TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Oil spill Dispersant ECC OSD 981 (ECC OSD 981)

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1 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD وزارة الدولة للبحث العلمى المعهد القومى لعلوم البحار و المصايد TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant ECC OSD 981 (ECC OSD 981) Prreeparred byy Prroff.. Drr.. Tarrek Otthman Said Prreessiideent t off NIIOF Prof.. Dr.. Mohamed A.. Shreadah March 2010

2 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR OIL SPILL DISPERSANT (ECC OSD 981) Sample: One sample of oil spill dispersant was received from the Egyptian Canadian Company for Chemical Industries (ECC OSD 981). Methods: 1. Methods were applied according to EPA 1993 in: Weber C.I. et al. (1993). Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms 4 th Ed). Environmental Monitoring Systems Laboratory, US Environmental Protection Agency, Cincinnati, OH. August 1993, EPA/600/4-90/027F. 2. The Determination of the efficiency index of oil spill dispersants. According to: Appendix A to WSL Report LR 448 (OP), revised February This specification relates to oil spill dispersants for application at sea or on beaches and to the procedures adopted in the UK before any dispersant can be so used. Physical and Chemical Properties properties stated in the safety data sheet of ECC OSD 981: From at 20 C: green liquid. Density at 20 C: 0.90 kg/l. Flash Point, PMCC: 24 0 C. K. Viscosity at 25 C: cst. K. Viscosity at 40 C: 8.5 cst. Solubility: Hydrocarbon Soluble

3 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Preparation of (ECC OSD 981)/Gulf of Suez crude oil mixture: ECC OSD 981 oil spill dispersant was first mixed with Suez I- TOXICITY TEST Gulf crude oil with a ratio of 1:10; W/W (g), then the mixture was shacked for 10 min. Serial dilutions of the Mixture (50-450ppm) were made to perform the test using seawater of the Eastern Harbour, Alexandria-Egypt. A) 96 hr Natural Phytoplankton Population Test Conditions 1. Date of the started test: 15/03/ Test Type Static method, non-renewal. 3. Sterilized natural seawater was used. 4. Salinity Temperature (20 ± 1 o C). 6. ph ( ±1.1). 7. Light Quality laboratory illumination 6000 Lux light. 8. Photoperiod 24 hr light, 0 hr dark. 9. Size of test vessel: 250 ml. 10. Volume of test solution: 100 ml. 11. The tested Organism: Tetraselmis chui (phytoplankton, Chlorophytes). 12. Total No. of organisms concentration in each flask (0.3x10 6 cell/ml). 13. The exposure time: 24hr, 48hr, 96hr. 14. No. of replicate test vessels per treatment Aeration: None. 16. Number of dilutions 6 plus one control. 17. Effect of measure: number of cell in ml.

4 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Results Table (1) Concentration of cells/ml (x10 6 ) compared to the control after 96 hours Average No. of Average No. of No. Concentration live cells/ml dead cells /ml Mortality % (ppm) X 10 6 X 10 6 after 96hr after 96hr 1 Control The initial count = 0.3X10 6 cell/ml Tetrasaelmis sp. Mortality % Concentration ppm Figure 1. Toxicity test for ECC OSD 981/Suez Gulf crude oil applied on Tetraselmis chui.

5 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD According to Figure 1 and Table 1, the LC50 for ECC OSD 981 is 160ppm applied on Phytoplankton (Tetraselmis chui). PHYTOPLANKTON (Tetraselmis chui) 96hr LC 50 ITEM CONCENTRATION 160ppm B) Shrimp Material and methods: Test conditions: 1. Date test started: 15/03/ Test Type Static method, non-renewal. 3. Sterilized natural seawater was used. 4. Salinity: (36 ). 5. Temperature (27 o C ± 1 o C). 6. ph: ( ±1.1). 7. Dissolved oxygen: mg/l. 8. Light Quality laboratory illumination 200 Lux light. 9. Photoperiod 16 hr light, 8 hr dark. 10. Size of test Glass enclosures: 30x30x30 cm 3 (25 liters net). 11. Volume of tested seawater samples of 19 liters. 12. Organisms type species Penaeus japonicus. 13. Mean weight: (0.03±0.008g). 14. Mean length: (1.2±0.012 cm). 15. Source: Natural resource at Eastern Harbour of Alexandria.

6 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Acclimation: the shrimps were acclimated for 3 days before the test. 17. Feeding: non. 18. Total No of organisms per test chamber = 10 organisms. 19. No. of replicate test chamber per treatment = Aeration: by air stone rate 250 bubbles/min. 21. Number of dilutions 10 plus one control. 22. Effect Measured Mortality: no movement for body of Penaeus japonicus. Results Table (2) Percentage of observed mortality after 96hrs for Penaeus japonicus No. Concentration (ppm) Mortality% 1 Control

7 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD hr LC 50 Toxicity test for Penaeus japonicus Penaeus japonicus Mortality % Concentration, ppm Figure (2) Toxicity test for ECC OSD 981/Suez Gulf crude oil mixture applied on Penaeus japonicus.

8 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD ITEM LC 50 Shrimp (Penaeus japonicus) Concentration 270 ppm C) Fish Material and Methods: Test conditions: 1. Date test started: 15/03/ Test Type Static method, non-renewal. 3. Sterilized natural water was used. 4. Salinity: (36 ). 5. Temperature (23 o C - 26 o C ± 1 o C). 6. ph ( ±1.1). 7. Dissolved oxygen:( ml/l). 8. Light Quality laboratory illumination 200 Lux light. 9. Photoperiod 16 hr light, 8 hr dark. 10. Size of test Glass enclosures-30x30x30cm (25 liters net. 11. Volume of test solution 19 liters. 12. Organisms type species (Liza ramada). 13. Mean weight :(0.61 ±0.04 g). 14. Mean length: (3.56 ±0.002 cm). 15. Source: Natural resource at Eastern Harbour of Alexandria. 16. Acclimation: Liza ramada was acclimated for 4 days before starting of test. 17. Feeding: non.

