Toxicological studies of Irgarol-1051 and its effects on fatty acid composition of Asian sea-bass, Lates calcarifer

Regional Studies in Marine Science 2 (2015) 171–176

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Toxicological studies of Irgarol-1051 and its effects on fatty acid composition of Asian sea-bass, Lates calcarifer Hassan Rashid Ali a,c,∗ , Marinah Mohd Ariffin b , Mohammed Ali Sheikh c , Noor Azhar Mohamed Shazili a , Zainudin Bachok a,b a

Institute of Oceanography and Environment, University of Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia

b

School of Marine and Environmental Sciences, University of Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia

c

Tropical Research Centre for Oceanography, Environment and Natural Resources, The State University of Zanzibar, P. O. Box 146, Zanzibar, Tanzania

highlights • • • •

Toxicological responses of Lates calcarifer was done using doses of Irgarol. Sublethal exposure (21 days) was established using 50%, 30% and 10% of LC50 value. Then liver was taken for measuring fatty acid composition from exposed fishes. Results reveal that Irgarol is toxic and may affects fatty acid content of the fishes.

article

info

Article history: Received 9 June 2015 Received in revised form 26 August 2015 Accepted 25 September 2015 Available online 8 October 2015 Keywords: Irgarol Antifouling Fatty acids composition Lates calcarifer Marine organisms

abstract The herbicides Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) is commonly used as an antifouling paint to the boats and ships however its toxicity on marine organisms using fatty acid composition as indicator has not been fully investigated. Previous investigations have identified environmental concentrations of these herbicides as being a threat to non-target organisms, such as coral-reef and fishes. Their individual toxicity has been assessed, but they can co-occur and interact, potentially increasing their toxicity and the threat posed to marine organism. For example in Malaysia coastal water, we reported the concentration up to 2021 ng/L at Klang port. This is more than 84-fold a maximum permissible concentration in water proposed by the Dutch National Institute of Public Health and the Environment which is 24 ng/L for Irgarol-1051. This paper explains the toxicological responses of Asian seabass, Lates Calcarifer,upon exposure to different concentrations of Irgarol in laboratory. The 96 h-LC50 ’s of Irgarol for acute exposure was found to be 0.535 ± 0.065 mg/L. Sublethal exposure, 21 days, was done using 50%, 30% and 10% of LC50 value. Further on liver was taken for fatty acid composition study. Results of fresh and control samples were not significantly different (P > 0.05) but dominated by Polyunsaturated Fatty Acids (PUFA), followed by Saturated Fatty Acids (SAFA) and Monounsaturated Fatty Acids (MUFA). The trends for other tested groups were significantly different (P < 0.05), with species suffered even at a low level of exposure. Results might reveal that Irgarol is toxic and may affect the marine organisms. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Since the restricted use of tributyltin (TBT) in many parts of the world marine paint companies have been developing alternative

∗ Corresponding author at: Tropical Research Centre for Oceanography, Environment and Natural Resources, The State University of Zanzibar, P. O. Box 146, Zanzibar, Tanzania. Tel.: +255 777667748 (Mobile). E-mail address: [email protected] (H.R. Ali). http://dx.doi.org/10.1016/j.rsma.2015.09.008 2352-4855/© 2015 Elsevier B.V. All rights reserved.

antifouling biocides to substitute TBT (Bao et al., 2011; Alzieu, 2000). At present, most of the alternative antifouling paints are based on copper (Cu) compounds such as cuprous oxide (Cu2 O) and copper thiocyanate (CuSCN), with supplementation of booster biocides to control Cu resistant fouling organisms (Voulvoulis et al., 2000). Irgarol 1051 is one among the most commonly used booster biocides nowadays (Konstantinou and Albanis, 2004). The production, sale and use of Irgarol have been increased considerably, leading to significant increases of its inputs into the marine environment (Bao et al., 2013). As a consequence, elevated

