1. Title: Toxicological studies of herbicide Diuron and its effects on fatty acid profile of Asian Sea-bass, Lates Calcarifer Presenter name: Dr. Hassan Rashid Ali Institution: State University of Zanzibar Zanzibar - Tanzania

2. Coral reefs are among of the world’s most valuable natural resources that provide food, medicines, ecological services and source of income for the local communities (Hughes et al., 2010). In the past few years, however, threats to coral reefs have been increasing through human activities events such as global warming and marine pollution (Scope, 2009). INTRODUCTION

3. These new chemicals have been reported to be dangerous to marine organisms’ especial in growth, survival and health of corals and fishes (Knutson et al., 2011; Sheikh et al., 2009; Owen et al., 2003) Following the restriction of usage of Tributyltin (TBT), the world has shifted to new antifouling such as Irgarol and Diuron as antifouling paints to the boats and ships (Gatidou et al., 2007; Dafforn et al., 2011) Irgarol Diuron

5. Diuron is categorized as priority hazardous substance by the European commission (Malato et al., 2002). Diuron is well known as photosynthetic inhibitor. Diuron of conc. (~0.05 µg/L) causes spinal deformity in early-life-stage of pink snapper Pagrus auratus (Gagnon and Rawson, 2009). Toxicity of Diuron:

6. Despite the extensive usage of Diuron as an antifouling paints in the world, up to 2013, there was almost no study on the occurrence of Diuron on marine ecosystem of Malaysia (we published a paper in 2014) Moreover, to our knowledge, no published study at all in the world showing the effects of Diuron on fatty acids compositions of Lates Calcarifer Scientific gap!!!

7. - main components of lipids - essential elements of cell membrane lipids - precursors of bioactive metabolites Fatty acid composition reflects organisms specific cellular physiological functions and physiological state (Sargent et al., 1990, 2002). Examining the lipid content and FA composition in organisms such as corals is a potential diagnostic indicator of their health (Bachok et al., 2006). Why fatty acid?

8. METHODOLOGY  Two months old Lates Calcarifer were obtained from a commercial fish farm in Setiu province, between April and May 2012.  The fish was acclimatized for 2 weeks in well aerated holding polyethylene tanks (500 L), containing natural seawater with a salinity of 30 ppt, under a natural photoperiod 12 h:12 h (light: dark) cycle.  The water in the tank was passed through a 1-mm filter, treated with UV-sterilized and refilled daily.  Fish were fed twice daily with standard accepted food, and starved 24 h before and during the chronic exposure experiment.  A stock solution of 1000 mg/l Diuron was prepared by using acetone and the working concentrations were made up by spiked the required concentrations to the sea water.

9. Experimental design

10. Extraction of fatty acid FAME n-Hexane, BF 3 & internal standard was added to the sample Flushed with N 2 Heated at 100˚C for 2 hr (Tsuchiya & Sakdullah, 2008) GC-FID Extraction & esterification + 4ml Hexane + 2ml, 14% BF3 in CH3OH One-step method

11. RESULTS & DISCUSSION

12. ConcentrationNumber of live fishes at% Alive at% Mortality at (ppm)24h48h72h96h24h48h72h96h24h48h72h96h 0 (control)10 100 0000 0 (check/ acetone)10 100 0000 110 7100 7000030 210 65100 6050004050 3108421008040200 6080 5107211007020100308090 79300 30001070100 Table 1: Toxicity testing of the Diuron to Lates Calcarifer (1 st replicate) LC 50 was calculated by different methods and the results are as follows:- DATE: 15/6/2012 TEST NUMBER: 1 DURATION: 96 HOURS SAMPLE: DIURON SPECIES: LATES CALCARIFER METHOD LC50 CONFIDENCE LIMITS LOWER UPPER SPAN BINOMIAL 2.000.000 5.000 5.000 MAA 1.754.508 3.730 3.221 PROBIT 1.676.945 2.318 1.373 SPEARMAN 1.799 1.103 2.936 1.833 Average1.807 Fig.16: % Mortality of Lates Calcarifer at 96h using Diuron concentrations (1 st replicate)

13. Table 2: Toxicity testing of the Diuron to Lates Calcarifer (2 nd replicate) ConcentrationNumber of live fishes at% Alive at% Mortality at (ppm)24h48h72h96h24h48h72h96h24h48h72h96h 0 (control)10 100 0000 0 (check/ acetone)10 100 0000 110 96100 9060001040 210 84100 8040002060 3108641008060400204060 597529070502010305080 793009030001070100 LC 50 was calculated by different methods and the results are as follows:- DATE: 15/6/2012 TEST NUMBER: 2 DURATION: 96 HOURS SAMPLE: DIURON SPECIES: LATES CALCARIFER METHOD LC50 CONFIDENCE LIMITS LOWER UPPER SPAN BINOMIAL 1.414.000 7.000 7.000 MAA 1.414 ******* ******* ******* PROBIT 1.563.470 2.422 1.952 SPEARMAN 1.414.662 3.022 2.360 Average 1.451 Fig.17: % Mortality of Lates Calcarifer at 96h using Diuron concentrations (2 nd replicate)

