TOXIC EFFECTS OF AZINPHOS METHYL AND AZINPHOS ETHYL ON GROWTH AND CHLOROPHYLL PIGMENTS PRODUCTION OF MARINE UNICELLULAR MICROALGAE TETRASELMIS SUECICA

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1 Proceedings of the 13 th International Conference of Environmental Science and Technology Athens, Greece, 5-7 September 2013 TOXIC EFFECTS OF AZINPHOS METHYL AND AZINPHOS ETHYL ON GROWTH AND CHLOROPHYLL PIGMENTS PRODUCTION OF MARINE UNICELLULAR MICROALGAE TETRASELMIS SUECICA A.S. PETSAS* 1,2, M.C. VAGI 1,2, M.D. PAVLAKI 1,3, N.M. SMARAGDAKI 1, M.N. KOSTOPOULOU 1 and T.D.LEKKAS 2 1 Department of Marine Sciences, Laboratory of Chemical Processes & Aquatic Toxicology, Faculty of Environment, University of the Aegean, University Hill, Mytilene, Greece 2 Department of Environmental Studies, Water & Air Quality Laboratory, Faculty of Environment, University of the Aegean, University Hill, Mytilene, Greece 3 Department of Biology & Center for Environmental and Marine Studies, University of Aveiro, , Aveiro, Portugal Corresponding Author: Tel: Fax: apetsas@env.aegean.gr. EXTENDED ABSTRACT Acute toxicity of two organophosphorus pesticides, azinphos methyl and azinphos ethyl to a non-target aquatic microorganism, the marine alga Tetraselmis suecica was studied. Bioassays were conducted to determine their toxic effects according to the OECD Guideline 201. The green unicellular alga was exposed to several concentrations of the selected compounds. Algal densities were determined by daily cell number counting of the tested microorganism. Specific growth rate (μ) and inhibition of algal growth as a reduction in specific growth rate, relative to control cultures grown under identical conditions (%I) were calculated for each treatment sample. Estimation of effective concentration that inhibits 50% microalgal growth at 96h (96h EC 50) values was performed. Furthermore chlorophyll pigments fractions were measured in the exponential phase after 96h exposure. The contents of acetone-soluble chlorophyll-a (Chl a), chlorophyll b (Chl b) and chlorophyll c (Chl c) that were contained in 10mL of culture medium were evaluated, whereas total chlorophyll (Chl tot) as the sum of Chl a, Chl b and Chl c was also evaluated. Statistical analysis of data was performed by using the software of the statistical package SPSS, version Toxicity data evaluated as percentages and arcsine transformed values (arcsine x ), were analyzed by using one-way analysis of variance (ANOVA). Variances were considered equal based on Kolmogorov-Smirnof test for homogeneity of variance. To determine the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) values ANOVA was carried out and the multiple comparisons Dunnett s Test Method was used to detect significant (Dunnett Method, p 0.05) differences between the data that followed a normal distribution. The maximum acceptable toxicant concentration (MATC), defined as a hypothetical threshold value, was estimated as the geometric mean of NOEC and LOEC. Experimental results revealed that azinphos ethyl is more toxic than azinphos methyl, while low concentrations of both of the examined pesticides did not affect the growth of T. suecica. On the contrary concentrations of azinphos methyl above 12.0mg L -1 and of azinphos ethyl above 3.0mg L -1 were found to have marked effects (p 0.05) on the growth of the target microorganism. Obtained data concerning the assessment of effect on the production of chlorophyll pigments confirmed the commonly accepted hypothesis of chlorophyll pigments content being proportional to growth rate of microalgal species.