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concentrations of many of Irgarol its degradation products have been detected worldwide in the coastal areas especially marinas and harbours with heavy boating activities (Hall et al., 1999; Konstantinou and Albanis, 2004). The highest concentrations of Irgarol in coastal marine waters were reported to be over 1 µg/L (Konstantinou and Albanis, 2004; Lam et al., 2005). In Malaysia it was reported a maximum concentration of Irgarol of 2021 ng/L at Klang west, cargo and commercial port (Ali et al., 2013). There is an increasing concern on the use of booster biocides, as they have not been fully verified to be less harmful to the environment than TBT (Yebra et al., 2004). So far there are limited data on the toxicity and persistence of these booster biocides as well as their potential risks on marine ecosystems due to their increased use. The mode of toxic action of Irgarol upon aquatic invertebrates is not well known. It has been reported that Irgarol at concentration of 10 and 50 µg/L could induce significant increase of larval malformation (mainly caused by skeletal alteration) and sperm fertilization rate respectively for the sea urchin Paracentrotus lividus (Manzo et al., 2006). It was hypothesized that Irgarol could interact with calcium homoeostasis in P. lividus (Manzo et al., 2006). Irgarol has been proved to inhibit ATP synthesis by inducing opening of small size pores in membrane of mitochondrial from rat liver (Bragadin et al., 2006). Irgarol was found to decrease normal chemoreceptive response and induce metabolic disturbance in the mud snail Ilyanassa obsolete, which was explained by the Irgarol’s negative effect on ATP synthesis (Bragadin et al., 2006). The toxicity of Irgarol 1051 to aquatic organisms has been comprehensively reviewed recently by Konstantinou and Albanis (2004). Most studies have focused on marine environments and, organisms (Scarlett et al., 1997; Liu et al., 1999; Biselli et al., 2000; Boxall et al., 2000; Sargent et al., 2000; Voulvoulis et al., 2000; Thomas et al., 2001; Gardinali et al., 2004; Manzo et al., 2008; Buma et al., 2009). However, there are limited data on the effects of Irgarol in relation to the fatty acid composition as a major of physiological health of marine organisms. The toxicity of these booster biocides on marine organisms, especially those from tropical and subtropical regions are still largely unknown, as most available and relevant toxicity data were generated with temperate species (Sheikh et al., 2009). For some biocides such as Irgarol there are limited toxicity data on marine organisms (Bao et al., 2011). As reported by our previous paper Malaysia Peninsular coastal water is contaminated by more than 1000 folds compared to the maximum permeable concentration of 24 ng/L (Ali et al., 2013). Thus increased an interest to know the toxicological effects of Irgarol to marine species and later fatty acid composition using liver samples was determined as a measure of physiological status of the organisms. Therefore, this paper presents the findings of median lethal concentration (96-h LC50 ) of Irgarol using Lates calcarifer (Siakap) and its fatty acid composition after sub-chronic exposure (21 days) using liver as a sample. We hypothesized that Irgarol can affect marine organisms if exposed to certain levels and there is a change in fatty acid composition of Lates calcarifer exposed in Irgarol even at short period of time. 2. Materials and methods 2.1. Experimental fish Two months old Lates calcarifer were obtained from a commercial fish farm in Setiu province, between April and May 2012. The fishes were 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 1mm filter, treated with UV-sterilized and refilled daily. Fishes were fed twice daily with standard accepted food. Fishes were starved 24 h before and during the chronic exposure experiment.