14. Table 3: Toxicity testing of the Diuron to Lates Calcarifer (3 rd replicate) ConcentrationNumber of live fishes at% Alive at% Mortality at (ppm)24h48h72h96h24h48h72h96h24h48h72h96h 0 (control)10 100 0000 0 (check/ acetone)10 100 0000 110 7100 7000030 210 64100 604000 60 3109531009050300105070 5964190604010 406090 78200802000 80100 LC 50 was calculated by different methods and the results are as follows:- DATE: 15/6/2012 TEST NUMBER: 3 DURATION: 96 HOURS SAMPLE: DIURON SPECIES: LATES CALCARIFER METHOD LC50 CONFIDENCE LIMITS LOWER UPPER SPAN BINOMIAL 1.592.000 5.000 5.000 MAA 1.626 ******* ******* ******* PROBIT 1.647.868 2.315 1.448 SPEARMAN 1.622.969 2.716 1.748 Average1.622 Fig.18: % Mortality of Lates Calcarifer at 96h using Diuron concentrations (3 rd replicate) Diuron Average LC 50 for all three replicates is 1.627±0.031

15. FATTY ACID COMPOSITION OF Lates Calcarifer

16. Table 4: Fatty acid composition (mg/g) dry weight of liver sample of Lates Calcarifer (Siakap) after 3 weeks exposure test NAMEFRESHCONTROL10%LC 50 DIURON30%LC 50 DIURON50%LC 50 DIURON SAFA C14:04.66 ab ± 0.364.96 a ± 1.464.18 ab ± 1.354.17 ab ± 1.911.78 cd ± 1.55 C16:035.9 a ± 11.630.9 ab ± 7.3127.0 ab ± 9.8427.1 ab ± 12.218.8 bc ± 4.30 C18:012.8 a ± 1.5613.3 a ± 3.2211.0 ab ± 4.3510.3 abc ± 4.679.34 abc ± 0.95 C20:00.001.19 a ± 0.600.00 MUFA C16:13.85 bc ± 0.826.68 a ± 2.335.58 ab ± 1.914.80 abc ± 2.072.20 c ± 1.92 C17:11.72 a ± 0.380.00 C18:1ω9c34.6 ab ± 0.8237.1 a ± 11.531.2 abc ± 11.827.4 abc ± 13.317.4 c ± 9.26 C18:1ω9t8.13 b ± 0.170.00 C20:11.32 bc ± 1.154.34 a ± 1.941.59 abc ± 0.152.80 abc ± 2.700.00 PUFA C18:3ω628.7 a ± 0.8024.9 ab ± 7.0319.3 bc ± 7.5417.8 bc ± 8.0811.9 cd ± 6.36 C18:3ω39.25 a ± 1.348.44 a ± 6.969.95 a ± 3.138.83 a ± 3.3810.1 a ± 3.05 C20:3ω36.48 a ± 2.505.37 a ± 1.931.06 b ± 0.931.11 b ± 0.981.69 b ± 0.35 C20:5ω37.73 a ± 0.026.33 a ± 0.203.84 cd ± 1.423.65 cd ± 1.642.61 d ± 1.44 C22:6ω36.40 ab ± 1.708.56 a ± 1.976.61 ab ± 2.885.56 ab ± 0.468.07 a ± 1.36 ΣSAFA53.4 a ± 13.450.4 ab ± 13.242.2 ab ± 11.941.6 ab ± 11.930.0 bc ± 8.62 ΣMUFA49.6 a ± 14.048.1 a ± 15.638.4 ab ± 13.435.0 ab ± 11.619.6 bc ± 7.62 ΣPUFA58.5 a ± 6.7653.6 ab ± 9.2340.8 bc ± 7.0537.0 bcd ± 6.5834.4 bcd ± 4.93 ΣFA161 a ± 11.8152 ab ± 12.0121 ab ± 10.2114 bc ± 9.4883.9 bcd ± 6.88 Values are means ± standard deviation (SD) for n=3. SAFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids. Means within rows followed by the same superscript(s) are not significant different (P > 0.05).

17. Figure 1: Fatty acid concentration of different classes and Total fatty acid (mg/g) dry weight from liver of Lates Calcarifer after exposed at different concentration of Diuron. Values are mean ± S.D, n = 3. In fresh and control samples PUFA >SAFA >MUFA group. In 10, 30 and 50%LC50 there was no clear trends. Diuron affects the species In most cases fatty acid decreased as the concentration dose increased.

18. Figure 2: Individual Fatty acid concentrations of Lates Calcarifer after exposed at different doses of Diuron.

19. This paper explains the toxicological responses of Lates Calcarifer upon exposure to different concentrations of Diuron in laboratory experiments. It can be concluded that; 1. Acute exposure of Lates Calcarifer has shown Diuron with 96h LC 50 value of 1.627±0.181mg/L. 2. Sub-lethal exposure to Lates Calcarifer (21 days) to different concentrations of Diuron had shown significant impact to the fatty acid composition of these organisms. 3. Fresh and control samples of Lates Calcarifer was dominated by PUFA followed by SAFA and then MUFA Summary and conclusions

20. 4. It was observed that tested species general health (observed in terms of ability of movements) was affected in exposures to all fractions of 96h LC 50 of Diuron, the severity of effects increasing with increase in test concentrations. 5. Results reveal that Diuron is toxic and reduce the fatty acid composition of the fish even at the low level of exposure (10 and 30% LC50 values) which are normal considered as safe exposure values to the organism.

21. THANK YOU SO MUCH FOR YOUR ATTENTION AHSANTENI SANA