Anatoxin-a, Cylindrospermopsin, Adda Microcystins/Nodularins, & Saxitoxins Report Project: City of Melbourne, Water Production Submitted to: Organization: Address: Email: Sample Receipt Date: Sample Condition: Report# Date Prepared: Prepared by: Shaniese Alexander City of Melbourne, Water Production 6055 Lake Washington Rd. Melbourne, FL 32934 shaniese.alexander@mlbfl.org 31 July 19 3.9 ºC upon arrival 190730_City of Melbourne, Water Production-SRaw 1 August 19 Kamil Cieslik Table 1: Samples analyzed Sample Identification Description/Site Collection Date Collection Time 19-3168 S. Raw Intake 30 July 19 1237 Analytes: Anatoxin-a (ANTX-A), Cylindrospermopsin (CYN), Adda Microcystins/Nodularins (Adda MCs/NODs), Saxitoxin (STX/PSTs) Sample Preparation Water Sample Freeze-Thaw The sample was received and inverted for 60 seconds to mix. A subset from the sample was removed prior to cell lysis for algal identification purposes. A second subset from the sample was transferred to a 15 mL vial. Three freeze-thaw cycles were employed prior to additional sample preparation and subsequent analysis. Page 1 of 4 Analytical Techniques Enzyme-Linked Immunosorbent Assay (ELISA) Adda MCs/NODs A microcystins/nodularins Adda ELISA (Abraxis) was utilized for the quantitative and sensitive congener-independent detection of Adda MCs/NODs (US EPA Method 546 & Ohio EPA DES 701.0). The current method reporting limit is 0.30 ng/mL (ppb) based on kit sensitivity, dilution factors, and initial demonstration of capability. STX A saxitoxin specific ELISA (Abraxis PN 52255B) was utilized for the detection and quantification of saxitoxin and related analogs (paralytic shellfish toxins – PSTs). The current method reporting limit is 0.05 ng/mL (ppb) based on kit sensitivity and dilution factors. Based on manufacture instructions, the STX ELISA is less cross-reactive to other PSTs and will likely underestimate total PSTs/Saxitoxins. Reported cross-reactivities are as follows: NEO (1.3%), dcSTX (29%), GTX2/3 (23%), GTX5 (23%), dcGTX2/3 (1.4%), dcNEO (0.6%) & GTX1/4 (<0.2%). Liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) ANTX-A & CYN A Waters XSelect HSS T3 2.1 x 150 mm, 3.5-µm column was used in separation with mobile phases (methanol and water) containing acetic acid. The [M+H]+ ion for ANTX-A (m/z 166) was fragmented and the product ions (m/z 91, 131, 149) were monitored. The [M+H]+ ion for CYN (m/z 416) was fragmented and the product ions (m/z 194, 274, 336) were monitored. The [M+H]+ ion for the internal standard [15N5]-Cylindrospermopsin (421 m/z) was fragmented and the product ion (341 m/z) was monitored. The [M+H]+ ion for the internal standard [13C4]Anatoxin-a (171 m/z) was fragmented and the product ion (153 m/z) was monitored. The internal standard method was utilized for all quantification. NA MDL MQL ND Blank LFB Abbreviations Not Applicable LFSM Method Detection Limit LFSMD Method Quantification Limit LD Not Detected above the MDL IS Regent Water free from interferences ― Lab Fortified Blank MRL Page 2 of 4 Lab Fortified Sample Matrix Lab Fortified Sample Matrix Duplicate Lab Duplicate Internal Standard Not Analyzed Method Reporting Limit Quality Control Table 2: QA/QC samples prepared for analyses. Analyte MC-LR CYN [15N5]-CYN ANTX-A [13C4]-ANTX-A STX Concentration (ng/mL) 1.0 0.1 1.0 0.1 1.0 0.2 Sample ID 19-3168 19-3168 19-3167 & 19-3168 19-3168 19-3167 & 19-3168 19-3168 QC Type LFSM LFSM IS LFSM IS LFSM Return 93% 112% 87 ± 10% 104% 90 ± 6% 85% Additional Quality Control/Quality Assurance checks included method blanks, LFBs, and standard curves. Table 3: Adda MC-ELISA Quality Control Value Table Date Analyzed: R2 value: %CV range STDs: LFB (1ppb) Recovery: %CV range LFB: Low CV (0.15 ppb) recovery: LRB Qualifier CL J 1 August 19 0.997 0.5-9.2% 91% 13.2% 113% <0.08 Requirement ≥0.98 ≤15% ±40% True Value <20% ±50% True Value <0.08 Pass/Fail PASS PASS PASS PASS PASS PASS Flag Analytical result is estimated due to ineffective quenching. Analyte was positively identified; the associated numerical value is estimated. PT The reported result is estimated because the sample was not analyzed within required holding time. B Analytical result is estimated. Analyte was detected in associated reagent blank as well as the samples. E Analytical result is estimated. Values achieved were outside calibration range. N Spiked sample control was outside limits T The reported result is estimated because the sample exceeded temperature threshold when received Page 3 of 4 Summary of Results Table 4: Summary of results in ng/mL Sample ID 19-3168 MRL (ng/mL) Analyst Initials Date Analyzed Adda MCs/NODs (ng/mL) CYN (ng/mL) ANTX-A (ng/mL) STX (ng/mL) ND ND ND ND 0.30 KC 8/1/19 0.05 MA 8/1/19 0.05 MA 8/1/19 0.05 KC 8/1/19 Interpretations: Adda MCs/NODs, CYN, ANTX-A, and STX were not detected in the submitted sample above the MRLs. Submitted by: Date: Mark T. Aubel, Ph.D. August 1, 2019 The results in this report relate only to the samples listed above. This report shall not be reproduced except in full without written approval of the laboratory. Page 4 of 4