2.2. Chemicals A stock solution of 1000 mg/L Irgarol was prepared by using acetone and the working concentrations were made up by spiked the required concentrations to the sea water. 2.3. Acute toxicity tests A static bioassay test was performed to determine the 96-h LC50 of Irgarol to Lates calcarifer, following the Standard Methods (APHA, 1995). After the acclimatization period, 2-month-old fish (6.29 ± 0.37 cm in length, 7.96 ± 0.56 g in weight) were transferred from the stock tank to the experimental aquaria. Ten fish were randomly placed in each plastic aquarium (51 × 25 × 30 cm) filled with 30 L of water. Fish were exposed to nominal concentrations of 0.3, 0.5, 1, 3, and 5 mg/L of Irgarol. Each concentration was done in three replicates. Control fish were held in a similar facility without exposure to Irgarol. The water quality characteristics were measured daily: dissolved oxygen (DO) 7.2 ± 0.5 mg/l, temperature 28.5 ± 0.4 °C and pH 7.69 ± 0.2. Water samples were taken at the beginning and at the end of the test for Irgarol analysis. Fish mortality was monitored at 24 h intervals. The criteria for death were no gill movement and no reaction to gentle prodding. Dead fish were immediately removed. Fish mortality (in percentage) from each aquarium was recorded at the intervals of 24, 48, 72 and 96 h (Table 2) and 96 h LC50 values were calculated using Probit Analysis method (Finney, 1971). Fish were not fed throughout the test period. 2.4. Sub-chronic exposure Fish were exposed to 10%, 30% and 50% of the 96 h LC50 values of Irgarol determined from the acute toxicity tests. The same procedures as in acute exposure were followed but the duration of exposure to sub-lethal concentrations was for 21 days. Each aquarium was filled with 30 L of natural sea water (salinity of 30 ppt), pumped continuously over a bio filter column at the rate of 4 L/min. The water was continuously aerated throughout the experiment. Three replicates were performed for test concentration and control. Fish were fed twice daily during the 21 days exposure period. The experimental water (50%) was changed every week to keep water quality within acceptable limits according to APHA (1995). Water quality (dissolved oxygen, temperature, pH and salinity) was measured daily and water chemistry (ammonia nitrogen, nitrite nitrogen and nitrate nitrogen) was measured twice weekly. The ammonia nitrogen and nitrite nitrogen levels were controlled not to exceed 0.2 mg/L. All chemical parameters were determined following the techniques of APHA (1995) using analytical grade reagents. The actual concentration of Irgarol was measured weekly before and after its addition to maintain concentrations at the nominal level. The water characteristics are shown in Table 1. Mortality and behaviour were observed daily in each concentration. One fish from each aquarium was sampled at the end (21 day) of the experiment for fatty acid analysis in the laboratory. 2.5. Analytical procedure Liver from fish was taken as samples for fatty acid composition analysis. The one step method (Abdulkadir and Tsuchiya, 2008) was used in this experiment to combine extraction and esterification processes using a single tube. Three replicates of each liver samples (200–300 mg) were mixed with 4 ml of hexane and 1 ml of internal standard solution in a 50 ml centrifuge tube. After adding 2 ml of 14% BF3 in methanol and a magnetic stirring bar, the head

H.R. Ali et al. / Regional Studies in Marine Science 2 (2015) 171–176 Table 1 Water quality characteristics during sub-chronic exposure to Lates calcarifer for 21 days. Parameters

Range

Mean ± S.D

Dissolved oxygen (mg/L) Temperature (°C) Salinity (%) pH Ammonia nitrogen (mg/L) Nitrite nitrogen (mg/L) Nitrate nitrogen (mg/L)

6.2–7.4 27.9–29.8 29.3–31.2 6.89–8.04 0.02–0.28 0.03–1.25 0.96–3.11

6.9 ± 0.3 28.9 ± 0.66 30.14 ± 0.65 7.76 ± 0.29 0.19 ± 0.12 0.07 ± 0.33 2.16 ± 0.44

