Luminescent Bacteria Test according ISO (-1, -2, -3) detecting and monitoring inhibitory effects in water samples. Work Shop

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1 Luminescent Bacteria Test according ISO (-1, -2, -3) detecting and monitoring inhibitory effects in water samples Work Shop Dr. Elmar Grabert, HACH-LANGE GmbH, Duesseldorf, Germany

2 In certain cases a harmful effect water testing is required, especially if you do not know what exactly to monitor Picture Copyright Nucleus CMS v3.22 Understand severity, area and duration of a pollution situation

3 Harmful Effect Analysis are done by biological testing A biological test system detects the effects of a sample on living structures. A biological test system does not detect the single components of a sample and their concentration. A biological test gives us information about the sample that could not be given by any other chemical or physical test system. The analytical system used depends on the analytical question analysis of single compounds sample analysis of harmful effects (toxicity) chemical test biological test chemical test

4 The single compounds of aqueous samples could only be detected by chemical test systems.

5 Harmful Effect Analysis Principle of all biological testings Noninhibitory Control 100 % Activity Sample 30 % Activity biological component: whole eco system; living organisms, living cells, active enzymes detection of biological activity 70 % Inhibition

6 Harmful effects of aqueous samples ( toxicity) could only be detected by biological test systems. ISO 7346 Brachydanio rerio ISO 6341 Daphnia magna ISO 8692 Desmodesmus subspicatus ISO Vibrio fischeri

7 Luminescent Bacteria Test System ISO 11348

8 Luminescent Bacteria Test is a biological test system that is internationally used and accepted and available as a validated standard method: ISO 11348, -1, -2, -3 Water Quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fisheri (Luminescent bacteria test)- First Edition Second Edition ISO describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fisheri (NRRL B-11777). The methods are applicable to: waste water aqueous extracts and leachates Fresh water (surface or ground waters) or salt and brackish waters, especially the monitoring of changes in inhibition towards bacteria, pore water, single substances diluted in water

9 LUMIStox 300 Laboratory System according to ISO ECLOX, portable Field Testing System Extremely robust LUMISsoft 4, PC software, evaluation for all luminescent bacteria test methods: The Luminescent Bacteria Preserved by freeze-drying. Immediately ready for use after minutes reactivation time. First results after 5 minutes, ISO results after 15 or 30 minutes incubation time. Shelf live: one year guaranteed at -18 C storage.

10 The LUMIStox 300 Cuvette luminometer with photomultiplier. Fulfils all requirements of the norm ISO , -2, -3 (Luminescent Bacteria Test). Designed for user convenience. Integrated evaluation for all luminescent bacteria test methods: GLvalue, ec20, ec50, Screening. Cuvette luminometer function for other luminometric methods. Automatic error compensation for coloured samples. ETV verified LUMIStherm Thermostate (15 C) designed for Luminiscent Bacteria Test Fulfils all requirements of the norm ISO , -2, -3

11 The LUMISsoft 4 PC software Organisation, execution and documentation of up to 18 samples in one test, really easy. Fulfils requirements of ISO , -2, -3 for the evaluation. Evaluation for all luminescent bacteria test methods: LID-value, ec20, ec50 ec 80, screening with and without colour correction. Concentration-effect plots. Control of analytical quality assurance of ISO requirements for accuracy and standard samples. Extensive documentation of sample treatment and test conditions according to GLP and national standards. Integrated database for the collection of results of e.g. similar type of samples. For PC with Windows and Vista. ETV verified

12 You will normally be in contact with luminescent bacteria in the same way you will be in contact with any harmless chemical reagent. The Luminescent Bacteria Preserved by liquid- or freeze-drying. Immediately ready for use after minutes reactivation time. First results after 5 minutes, ISO compatible results after 15 or 30 minutes incubation time. Shelf live: one year guaranteed at -18 C storage. ETV verified

13 ECLOX TM Extremely robust Portable small foot print, complete, no need for wall power and power set,. For prtable and lab use, can be combined with a LUMIStherm and LUMISsoft to make ISO equivalent data. ETV verified

14 Luminescent Bacteria

15 Luminescent bacteria are living free in salt-water, in the intestine of fish, and in a very special ecological niche: the luminescent organs of deep-sea fishes Sprats, after 3 days at approx. 15 C in 3.5% NaCl solution. Picture was taken in the dark. Dragfish not really

16 Aliivibrio fischeri or Vibrio fischeri or Photobacterium fischeri NRRL B wrongly named Photobacterium phosphoreum NRRL = Nothern Regional Research Laboratory ( U.S. Department of Agriculture, Peoria, Illinois, USA) Isolated from seawater and marine animals. Non-Pathogen Gram-negative polar flagella slightly curved rod optional aerob Oxidase positive fermentation no pigments marine Enterobacteria, need 2% bis 4 % NaCl for living luminescence when oxygen is available

17 luxr luxi luxc luxd luxa luxb luxe luxg promotor regulator LuxR autoinducer N-3-(oxohexanoyl) L-homoserin-lacton - ß- subunit of luciferase enzymes for synthesis of long chain aldehyde? Genetics, regulation and biochemistry are in the focus of scientists for many years. Many questions are answered. reductase transferase synthetase luciferase O 2 FMN H 2 luciferase luciferase FMN H 2 FMN H 2 O 2 without luciferase O 2 without l. ch. aldehyde l. ch. aldehyde luciferase luciferase FMN FMN H 2 O 2 l. ch. aldehyde luciferase l ch. C-acid FMNH 2 + O 2 + R-COH -> FMN + R-COOH + H 2 O + light