9 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Total No of organisms per test chamber = 10 Liza ramada. 19. No. of replicate test chamber per treatment = Aeration: by air stone rate 250 bubbles/min. 21. Number of dilutions 8 plus one control. 22. Clean of aquaria: by siphoning. 23. Effect of Measured Mortality: no movement of body of animal. Toxicity test for fish (Liza ramada)

10 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Results Table (3): percentage of observed mortality on 96 hours No. Concentration (ppm) % of observed mortality Control Liza ramada Mortality % Concentration, ppm Figure 3. Toxicity test for ECC OSD 981 applied on Liza ramada.

11 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD ITEM LC50 50 Fish (Liza ramada) Concentration ppm 220ppm II. Efficiency test for Oil spill Dispersant-ECC OSD 981 Preparation of dispersed oil: The stock of dispersed oil was prepared by adding 5 ml (4.17g) of Suez Gulf crude oil to 25o ml of natural seawater. Two ml of diluted ECC OSD 981 was added to the crude oil, the mixture was shacked for 10 minutes. The concentrations of the dispersed oil for the range (g/ml) of chloroform were measured at 580nm using UV/V Spectrophotometer (Table 4 and Figure 4). Table 4. Absorbance reading for different concentrations of Suez Gulf crude oil using UV/V Spectrophotometer at λ = 580 nm Wt.(g) Abs

12 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Absorbance y = x R 2 = wt., g Figure 4. Calibration Curve for standard Suez Gulf crude oil Slope = 4.62 F = Efficiency % = weigt of oil in 50ml sample of oily water X 500 Weight of test oil added to the 250 ml separating funnel Efficiency = X Efficiency % = 92% III. BIODEGRADTION TEST A thorough sight on data of Table (5) and Figure (5) indicated that ECC OSD 981 is biodegradable with 64% degradation rate after 96hr. Table 5. Biodegradation test for ECC OSD 981 at 96hr Time hr Absorbance Conc. (ppm) Residual Conc. (mg/l) Degradation %

13 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Residual Concentration (ppm) time (hr) Figure (5) Biodegradation test for Oil Dispersant (ECC OSD 981) after 96hr Conclusion: Gulf of Suez crude oil was treated by (ECC OSD 981) and tested for toxicity to natural phytoplankton population, fish and shrimp. The 96 hr EC 50 of treated oil were 160ppm for natural phytoplankton population, 220ppm for fish and 270ppm for shrimp. Indicating lower toxicity limits within a concentration range lower than 160ppm in a ratio of 1:10 of concentrated ECC OSD 981 mixed with Suez Gulf Crude oil; w/w). The efficiency test indicated that ECC OSD 981 is highly efficient for treatment of crude oil with efficiency equal 92%,

14 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD higher than the acceptable limit of 60% (recommended by EPA 1993). The biodegradation test indicated that the maximum degradation rate of ECC OSD 981 was 83% after 96hr. This clearly indicates that the biodegradation rate for ECC OSD 981 is considered to be biodegradable, lesstoxic and highly efficient in the treatment process (Table 6). Table 6 TEST Result For ECC OSD 981 Acceptable limits Comment EFFICIENCY TOXICITY for Suez Gulf crude oil and ECC OSD % ppm for different biota samples > 60% (EPA 1993) Should not be significantly greater than the toxicity for Suez Gulf crude oil alone which equal up to 1000 mg/l (UNEP 1984) ECC OSD 981 (1:10, W:W) is highly efficient for treatment of crude oil Concentrated ECC OSD 981; Suez Gulf Crude oil (1:10, W:W)is Non-toxic BIODEGRADATION 83% acceptable environmental impact (UNEP 1984) Biodegradability of ECC OSD 981 (1:10, W:W)is very high

15 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD Table 6 clearly indicates that ECC OSD 981 is highly efficient, easily biodegradable and lesstoxic in the mixing ratio (1:10, W:W)with crude oil. References Appendix A to WSL Report LR 448 (OP), revised February "The determination of the efficiency index of oil spill dispersants". EPA 1993: Impact of oil and related chemicals on the marine environment. Coastal House Publication LTD GraphPad Prism, 1999,version 3.02, April 25, 2000 GraphPad Software Inc., San Diego CA, UNEP 1984: Combating oil pollution in the Kuwait Action Plan Region. UNEP Regional Seas Reports and studies No 44, pp: UNEP. 1989: Test of the acute lethal toxicity pollutants to marine fish and invertebrates. Reference methods used for marine pollution studies.no.43, 27pp. UNEP. 1989: Methods of the toxicity of pollutants to marine phytoplanktonic and zooplanktonic organisms. Reference methods used for marine pollution studies.no.44, 24pp. Weber C. 1993: Methods for measuring the acute toxicity of effluents and receiving waters freshwater and marine organisms EMSL.257pp. Prroff.. Drr.. Tarreek Otthman Said Prreessiideent t off NIIOF Proff.. Mohamed A.. Shreadah Allexandriia March 2010

16 TOXICITY, EFFICIENCY AND BIODEGRADTION TEST FOR Oil spill Dispersant-ECC OSD