space of tube was flushed with nitrogen gas and then closed tightly with a Teflon-lined screw-cap. The capped tube was heated on a hot plate at 100 °C for 120 min under continuous stirring. After cooling to room temperature, 1 ml of hexane was added followed by 2 ml of distilled water. The tube was then shaken vigorously for 1 min and centrifuged for 3 min at 2500 rpm (650 × g). Of the two phases which formed, the upper phase was hexane layer containing the FAMEs. Finally, ∼1–2 ml of the hexane layer was transferred using a Pasteur pipette into a clean sample vial to be injected into the GC-FID for FAME analysis. Fatty acid concentrations (CFA, mg/g of dry sample) were calculated by comparing the peak area of fatty acid in the sample with the peak area of internal standard as follows: CFA = AS /AIS × CIS /WS where; AS = peak area of fatty acid in the sample in chromatogram AIS = peak area of internal standard in chromatogram CIS = concentration of internal standard (mg) WS = weight of sample (g). Qualitatively (as a percentage), composition of individual fatty acids were calculated by comparing the peak area of each fatty acid with the total peak area of all fatty acids in the sample. 2.6. Gas chromatography conditions The FAMEs were separated and quantified using a gas chromatograph (GC 14-B Shimadzu) equipped with flame ionization detector. Separation was performed with an FFAP-polar capillary column (30 m × 0.32 mm internal diameter, 0.25 µm film thickness). Hydrogen was used as a carrier gas. After injection at 60 °C, the oven temperature was raised to 150 °C at a rate 40 °C min−1 , then to 230 °C at 3 °C min−1 , and finally held constant for 30 min. The flame ionization was held at 240 °C. FAME peaks were identified by comparing their retention times with those of authentic standards (Supelco Inc.,). Fatty acids were designated as an n : pωx, where n is the number of carbon atoms in the aliphatic chain, p is the number of double bonds and x is the position of the first double bond from the terminal methyl group. The analytical precision for samples was generally <5% for total amounts and major components of FAMEs. 2.7. Data analysis The 96-h LC50 value was determined using the probit analysis method (Finney, 1971). The no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) were determined by hypothesis testing of mortality on the acute toxicity test. The fatty acid results were calculated and presented as mean ± standard deviation. Significant differences among the mean value of treatments were treated by One-way Analysis of Variance (ANOVA) followed by Least Significant Different (L.S.D). Two-way Analysis of Variance was used to support one way Analysis of Variance to get the F-probability interactions between chemicals, concentrations and/or species. Duncan’s multiple range test (P < 0.05) were calculated.

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3. Results 3.1. Visual observation Acute exposure: control fish swam normally with no sign of any abnormality throughout the 96 h test period. Some treated fish exhibited erratic swimming and excessive mucus production on the opercula surface. Hyperventilation was observed after 48 h and became more severe within the 96 h of exposure in some fish (at 3 and 5 ppm Irgarol concentrations) before death occurred. The dead fish from different concentrations were recorded in Table 2 as percentage mortality rate for Irgarol exposure for interval of 24, 48, 72 and 96 h. Table 3 present the calculated LC50 (95% C.I) value of Irgarol from different methods. In both cases death did not occur in the controls and acetone (check tanks). The 96-h LC50 of Irgarol was 0.535 ± 0.011 mg/L (Mean ± SD) as presented in Table 3. Sub-chronic exposure: Fishes were exposed at 50%, 30% and 10% of the Irgarol 96 h LC50 value (0.535 ± 0.011 mg/L) obtained from acute toxicity tests. The sub-chronic exposure results were 0.26, 0.16 and 0.05 mg/L for 50%, 30% and 10% respectively. Control fish swam normally with no sign of abnormality. Death did not occur in either control or treated fish during the exposure period. 3.2. Fatty acid From sub-chronic exposure test of Lates calcarifer, one fish from each replicate tank was examined for fatty acid composition in the laboratory. The results are presented in Table 4. 4. Discussion 4.1. Toxicity testing The 96 h LC50 values of Irgarol was 0.535 ± 0.011 mg/L for Lates calcarifer used in this study; however to our knowledge no study published yet reporting the toxicity effects on the Lates calcarifer using Irgarol. Only a few studies have studied the toxicity of Irgarol using other species. Bao et al. (2011) reported 96 h LC50’s of 23 µg/L for Irgarol for Synechococcus sp., much lower than the 72 h EC50’s of 0.16 µg/L for Irgarol reported by Devilla et al. (2005). This might be due to different Synechococcus species used in those two separate studies. Our results in this study are very far from Maximum Permissible Concentrations (MPC) of 24 ng/L for Irgarol (Giacomazzi and Cochet, 2004). Irgarol is photosynthetic system II (PSII) inhibitors (Jones and Kerswell, 2003), and is highly toxic to autotrophic aquatic species such as cyanobacteria, algae, macrophytes and symbiotic dinoflagellates in corals, with acute EC50’s on growth generally range from ng/L to µ g/L levels; however, they are far less toxic to crustaceans and fish with acute LC50’s generally at mg/L level (Hall et al., 1999; Zhang et al., 2008). The results of this study were also consistent with those findings. Irgarol could affect the photosynthesis of the symbiotic dinoflagellates in corals at levels of <1 µg/L (Jones and Kerswell, 2003) suggesting that, at its highest detectable concentrations (>1 µg/L), could cause severe impacts on the growth of microalgae and corals in the marine ecosystem. Moreover, the findings of the current study supported previous studies of Jones and Kerswell (2003) and Chesworth et al. (2004) which showed that Irgarol was generally toxic for the tested species. In essence, Irgarol might have a greater affinity (i.e. higher log Kow) to its target site (quinine- B binding site) and organism’s lipid tissues. Therefore, Irgarol could be taken up more readily, accumulate more effectively in the organism, and thus exhibit a greater toxic potential (Chesworth et al., 2004).