2 KEYWORDS: Organophosphorus pesticides; Tetraselmis suecica; Toxicity test; Alga; Bioassays; Ecotoxicology; Effective Concentration; Pigment biomarker; Chlorophylls. 1. INTRODUCTION Azinphos methyl (S-3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-ylmethyl O,O-dimethyl phosphorodithioate) and the analogous diethyl ester, azinphos ethyl (S-3,4-dihydro-4- oxo-1,2,3-benzotriazin-3-ylmethyl O,O-diethyl phosphorodithioate) are two phosphorus containing pesticides. Both of them are classified as organothiophosphate acaricides or as benzotriazine organothiophosphate insecticides and in any case they belong to the group of organophosphorus pesticides. Target pollutants are widely used as non-systemic agricultural insecticides and acaricides used on a wide range of crops as broad spectrum pesticides. These compounds are extremely potent systemic toxicants via ingestion, inhalation and skin contact and they share a common mechanism of toxicity; they affect the nervous system by inhibiting acetylcholinesterase (anti-ches). Target chemicals, like many other pesticides, are compounds that though they are used in agriculture worldwide they are also considered to be potential contaminants for surface and ground water. Paradoxically, they do not always remain in agricultural soils where they are applied, but some times they find their way into aquatic systems through leaching, surface run-off, spray-drift, soil erosion and volatilization. Therefore aquatic environments receive direct and indirect inputs of azinphos methyl and azinphos ethyl (Lekkas et al., 2004), inevitably exposing microorganisms to it (Vagi et al., 2005). Ecotoxicity data for the selected molecules towards non-target aquatic microorganisms are limited (Thurberg et al., 1973). Published data regarding marine or estuarine microorganisms are even scarcer than the ones regarding freshwater micro-species. It is known that marine microalgae are important inhabitants of aqueous ecosystems, where they fulfill critical roles in primary productivity, nutrient cycling and decomposition. The objectives of this study were: (i) to evaluate their toxicity towards non-target marine microorganisms, such as Tetraselmis suecica, and (ii) to investigate the possibility of using the parameter of chlorophyll pigments production as a biomarker of their exposure. METHODS AND MATERIALS 2.1 Test organism and culture conditions Tetraselmis is a genus of a marine, motile, green phytoplankton (Prasinophyceae). Bioassays were performed using the unicellular marine microalgae T. suecica as test organism, which is a strain of phytoplankton commonly cultivated in shellfish husbandries (Fábregas et al., 2001) and has been routinely and successfully cultivated at the laboratory for more than 10 years. Moreover this species was chosen because its response in toxicity tests is highly reproducible (Vagi et al., 2005). Unialgal cultures of T. suecica were maintained in liquid f/2 growth medium as recommended by Provasoli- Guillard National Center for Culture of Marine Phytoplankton (PGNCCMP, 2013). Salinity of the seawater was 35.0±0.1 and the initial ph of the cultures was 8.0±0.1 ((OECD, 2004). The cultures were incubated under continuous illumination with cool-white fluorescent lights emitting a radiant energy of 4300Lux equivalent to 12.9W m -2. Temperature was maintained stable in a temperature controlled growth chamber at 20.0±0.3 o C (Vagi et al., 2005). 2.2 Test chemicals, reagents and standards Organophosphorus pesticides were of analytical grade (98.5% for azinphos methyl and 99.4% azinphos ethyl) and obtained from Dr Ehrenstorfer (Germany). Chemical structure and main physicochemical properties of the target compounds are shown in Table 1. Due to their low water solubility, an appropriate organic solvent was used for the preparation of their independent stock solutions that were stored at -18 o C. Therefore, acetone was

3 used as the carrier solvent of the compound to the bioassays, since previous experiments proved that this solvent up to a final concentration of 0.5μL ml -1 in f/2 medium did not affect the growth rate of the tested alga (Vagi et al., 2005). Organic free water was prepared with a Milli-Q/Milli-Ro system (Millipore Corp., USA). GF/F filters with 0.45μm porosity were supplied by Whatman (UK). Table 1. Chemical structure and physicochemical properties of selected pesticides (Tomlin, 1997). Compound Chemical structure (IUPAC Name) Solubility in water Log K ow Azinphos Methyl 28-33mg L -1 (at 20 o C) 2.96 S-3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-ylmethyl O,Odimethyl phosphorodithioate Azinphos Ethyl 4-5mg L -1 (at 20 o C) 3.18 S-3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-ylmethyl O,Odiethyl phosphorodithioate 2.3 Acute toxicity test and pesticide s treatment Bioassays were performed according the OECD Guideline 201 for testing the effects of chemicals on alga growth inhibition test (OECD, 2004), with some modifications. Detailed information about the acute toxicity test protocol have been reported by Vagi et al. (2005). In brief cells in the exponential phase of growth were collected from stock cultures (called as pre-cultures and incubated under the previous mentioned conditions) and thus used as the inoculum. Initial algal density of 1x10 5 cells ml -1 in each one of the experimental treatments was