Contents. Clare Askem Cefas. Culturing Ceramium. Static tests Results with Ciprofloxacin and Triclosan Flow through set up Conclusions

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1 Growth inhibition test with Ceramium tenuicorne (ISO 10710:2010): our findings from using the OECD protocol with various chemicals, static and flow through systems. Clare Askem Cefas Contents Culturing Ceramium Overview of the OECD protocol Static tests Results with Ciprofloxacin and Triclosan Flow through set up Conclusions

2 Ceramium tenuicorne Ceramium tenuicorne is a red macroalgae. Found in temperate regions. Brackish and marine costal environments. Ceramium strictum (Marine, isolated in 1973*) and Ceramium tenuicorne (Brackish, isolated in 1995**) are now considered to be the same species and referred to as Ceramium tenuicorne. *Dr Jan Rueness, **Dr Britta Eklund Ceramium culturing at Cefas Clones obtained from Dr Britta Eklund, Stockholm University, Sweden. Originally at 20ppt salinity, acclimated to Lowestoft seawater (approx 29 32ppt) by 3ppt every few days. 20ppt Day 1 23ppt Day 3 Salinity adjustments over several days 26ppt Day 6 29ppt Day 9

3 Creation of Nutrient solutions Nutrient Chemical To make solution To make nutrient enriched seawater Nitrogen NaNO3 4.19g in 100ml R.O Water Use 0.5ml per L seawater Phosphorus KH2PO4 680mg in 100ml R.O Water Use 0.5ml per L seawater Iron FeCl3.6H2O 100mg in 100ml R.O Water Use 0.5ml per L seawater Trace elements All in 1000ml R.O Water: Dilute by 10% then add 0.5ml per L seawater Na2EDTA 6000mg MnCl2.H2O 904mg ZnSO4.7H2O 250mg Na2MoO4.2H2O 130mg CoCl2 3mg CuSO4.5H2O 4mg Vitamin solution All in 100ml R.O Water: Use 0.5ml per L seawater Thiamin B1 10mg Cyanocobalamin B12 0.1mg Biotin 0.1mg Ceramium culturing at CEFAS Select a Growing tip Cut aseptically 1.2 to 0.6 mm to fork Culture conditions ph : 6.5 to 9.5 Salinity : ppt Temperature : 22 C ± 2 Lux : 1800 ± 300 light:dark 16 h : 8 h

4 Ceramium growth of culture 1mm 1mm 7 days Growth in 7 days creates about 4 branchings to give 12 tips. Within 2 3 weeks there is enough tips on 1 plant for 1 test. OECD protocol 10710:2010 Water quality Growth inhibition test with the marine and brackish water macroalga Ceramium tenuicorne. 1. To test chemicals or water samples 2. 7 day test 3. Tips measured at start and end of test 4. Growth inhibition determined as a reduction on growth rate relative to control 5. Endpoints EC50, NOEC and LOEC

5 Test conditions Natural or artificial seawater for media, enriched with nutrients. Temperature 22 C ± 2 ph 6.5 to 9.5 Lux 3600 at 14 h light: 10 h darkness Salinity depending on purpose Reference test with 3,5 dichlorophenol or Zinc sulphate alongside test item Method Test concentration series prepared (mg/l or %) ml of each concentration, per replicate, dispensed into petri dishes or 6 well plates Add tips then incubate

6 Measure using eyepiece graticule or image analysis software Measure from 1 st fork to longest tip Analysis with statistical package eg: CETIS Validity criteria: Validity a) the increase in length in 7 d in the controls has increased by a factor of more than three compared to the starting length b) the coefficient of variation of the control growth rates does not exceed 30 %; c) the control ph has not changed to become lower than 6,5 or higher than 9,5.

7 Our experiences Culturing Poor growth spindly due to too high salinity. Best growth for long term at 26ppt salinity. Poor growth of Ceramium Healthy Ceramium Poor Growth examples Bleaching and spindly : too much light Claws not developing : light too strong

8 Hints and tips Cutting tips can be done with scissors or scalpel Easiest to cut leaving a long tail for handling Measure and cut in the same dish Preserve in formalin for measurement later on Adapt culture to test conditions at least 1 week before test Static tests Standard test conditions and static renewal Temperature changes Salinity changes 22 C 15 C 26ppt 33ppt Cultures with artificial seawater and natural seawater and at varying parameters

9 Reference tests Concentration range: , , 0.01, 0.022, 0.046, 0.1, 0.22, 0.46 mg/l Zinc sulphate. Reference tests 7 day EC50 Growth inhibition results: Zinc sulphate = mg/l to mg/l Zinc sulphate at 15 C = mg/l 1mm Zinc sulphate 0.46mg/l Here you can see the difference in growth between control and top concentration for the zinc reference tests. 1mm Control Ciprofloxacin Fluoroquinalone antibiotic used to treat various infections such as urinary tract, respiratory, typhoid fever and gonorrhea. Species tested: Fucus vesiculosus Brown macroalgae Skeletonema costatum Diatom Isochrysis galbana Green Algae Crassostrea gigas Pacific oyster

10 Ciprofloxacin tests summary Ciprofloxacin results Key: CI = confidence intervals; EC50 = 50% effective concentration; EC10 = 10% effective concentration; LOEC = Lowest Observed Effect Concentration; NOEC = No Observed Effect Concentration.

11 Triclosan Antimicrobial; used in soaps, deodorants, toothpastes and cleaning products. Species tested: Fucus vesiculosus Brown macroalgae Isochrysis galbana Green Algae Crassostrea gigas Pacific oyster Triclosan results Organism Test Toxicity as nominal concentration (confidence limits) Ceramium tenuicorne Isochrysis galbana Crassostrea gigas 7 day growth inhibition (22 C) 72 hour growth (20 C) 24 hour embryo development (24 C) EC50 mg/l EC10 mg/l NOEC mg/l LOEC mg/l ( N/A) (N/A ) > ( ) ( ) ( ) Fucus vesiculosus 96 hour average frond length (20 C) 0.52 ( ) 0.17 ( ) Fucus vesiculosus 96 hour germination rate (20 C) 0.79 ( ) 0.43 ( )

12 Flow through system Beakers Flow meters Peristaltic pump Stock bottles Flow through system Pods attached to skewer Pods suspended in beaker Beaker Water pipe with dosed water

13 Conclusions Ceramium easy to culture Straightforward assay Uses general lab equipment Basic or complex: eyepiece or image analysis More data for marine risk assessment Ecotox A useful addition to the ecotox toolbox! Thank you Britta Eklund, University of Stockholm CEFAS Collegues; Tom Fisher, Chris Martin, Thomas Mc Gowan, Leah Nelson, Andy Smith, Ruth Wortham.