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Table 2 Cumulative percentage mortality data of Lates Calcarifer (n = 10) in acute exposure test to Irgarol-1051. Concentration (ppm)

% Mortality (1st replicate)

0 (control) 0 (check/acetone) 0.3 0.5 1 3 5

% Mortality (2nd replicate)

% Mortality (3rd replicate)

24 h

48 h

72 h

96 h

24 h

48 h

72 h

96 h

24 h

48 h

72 h

96 h

0 0 0 0 0 50 100

0 0 0 0 0 80 100

0 0 10 20 40 90 100

0 0 30 50 70 90 100

0 0 0 0 0 30 90

0 0 0 20 30 60 100

0 0 10 30 50 70 100

0 0 20 60 80 80 100

0 0 0 0 0 40 100

0 0 0 0 20 60 100

0 0 20 30 50 80 100

0 0 30 40 70 90 100

Table 3 Calculated LC50 (95% C.I) value of Irgarol (mg/L) from different method. Method

1st replicate LC50

2nd replicate LC50

3rd replicate LC50

Mean

SD

Binomial MAA Probit Spearman Mean average

0.5 0.523 0.535 0.523

0.440 0.476 0.527 0.473

0.628 0.598 0.588 0.608

0.523 0.532 0.550 0.535 0.535

0.096 0.062 0.033 0.068 0.011

Table 4 Fatty acid composition (mg/g) dry weight of liver sample of Lates calcarifer (Siakap) after 3 weeks exposure test. Name

Fresh

Control

10%LC50 Irgarol

30%LC50 Irgarol

50%LC50 Irgarol

SAFA C14:0 C16:0 C18:0 C20:0

4.66ab ± 0.36 35.9a ± 11.6 12.8a ± 1.56 0.00

4.96a ± 1.46 30.9ab ± 7.31 13.3a ± 3.22 1.19a ± 0.60

3.84abc ± 2.23 24.8abc ± 0.62 6.17bcd ± 0.11 0.59b ± 0.16

2.46bcd ± 0.71 16.7bc ± 0.63 6.01cd ± 0.12 0.00

0.86d ± 0.04 9.43c ± 0.33 3.17d ± 0.44 0.00

MUFA C16:1 C17:1 C18 : 1ω9c C18 : 1ω9t C20 : 1

3.85bc ± 0.82 1.72a ± 0.38 34.6ab ± 0.82 8.13b ± 0.17 1.32bc ± 1.15

6.68a ± 2.33 0.00 37.1a ± 11.5 0.00 4.34a ± 1.94

4.00abc ± 1.68 0.00 20.5bc ± 3.51 0.00 2.24abc ± 0.23

3.63bc ± 0.15 0.00 1.73d ± 0.18 14.8a ± 4.36 0.00

2.27c ± 0.48 0.00 0.00 9.28b ± 0.47 0.00

PUFA C18 : C18 : C20 : C20 : C22 :