set (Vagi et al., 2005). The experimental design and test conditions were identical for the two selected toxicants. Each chemical bioassay included the below described treatments: a control (C), a control containing acetone as carrier solvent of the organic toxicants, in concentration 0.05% (C+A), and various toxicant exposure concentrations of each toxicant (in mg L -1 ), following the results of preliminary range-finding experiments conducted for each compound previously (Vagi et al., 2005). More specific test organisms were exposed to the concentration series of 10.0; 11.0; 12.0; 13.0; 14.0; 15.0 and 16.0mg L -1 of azinphos methyl and 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5 and 5.0mg L -1 of azinphos ethyl. The treatment solutions were made by adding appropriate amounts of each toxicant to the 100mL of algal nutrient medium. Each treatment contained three replicate flasks. The environmental conditions during the experiments were the same as the growth conditions stated in paragraph 2.1. The test vessels containing the cultures during the course of the experiments were gently shaken by hand once per day in order to keep the cells in free suspension and to facilitate CO 2 mass transfer from air to water, and in turn reduce ph shift. Thus variations in ph during the 96h of incubation were within the limit of ±1.0 unit. Cell densities were assessed daily by microscope counting using an improved Neubauer

4 haemacytometer. Lugol solution was added to the samples (ratio of lugol /culture media: 1/10 v/v) in order to prevent the natural movement of T. suecica cells. 2.4 Growth inhibition calculations Acquired values were plotted against time for the appropriate construction of growth curves. Average specific growth rate (μ) for each pesticide tested was calculated according to the Equation (1): lnn t lnn o μ (1) t t o where t o is the time of test start (t o=0h), t is the time of test termination (t=96h), while N t and N o are the initial and final cell densities at times t and t o respectively. Growth inhibition of the alga was used as the end point of the bioassays and was estimated from the relationship described by Equation (2): %Inhibition μcontrol μtoxicant % I ( ) 100 (2) μ where μ toxicant is the algal growth rate in the presence of the tested compound, and μ control is the growth rate in the untreated control (Nyholm and Kallqvist, 1989). Furthermore the no observed effect concentration (NOEC) was defined as the highest tested concentration of toxicant below which no reduction in reproduction was observed after a 96h exposure period, while the lowest observed effect concentration (LOEC) was defined as the lowest tested concentration of the chemical at which reduction of algal growth was observed after a 96h exposure period. The maximum acceptable toxicant concentration (MATC) was defined as a hypothetical threshold concentration that was calculated as the geometric mean between the NOEC and LOEC concentration. 2.5 Determination of photosynthetic pigments The effect of toxic substances azinphos methyl and azinphos ethyl on the photosynthesis of algae was measured by using pigments such as chlorophyll. At the end of incubation (96h) cells of the microalga T. suecica which have been treated with different concentrations of azinphos methyl or azinphos ethyl and contained in 10mL of culture medium were collected by filtration under vacuum on 0.45μm porosity glass filters (GF/F, Whatman, England). The contents of acetone-soluble chlorophyll-a (Chl a), chlorophyll b (Chl b) and chlorophyll c (Chl c) were calculated according to the relationships of Strickland and Parsons (1972), using Equations (3), (4) and (5) respectively: Chl a = 11.6 E E E 630 (3) Chl b = 20.7 E E E 630 (4) and Chl c = 55 E E E 645 (5) where E 630, E 645, and E 665 are the absorbancies of the acetone extracts at wavelengths indicated by the subscripts, whereas total chlorophyll (Chl tot) as the sum of Chl a, Chl b and Chl c was evaluated as well. 2.6 Data reliability and statistical analysis Independent experiments were repeated three times and each sample (treatment and/or control culture) was repeated three times. Statistical analysis of data obtained from the acute toxicity experiments, was performed by using the software of the statistical package SPSS, version Toxicity data calculated as percentages and arcsine transformed values (arcsine x ), were analyzed by using one-way analysis of variance (ANOVA). Variances were considered equal based on Kolmogorov-Smirnof test for homogeneity of variance. To determine the NOEC and LOEC values ANOVA was carried out and the multiple comparisons Dunnett s Test Method was used to detect differences between the data that followed a normal distribution. Finally the EC 50 value (pesticide concentration required to cause a 50% reduction in growth) was estimated using linear regression control