18 a living luminescent bacteria cell: CO 2 catabolic pathway [H] [H] electron transport system nutrients ATP monomers: amino acids carbo hydrates nucleotids polymers: proteins polysaccharides polynucleotides anabolic pathway Bioluminescence as an indicator of toxicity Luminescent bacteria, e.g. Vibrio fisheri NRRL B-11177, naturally emit light, called bioluminescence. The enzyme involved is bacterial luciferase which catalyses the following reaction: O 2 FMNH 2 + O 2 + R-COH -> FMN + R-COOH + H 2 O + Light

19 effects of toxins: damage or inhibition catabolic pathway electron transport system Non polar organics e.g. Toluene Oxidative phosphorylation uncouplers e.g. pentachlorophenol Respiratory blockers e.g. cyanide Aldehydes, Cationic agents, Heavy metals Phenols and Phenolics monomers: amino acids carbo hydrates nucleotids polymers: proteins polysaccharides polynucleotides anabolic pathway Membrane inhibitors e.g. chlorine Polar agents EDTA, solvents, alcohols Where toxins attack Bioluminescence is directly linked to the vitality, the metabolic status, of the cell. A toxic substance will cause changes to the cellular state cell wall cell membrane the electron transport system enzymes cytoplasmic constituents which are rapidly reflected in a decrease in bioluminescence

20 Results

21 Light reduction depends on concentration of toxins % inhibition toxic sample non toxic sample 0, The reduction of bioluminescence (the decrease of light) can be measured in a luminometer: The stronger the toxic effect of a sample - or in case of a single chemical compound the higher its concentration - the stronger is the resulting light reduction concentration (%) dilution factor The primary result is expressed as percentage inhibition of the luminescence. Results according to ISO standard are LID or ECxx values.

22 80 % inhibition toxic sample Expression of results: LID: lowest ineffective dilution 20 non toxic sample The lowest dilution factor at which the inhibitory effect is lower than 20 % is called LID LID Dilution factor A high value indicates a high toxicity, this is very obvious when using water samples.

23 80 toxic sample Expression of results: % inhibition EC 50 EC XX: The concentration of the sample that causes exactly XX % inhibition. 20 non toxic sample XX = 20 = EC20 XX = 50 = EC50 0, concentration (%) A low value indicates a high toxicity, obvious if used for e.g. chemicals

24 % 80 i n h i 60 b i t 40 i o n 20 % 80 i n h i 60 b i t 40 i o n minutes 10 minutes 0, concentration (%) 30 minutes 10 minutes The result depends on the incubaton time: For most organic compounds the results archieved after 10 or 30 minutes incubation time are the same. For heavy metal ions the results after 30 minutes incubation time are much higher than after 10 minutes. 0, concentration (%)

25 Luminescent Bacteria Test has been used since more than 40 years in many applications and environments. More than thousand single component have been tested and the results are published by: Kaiser, K. L. E. and Ribo, J. M. (1988), Photobacterium phosphoreum toxicity bioassay. II. Toxicity data compilation. Environ. Toxicol. Water Qual., 3: % inhibition chloramine B lead-ii-nitrate 2,6-dimethylphenole potassium peroxy disulphate 0 0,1 1, concentration mg/l Philipus Aureolus Paracelsus: Poison is in everything, and no thing is without poison. The dosage makes it either a poison or a remedy Sensitivity is typically in the ppm (mg/l) range

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27 Procedure

28 ISO Simplifyied Screening 1. Prepare sample 2. Prepare bacteria solution 3. Prepare sample dilutions 4. Measure start bacteria light 5. Add sample + dilutions to bacteria 6. Wait incubation time 7. Measure end bacteria light 1. Prepare sample 2. Prepare bacteria solution 3. Prepare sample dilutions 4. Add sample to bacteria bacteria 5. Wait incubation time 6. Measure 7. Read result (% inhibition) 8. Read result (% inhibition, LID, EC)

29 ISO Prepare sample Preservation Cooling 0-5 C: less than 2 days in the dark Freeze - 18 C: up to 2 month. Activities of preservation must be documentated. Sample pretreatment freezed samples must be defrosted completly. Homogenize the defrosted sample. ph-value of the sample Measure ph and documente the value Correct ph with HCl or NaOH to ph 7.0 +/- 0.2 if the value is not between ph 6.5 and 8.0. Choose the concentration of the hydrochloric acid or the sodium hydroxide to restrict the volume added to not more than 5 % of total volume.

30 ISO Prepare bacteria solution

31 ISO Prepare sample dilutions LID

32 ISO Prepare sample dilutions

33 ISO Prepare sample dilutions

34 ISO Measure start bacteria light, Add Sample, Wait incubation time

35 ISO Measure end bacteria light

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37 Interferences

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39 The influence of nutrients DIN EN ISO , -2, -3 : 1998 Interferences An organic contamination of the sample by readily biodegradable nutrients (e.g. urea, peptone, yeast extract usually >= 100 mg/l) may cause a pollutant-independent reduction in bioluminescence.

40 LID versus COD N = 1867 The influence of nutrients R 2 = 0,1261 GL COD Datensammlung Bioteste : Texte 9/98 Umweltbundesamt

41 Gracias