28.7a ± 0.80 9.25a ± 1.34 6.48a ± 2.50 7.73a ± 0.02 6.40ab ± 1.70

24.9ab ± 7.03 8.44a ± 6.96 5.37a ± 1.93 6.33a ± 0.20 8.56a ± 1.97

18.2bc ± 2.11 9.25a ± 0.75 0.00 4.64bc ± 0.39 7.71a ± 0.96

7.77d ± 0.42 10.4a ± 0.42 0.61b ± 0.01 5.59ab ± 0.68 7.06a ± 0.54

4.33d ± 0.12 6.87a ± 0.45 0.00 3.15cd ± 0.10 3.81b ± 0.10

53.4a ± 13.4 49.6a ± 14.0 58.5a ± 6.76 161a ± 11.8

50.4ab ± 13.2 48.1a ± 15.6 53.6ab ± 9.23 152ab ± 12.0

35.4bc ± 10.9 26.8bc ± 8.65 39.8bc ± 6.77 102bc ± 8.22

25.1bc 20.2bc 31.4cd 76.7cd

13.5c ± 4.26 11.5c ± 4.02 18.2d ± 2.48 43.2d ± 3.38

3ω6 3ω3 3ω3 5ω3 6ω3

Σ SAFA Σ MUFA Σ PUFA Σ FA

± 7.35 ± 6.22 ± 3.64 ± 5.51

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).

4.2. Fatty acid composition The average concentrations and their standard deviation of saturated fatty acids (SAFA), monounsaturated fatty acids (MUFA) and Polyunsaturated fatty acids (PUFA) in the Lates calcarifer is presented in Table 4. In fresh and control samples, PUFA group is dominant followed by SAFA and then MUFA group. It shows Σ PUFA 58.5 ± 6.76 (36.2%) and 53.6 ± 9.23 (35.2%) mg/g dry weight, followed by SAFA group, 53.4 ± 13.4 (33.0%) and 50.4±13.2 (33.1%) mg/g dry weight and MUFA group, 49.6 ± 14.0 (30.7%) and 48.1 ± 15.6 (31.6%) mg/g dry weight for fresh and control samples respectively (Table 4). The trend for other fatty acids in Lates calcarifer exposed with different doses of Irgarol from the groups of 50%, 30% and 10% of 96 h LC50 agreed with that of control and fresh sample; i.e. PUFA > SAFA > MUFA. It is interesting to note that in Lates calcarifer (Fig. 1) the MUFA groups contributed less in the total FA among all exposed groups.

4.2.1. Saturated Fatty Acids (SAFA) The Lates calcarifer is enriched in SAFA and the Total SAFA are not significant different (P > 0.05) between fresh and control groups, however the other groups and individual SAFA were significant difference (P < 0.05) in Lates calcarifer (Table 4). Of the SAFAs, Palmitic acid (C16:0) was found predominant in the fresh samples (35.9 ± 11.6 mg/g, 67.2%), followed by control samples (30.9 ± 7.31 mg/g, 61.3%) dry weight of the liver of Lates calcarifer. It was also reported that Palmitic acid was the predominant in SAFA group as found in freshwater channel catfish (Ictalurus punctatus) (19.2%) (Sathivel et al., 2002), and in freshwater rainbow trout (Oncorhynchus mykiss) (21.3%) (Ibrahim Haliloglu et al., 2004). The fresh water species domination on SAFA fatty acids is also reported in the study of Jabeen and Chaudhry (2011). Irgarol was seen to be toxic since it affected the fatty acid concentrations more in all SAFA groups. As expected, individual fatty acids from 50% groups and 500 µg/L were much affected than the rest of the groups for Irgarol (Fig. 2).