5 analysis of transformed pesticide concentration as logarithm data versus percent inhibition (Nyholm and Kallqvist, 1989; OECD, 2004). 3. RESULTS AND DISCUSSION 3.1 Acute toxicity of pesticides tested on growth of marine alga T. suecica Cultured in different concentrations of azinphos methyl and azinphos ethyl the algal growth curves of T. suecica were derived. In Figure 1 the growth curves of test organism are depicted, during its 96h exposure to azinphos methyl for the range of concentrations 10-13mg L -1. The results contained in the chart indicated that cells in the presence of azinphos methyl grew slower than those in control group. The same pattern was observed in azinphos ethyl treated mediums. Therefore, it was demonstrated that the two selected toxic agents, azinphos methyl and azinphos ethyl, can inhibit growth of T. suecica at the concentration ranges tested. Cell density, cell/ml Control Control+Acetone Cell density, cell/ml Control 10mg/L 11mg/L 12mg/L 13mg/L Time, h Time, h (a) (b) Figure 1. Growth curves of Tetraselmis suecica of (a) control and acetone control, (b) 96h exposure to several treatments of azinphos methyl. Mean specific growth rate (μ) of exposed marine alga when incubated into culture mediums containing the studied toxicants was calculated (not presented) by using Equation (1) for each of the three replicated bioassays conducted and for each level of exposure concentration. Acquired data showed that acetone controls containing the carrier solvent did not differ significantly from blank controls, illustrating that acceptance criteria that were set up according the applied OECD Test No 201, were fulfilled and hence this solvent could be used in performed bioassays (Figure 1a). On the contrary it could be observed that both of the studied organophosphorus compounds consistently inhibited the algal population growth, whereas μ values decreased when concentration of examined pollutants increased. More specifically, in the case of azinphos methyl the specific growth rate became significantly (Dunnett Method, p 0.05) lower with the increase of its concentration above 12.0mg L -1. At higher exposure concentrations severe reduction in growth of tested microorganisms occurred, whereas lethal effects were observed as well (Figure 1b). In the case of azinphos ethyl obtained values of μ indicated that significant (p 0.05) reduction of the algal densities occurred in treatment levels above 3.0mg L -1. Pesticide concentrations of 3.5, 4.0, and 4.5mg L -1 significantly decreased T. suecica densities after 96h of exposure, while 5.0mg L -1 was found lethal. The percentage of inhibition data relative to growth in untreated controls (%I) was estimated by following Equation (2) and typical sigmoid form (S-shape) concentration-

6 response curves of studied insecticides to T. suecica were plotted (not shown). Obtained %I estimations were linearly related to transformed pesticide concentration levels by logarithmic conversion (logc). High values of correlation coefficients showed that data fitted satisfactory the linear model for both of the tested substances. According to the acquired EC 20 and EC 50 results of present study (not presented) it was confirmed that azinphos methyl is less toxic towards the target marine microalgae than azinphos ethyl. This observation is in agreement with previously reported findings of long range toxicity experiments of azinphos methyl and azinphos ethyl towards T. suecica (Vagi et al., 2005). Moreover, results of this survey and in accordance with other published data concerning the toxicity of organophosphates towards microalgae (Li et. al., 2005). The available data in literature involving the ecotoxicological effects of organophosphorus pesticides towards non-target algae revealed a wide range variation depending on the organophosphorus compound and the tested species. Studies that have been conducted with such chemicals towards several algal strains showed a higher or a lower toxicity than the ones obtained in current survey, but the majority of the relevant published studies suggested that azinphos methyl and azinphos ethyl are relatively more toxic to algae compared to other organophosphorus pesticides (Vagi et al., 2005; Ma et al. 2004; 2005). For instance according to Ma et al. (2004) after 96h of exposure to ethephon an EC 50 equal to mg L -1 was obtained for Chlorella pyrenoidosa, while NOEC and LOEC values were reported 2mg L -1 and 5mg L -1 respectively, indicating a lower toxicity to this insecticide (Ma et al. 2004). Another study where considerably lower toxicity values were observed is the case of four organophosphates diazinon, methidathion, phoxim and thionazin when tested to 5 species of freshwater green algae, Raphidocelis subcapitata (also known as Selenastrum capricornutum), Scenedesmus quadricauda, Scenedesmus obliquus, Chlorella vulgaris and Chlorella pyrenoidosa (Ma et al. 2005). In this point it must be mentioned that when comparing chemicals toxicity the sensitivity of the exposed organism should also be taken into consideration. Even though it is well known that freshwater and marine organisms (among which the tested microalgae are included), exhibit different sensitivities to toxicants, however this is the only available comparison that can be done, since there are generally fewer toxicity data of azinphos methyl and azinphos ethyl available for saltwater species than for freshwater species. Finally T. suecica is considered to be quite sensitive to several organophosphorus insecticides, such as fenthion, parathion ethyl, parathion methyl, disulfoton and dimethoate (Vagi et al., 2005). Compared with the above mentioned pesticides azinphos methyl exhibited moderate toxicity, whereas azinphos ethyl was one of most toxic ones towards the afore mentioned marine miroalgal species.

