CyanoSurvey Toxicity Methods Comparison Andrew Humpage, Melody Lau, Virginie Gaget, Barbara Sendall, Somprasong Laingam
Friday, 24th August 2012 Assessing the Risk
• Effec ve Public Health Risk Management depends upon accurate Hazard Assessment
• Accurate Hazard Assessment depends upon accurate, reliable data that are representa ve of the real world situa on – Representa ve sampling – Accurate and complete quan fica on of toxins – Adequate toxic potency assessment
• These stages are o en dissociated, leading to poten al for misunderstandings and false assump ons
CyanoSurvey Sampling Sites – 61 in all
Plus 36 “Ad hoc” samples obtained from routine Biology Services submissions.
For genetic taxonomy, these have been supplemented with AWQC culture collection samples from 37 sites.
29 CyanoSurvey samples used for toxicity methods comparison.
Page 3 Sample processing
10L water sample
Inspect – rough cell count & ID
Filter through GFC to concentrate Submit to Biology Services
Freeze-Dry Culture
Toxin/Toxicity/Tox Gene Analysis Accurate cell count & ID
Page 4 Toxin/toxicity detec on method comparison Assay Class Type Endpoint Detects
Microscopy Morphological recogni on Known toxin producers
ELISA An body binding MC/NOD, CYN, STX
Toxin genes Primer binding, PCR amplifica on Cyano16S, MC, NOD, CYN, STX, ANTX-a
Analy cal HPLC UV absorbance MC, NOD LC-MS Molecular fragmenta on CYN, STX Toxicity PP2A Enzyme inhibi on MC, NOD PSI Interference with ribosomal transla on CYN, Limnothrixin*
Neuro-2A Cell preserva on by Na channel STX (nerve cells) blockade Vero Mitochondria-dep. metabolism Anabaena 131 toxin**, non-specific (kidney cells) (Resazurin) Cell death (cell count) Anabaena 131 toxin**, non-specific
Cell cycle (G1/G2 ra o) Non-specific *Bernard et al 2011 Env Tox 26: 260-270; Humpage et al 2011 Water Res 46: 1576-1583; Whan et al, this meeting. Page 5 **Froscio et al 2011 Toxicon 58: 689-692 Assay Methods & Valida on
• All assays were “internally validated” using standards, spiking experiments, matrix controls, etc. • Cell counts and HPLC/LC-MS methods were commercial, NATA-accredited. • Cell counts and HPLC/LC-MS were run as singlet samples as per standard prac ce. • ELISA & Toxicity Assays were run on 2 days as independent experiments with repeats if reps did not agree. • Toxin genes were determined at 3 dilu ons of the extract, giving effec ve concentra on factors of 1.5x, 15x and 150x • Sample prepara on/concentra on was iden cal for most assays and all comparisons (most analyses done on extracts of cell concentrates).
Page 6 Cell counts and species iden fica on Microcys s Microcys s Cylindrospermopsis Anabaena Aphanizomenon ovalisporum raciborskii
Sample Name/
aeruginosa flos-aquae
circinalis Main other species from the most to the least abundant (cells/mL) Loca on
Palm Valley 88800 Merismopedia (69800), Planktolyngbya (9500), Anabaena bergii (1760) Angourie NSW 320 109000 151000 Planktolyngbya (98000), Leslie Dam QLD 16900 14000 Aphanocapsa (255000), Planktolyngbya minor (154000),Planktolyngbya (9590) Pindari Dam 1040 40000 Kangaroo Creek SA 299000 394000 655 Anabaena crassa (10000) Welltree Billabong 24 Aphanocapsa (1510000), Coelosphaerium (2090) Harding Dam 1860 Merismopedia (24700), Aphanizomenon (14700), Aphanocapsa (6610), Pseudanabaena (1940) Mount Bold 183000 17500 1300 Wyangala Dam 76500 Anabaena coiled_spp (11200) Taylor Lake 486 95 1200 Penrith Lake 120000 Limnotrix (1660) Brunswick 6E+06 1.2E+07 Planktolyngbya minor (1360000 Corowa 63 830 723 Aphanocapsa (15700), Snowella (7700), Aphanizomenon (2900), Anabaena straight_spp (1650) Tocumwal 541 63 675 769 Aphanocapsa (68000), Snowella (14500), Planktolyngbya (cells/ml) (5500), Anabaena aphanizomenoides (1280) Moama 636 294 1280 Aphanocapsa (114000), Planktolyngbya (14300), Anabaena straight_spp (1570), Aphanizomenon (1330) Toolebuc 185 Aphanocapsa (7000), Planktolyngbya (1080) Banyan Farm Curlwaa Aphanocapsa (31700) Hume reservoir 924 2610 198 175 Aphanocapsa (9000), Snowella (5000), Aphanothece (4250) Ellery Creek Synechocys s (5440000) Myponga Boat Ramp 143 Burrendong Dam 827 Burrinjuck Dam 840 3180 Carcoar Dam 75 975 7300 Angaston 9E+06 Tychonema (726000), Planktolyngbya minor (551000), Aphanocapsa (256000) Clarrie Hall 16200 Murray Bridge WWTP 462000 Malpas 348000 21400 34300 Pseudanabaena (2020) Penrith Lakes 1250 346000 Aphanocapsa (69900), Pseudanabaena (20800)