H.R. Ali et al. / Regional Studies in Marine Science 2 (2015) 171–176

175

200 Concentration (mg/g) dry weight

180 160 140 120 100 80 60 40 20 0 FRESH

CONTROL

10%LC50

30%LC50

50%LC50

Fig. 1. Fatty acid concentration of different classes and Total fatty acid (mg/g) dry weight from liver of Lates Calcarifer after exposed at different concentrations of Irgarol. Values are mean ± S.D, n = 3.

Fig. 2. Individual Fatty acid concentrations of Lates calcarifer after exposed in different doses of Irgarol.

4.2.2. Monounsaturated fatty acids (MUFA) The Σ MUFA were not significantly different between Fresh (49.6 ± 14.0, 30.7%) and control (48.1 ± 15.6, 31.6%) groups of Lates calcarifer (P > 0.05) in Table 4. In Lates calcarifer, oleic acid (C18 : 1ω-9c) was the most prevalent MUFA and it was higher (P < 0.001) in all experimental fish except for 50%LC50-Irgarol where it was not detected (Fig. 2). Ho and Paul (2009) found about 8% only of oleic acid in Total fatty acid in Asian sea bass. However, Steiner-Asiedu et al. (1991) found that freshwater tilapia (Tilapia sp.) had significantly higher oleic acid levels than flat sardine (Sardinella sp.) and sea bream (Dentex sp.). The level of oleic acid in American freshwater channel catfish flesh was also about 50% compared to less than 1% in sardine and sea mullet (Ackman, 1994). Irgarol affected more in MUFA concentrations (Fig. 2) of Lates calcarifer. 4.2.3. Polyunsaturated Fatty Acids (PUFA) The Σ PUFA in Lates calcarifer were not significantly different among fresh, control and acetone samples (P > 0.05). The PUFA is pronounced more in average for Lates calcarifer in most of experimental groups (Fig. 1). γ -Linolenic acid (C18 : 3ω6) was the major PUFA (49.1% Fresh sample), followed by α -Linolenic acid (C18 : 3ω3) (15.8% fresh sample) in Lates calcarifer (Fig. 2). The other fatty acids, cis-11, 14, 17-Eicosatrienoic Acid (C20 : 3ω3), cis-5,8,11,14,17-Eicosapentaenoic Acid (C20 : 5ω3) and DHA (C22 : 6ω3) showed significant contribution on total PUFA of Lates

calcarifer (Table 4). The higher levels of omega 3 and 6 in this species were not surprising because seawater organisms obtain their omega-3 and 6 FA from oceanic plankton (Steffens, 1997) or are fed fishmeal containing these FA. The tendency of Irgarol to disturb the organisms is also pronounced in PUFA groups of tested organism (Fig. 2). The results showed an inverse relationship between the dose of chemical used and activities of the Lates calcarifer in the tanks. It is clear that, as the concentration of the Irgarol increased in different exposed tanks there was a decrease in activities in Lates calcarifer. 5. Summary and conclusions This paper explains the toxicological responses of Lates Calcarifer upon exposure to different concentrations of Irgarol in laboratory experiments. It can be concluded that; acute exposure of Lates calcarifer has shown Irgarol with 96 h LC50 value of 0.535 ± 0.011 mg/L while sub-lethal exposure to Lates Calcarifer (21 days) to different concentrations of Irgarol had shown significant impact to the fatty acid composition of these organisms. Moreover, fresh and control samples of Lates calcarifer was dominated by PUFA followed by SAFA and then MUFA. It was also observed that test species general health (observed in terms of ability of movements) was affected in exposures to all fractions of 96 h LC50 of Irgarol, the severity of effects increasing with increase in test concentrations. Finally results reveal that Irgarol is toxic and

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