobacterial isolates were made unialgal and maintained as live cultures in the culture collection. This collection served as a resource for evaluation of BMAA production within and between taxonomic groups. The extent and diversity of the culture collection was ideally suited to serve this purpose and therefore achieved the stated objective. BMAA analysis was optimized using a commercial chloroformate derivitization kit (EZ:faast) with gas chromatography - mass spectrometry (GC/MS) and high performance liquid chromatography - mass spectrometry (LC-MS) analsyis of derivatized amino acids. An in-house BMAA analytical system (LC-MS) was installed and validated using a chloroformate pre-derivatized BMAA standard (Sigma). The resulting molecule was quantified against three internal standards: homoarginine, methionine-D3 and homophenylalanine. The BMAA standard was derivatized in varying concentrations on one day and injected in triplicate to assess machine reproducibility and on 3 consecutive days in order to analyse derivatization reproducibility. From the information obtained a calibration curve was constructed based on the representative molecular ion (m/z = 333) for quantification. Multiple user, delayed derivitization and delayed analysis assessments were conducted to verify the system and determine how robust the developed methods were. Analytical stability was excellent with insignificant variation in individual quantification runs on a given sample. Derivitization reproducibility across the concentration range used for the calibration curve was acceptable (N = 5; r2 = 0.985). The GC-MS BMAA detection developed for this project (Esterhuizen & Downing, 2008) was 15 times more sensitive than previously published fluorescent 6-aminoquinolyl -N-hydroxysuccinimidyl carbamate-derivatized BMAA detection methods and the LC-MS BMAA detection method is 50-fold more sensitive. The lower limit of sensitivity for detection was below 100 pg per injection, and quantification was possible at 148 pg. BMAA was detected in 97% of all culture collection strains examined. All taxonomic divisions contained BMAA producing strains. BMAA was observed in both the unbound and protein associated forms but to different extents in different strains. The ratio of free to bound BMAA appeared to remain constant for any given strain. No taxonomic or geographic basis for BMAA production or the ratio of free to bound BMAA was observed. A concentration protocol based on a strong cation exchanger with hydrophobic interaction was developed for extraction and concentration of BMAA from raw water samples. Recovery of BMAA from sterile distilled water samples of either one or 20 L, spiked with between 23.8 ng L-1 and 1.14 ng L-1, ranged from 90 to 115% indicating complete recovery (SD at sample concentrations = 8%). Dam water collected during an Anabaena bloom was pre-filtered and concentrated as previously described. The sample was evaluated for the presence of extracellular BMAA. From 20 L of water, no BMAA was detected (less than 500 pg l-1). Filtrates were then hydrolysed in a non-quantitative manner and tested for BMAA using LC-MS with pre-derivitization. BMAA found in filtrate was in excess of 17 g L-1, and possibly as much as 40 g L-1 intracellular BMAA. 25 L of water from a second dam was concentrated and tested for the presence of BMAA. Again no BMAA could be detected. A third dam with a Microcystis bloom also contained no extracellular BMAA in two separate samples collected from the dam on two separate occasions. Biomass collected from the dam was positive for BMAA on both occasions. These data suggest that BMAA is not released into the environment, or is rapidly degraded in, or removed from the environment. The project yielded a reproducible and sensitive method for BMAA analysis from water or biomass and an efficient and simple method for concentration of BMAA from large water samples prior to analysis. exposure to BMAA via drinking water supplies prompted the establishment of local analytical capacity for the neurotoxin, urgent verification of the production of BMAA by free-living cyanobacteria, the evaluation of the distribution of BMAA producing free-living cyanobacteria in South Africa and the extent of BMAA contamination of surface waters. In the case of freshwater cyanobacteria BMAA would be released into raw water which may be used as a potable water source. The efficacy of current treatment systems for BMAA removal is unknown. The potential risk to consumers was therefore also unknown but could be determined by addressing the extent of raw water contamination and treatment efficacy. Since biotoxins are often present in low concentrations, large volumes of water needed to be concentrated to be able to quantify BMAA. In order to assess the extent of free BMAA contamination of water, a concentration method needed to be developed. A culture collection of South African cyanobacterial isolates was established, expanded and maintained as part of this project. Over 100 impoundments were sampled over a period of two years and all culturable cyanobacterial isolates were made unialgal and maintained as live cultures in the culture collection. This collection served as a resource for evaluation of BMAA production within and between taxonomic groups. The extent and diversity of the culture collection was ideally suited to serve this purpose and therefore achieved the stated objective. BMAA analysis was optimized using a commercial chloroformate derivitization kit (EZ:faast) with gas chromatography - mass spectrometry (GC/MS) and high performance liquid chromatography - mass spectrometry (LC-MS) analysis of derivatized amino acids. An in-house BMAA analytical system (LC-MS) was installed and validated using a chloroformate pre-derivatized BMAA standard (Sigma). The resulting molecule was quantified against three internal standards: homoarginine, methionine-D3 and homophenylalanine. The BMAA standard was derivatized in varying concentrations and injected in triplicate to assess machine reproducibility and on 3 consecutive days in order to analyse derivatization reproducibility. Analytical stability was excellent with insignificant variation in individual quantification runs on a given sample. Derivitization reproducibility across the concentration range used for the calibration curve was acceptable. The GC-MS BMAA detection developed for this project was 15 times more sensitive than previously published fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate-derivatized BMAA detection methods and the LC-MS BMAA detection method is 50-fold more sensitive. The lower limit of sensitivity for detection was below 100 pg per injection, and quantification was possible at 148 pg. BMAA was detected in 97% of all culture collection strains examined. All taxonomic divisions contained BMAAproducing strains. BMAA was observed in both the unbound and protein associated forms but to different extents in different strains. The ratio of free to bound BMAA appeared to remain constant for any given strain. No taxonomic or geographic basis for BMAA production or the ratio of free to bound BMAA was observed. A concentration protocol based on a strong cation exchanger with hydrophobic interaction was developed for extraction and concentration of BMAA from raw water samples. Recovery of BMAA from sterile distilled water samples ranged from 90 to 115% indicating complete recovery. Dam water collected during an Anabaena bloom was pre-filtered and concentrated as previously described. The sample was evaluated for the presence of extracellular BMAA; no BMAA was detected from 20 L of water. Filtrates were then hydrolysed and tested for BMAA, whereupon >17 - 40 g L-1 intracellular BMAA was found. These results were repeated using two more dams and a Microcystis bloom. These data suggest that BMAA is not released into the environment, or is rapidly degraded in, or removed from the environment.
Date Published:01/01/2011
Document Type:Research Report
Document Subjects:Drinking water - Water treatment
Document Keywords:Environment, Water Quality
Document Format:Report
Document File Type:pdf
Research Report Type:Standard
WRC Report No:1719/1/10
ISBN No:978-1-4312-0060-3
Authors:Downing TG
Project Leader:Downing TG
Project No:K5/1719
Originator:WRC
Organizations:Nelson Mandela Metropolitan University
Document Size:1 148 KB
Attachments:EXECUTIVE SUMMARY 1719.pdf
TABLE OF CONTENT 1719.pdf
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