< 1000 cells/mL 1001-10000 cells/mL >10000 cells/mL Page 7 Saxitoxins (strong +ve, weak +ve, -ve)
ELISA Toxicity Toxin gene Analysis Microscopy Cell Quota STX LC-MS Anabaena Sample Name Conc factor for STX ug/L STX ug/L Neuro-2A PSTs PCR ELISA fg LC-MS fg Extract result as STXeq ug/L circinalis (cells/ extracts (Raw Water) (Cell extracts) raw (div. Conc) (ug/L) (conc factor) STX/cell STXeq/cell (total ug/L) ml)
Malpas 512 5.6 ± 1.1 864.87 ± 20.81 1.69 438.88 ± 106.55 1.5x 2130 (20960) 348000 16 12
Kangaroo Creek SA 0 3.2 ± 0.4 1.5x 299000 11
Mount Bold 607 5.9 ± 1.5 1883.29 ± 85.47 3.101 808.86 ± 99.56 1.5x 183000 32 Clarrie Hall 2909 0.03 ± 0.003 6.25 ± 0.01 0.002 Myponga Boat Ramp 4630 72.12 ± 69.10 0.016 1.5x 276 (2118) 143 108 416 Carcoar Dam 3320 0.91 ± 0.23 0.000 15x 75 3.6 Corowa 495 0.91 ± 0.01 0.002 63 29 Keepit Dam 2310 12.02 ± 0.90 0.005 Murray Bridge WWTP 783 0.1 ± 0.1 <0.02 150x Page 8 Conclusions - STX • Good +/- agreement between methods when A. circinalis >20,000 cells/ml • ~3-4-fold varia on in quan ta ve toxin determina ons • Calculated cell quotas highly variable but generally << 150 fg/cell assumed in ADWG • Possible false posi ves in ELISA with very concentrated material • But if a strong effect in the ELISA from concentrated material, correlates with gene and LC-MS • LOD for ELISA 0.05-0.1 µg STX eq/L rather than 0.02 µg STX/L • Neuroblastoma assay too insensi ve and complicated for rou ne use • Evidence for an STX producer in most concentrated samples (probably low numbers of Anabaena?) – ubiquitous occurrence. Page 9 Conclusions - MC • Good +/- agreement between ELISA/PP2A/gene, although some apparent false –ve in gene analyses • Generally reasonable quan ta ve agreement (± 2-3-fold) between ELISA, PP2A and HPLC, while cell quota varied ~6-fold. • One of 4 HPLC analyses false nega ve when ELISA, PP2A and gene all +ve • M. flos-aquae seems to be a fairly consistent MC producer (toxicity highly variable) • Evidence for an MC producer in most concentrated samples (probably low numbers of Microcys s?) – ubiquitous occurrence. Page 10 Conclusions - NOD • Gene detected in 4 samples • Not confirmed by MC ELISA, PP2A or HPLC • No Nodularia observed Page 11 Conclusions - CYN • Good quan ta ve correla on for toxin (generally <2-fold varia on) but not cells when C. raciborskii > 1,500 cell/ml • Hence, cell quota varied ~60-fold • One possible false nega ve in gene detec on • Possible evidence in 4 samples for Limnothrixin (PSI >> ELISA) though no Limnothrix cells detected • Evidence for a CYN producer in most concentrated samples (probably low numbers of Cylindrospermopsis?) – ubiquitous occurrence. Page 12 Conclusions - Toxicity • Cytotoxicity, enhancement of cellular metabolism, and cell cycle blockade seen with many concentrated samples • Some effects probably caused by CYN when present (3 samples) • Some effects are similar to those of Anabaena 131R • Significance currently unknown Page 13 Conclusions - Overall • While +/- agreement was good between methods for most toxins when cells numbers were high, actual quan fica on was quite variable • variability: STX > MC > CYN. Due to toxin mixtures? • do not rely on a single method, understand strengths/weaknesses • cell counts are not a quan ta ve measure of toxicity (OK as indicator of maximum likely toxicity if right species included) • however, assuming worst case may be “safe” but poten ally costly in terms of unnecessary closures/remedial ac ons Page 14 Conclusions - Overall • Sample concentra on allowed detec on of very low levels of all toxins even when producer cells not observed in cell counts • ubiquitous low level distribu on of toxin producers even in loca ons without a history of these species • poten al for “surprise” blooms when condi ons change Page 15 A Ques on of Balance • Sampling: – Maximise for representa ve hazard iden fica on • Quan fica on: – Minimise cost so as to maximise sampling – Maximise rapidity, comprehensiveness – Adequate accuracy, precision, sensi vity – Use screening assays to filter samples for detailed analysis • Toxicology: – Validate use of biological assays for toxicity assessment and detec on (eg. EU REACH & US ToxCast programs) • Overall: – Communicate assump ons/compromises/uncertain es to risk managers Thank you Page 17