7 3.2 Toxicity of pesticides tested on chlorophyll pigments production of marine alga T. suecica Effect of azinphos methyl and azinphos ethyl on the production of each pigment was assessed and results were expressed either as pigment concentration in the culture medium (Chl in μg L -1 ) or as chlorophyll content per cell exposed (Chl in pg cell -1 ). Percentage inhibition (%I) of chlorophyll pigments production of marine alga T. suecica as a reduction in concentration of each pigment content, relative to control cultures grown under identical conditions was estimated as well. From the derived experimental data (not presented) it became obvious that azinphos methyl and azinphos ethyl diminished the contents of photosynthetic pigments (Chl a, Chl b, Chl c and Chl tot). Statistical significant difference as compared to the controls occurred in photosynthetic activity of T. suecica cells that were exposed to concentration of azinphos methyl and azinphos ethyl above 12.0mg L -1 and 3.0mg L -1 correspondingly. These observations are in accordance with the results obtained from the performed acute toxicity tests of current study. Values of the ratio of chlorophyll-a/chlorophyll-b (Chl a/chl b) concentrations were also calculated and results pointed out that, whereas cell density decreased with increasing exposure treatments of azinphos methyl and azinphos ethyl, values of Chl a/chl b ratio remained stable or increased, suggesting that the biomass of algae was affected by the organophosphorus insecticides much more strongly than the structure of the chlorophyll body. This data are in accordance with those of Li et al. (2005), who reported that cypermethrin induced a drastic decrease on the growth and photosynthesis of Scenedesmus obliquus and that production of each one chlorophyll pigment separately was more sensitive to cypermethrin than the ratio of Chl a/chl b. Linear correlations between cell density and chlorophyll pigments concentrations of Chl a, Chl b, Chl c and Chl tot were evaluated, which resulted in high values of correlation coefficients. This fact indicated that the use of chlorophyll measurements to estimate biomass concentration was reliable and validated the possibility of using cell chlorophyll content to asses the state of the cells after 96h exposure to azinphos methyl and azinphos ethyl, as previously described by other authors for other cases of bioassays. These results confirmed the commonly accepted hypothesis of chlorophyll pigments content being proportional to growth rate of microalgal species and therefore apply for toxicity assessment of azinphos methyl and azinphos ethyl on marine phytoplanktonic species such as T. suecica. Chlorophyll pigments have often been used as biomarkers of exposure to pesticides in plants including algae (Li et al., 2005). Test organism, T. suecica Kylin (Buthch) is an algal strain that its mass and its biochemical composition, mainly in protein, chlorophyll a and RNA content has shown great variabilities, which are related to changes in nutrient concentrations and that phenomenon has a marked effect on the nutritive value of this microalga as feed in marine culture. According to the published data these observed changes in chlorophyll-a level either in the stationary or in logarithmic phase of growth were related to nitrogen depletion (Fábregas et al., 2001). Azinphos methyl and azinphos ethyl are not expected to be direct inhibitors of pigment synthesis nor to induce a direct oxidative stress as a consequence of their biochemical mode of action that would destroy chlorophyll pigments. However, pigment content may change in response to the cascade of events following contamination with the pesticide, regardless its different mode of action that is anti-ches. 4. CONCLUSIONS The acute toxicity of two acetylcholinesterase-inhibiting insecticides azinphos methyl and azinphos ethyl to the marine green alga Tetraselmis suecica was evaluated. Based on the results of the current study it appeared that azinphos ethyl was found to be highly toxic to the marine microalgal strain T. suecica, whereas the analogous dimethyl ester, azinphos methyl was found less toxic. Experimental data revealed that both of the

8 examined organophosphorus pesticides had marked effects on the growth of the tested alga and treatment concentrations above 12.0mg L -1 for azinphos methyl and above 3.0mg L -1 for azinphos ethyl strongly affected algal densities and significantly (Dunnett Method, p 0.05) decreased specific growth rate values. The finding that reduction of chlorophyll pigments production was observed due to exposure to azinphos methyl and azinphos ethyl indicated that this parameter could be used as a pollution biomarker. Although the effects on algal growth rate and on photosynthesis of chlorophyll pigments were significant at concentrations higher than are possible under field conditions and the fact that the cell density used in the performed bioassays were several orders of magnitude higher than is found in the natural ecosystems, however it can be expected that the organophosphorus insecticides may impose similar effects on the natural population of the studied alga. Finally, considering the fact that a chemical will rarely be found alone in the environment but commonly in combination with other chemicals, further studies with mixtures should be conducted so as toxicity evaluation of azinphos methyl and azinphos ethyl and their degradation compounds will be assessed in a more realistic way. REFERENCES 1. Couderchet M. and Vernet G., (2003). Pigments as biomarkers of exposure to the vineyard herbicide flazasulfuron in freshwater algae. Ecotoxicol. Environ. Saf., 55, Fábregas J., Otero A., Dominguez A. And Patiño M., (2001). Growth Rate of the microalga Tetraselmis suecica changes the biochemical composition of Artemia species. Mar. Biotechnol., 3(3), Lekkas T., Kolokythas G., Nikolaou A., Kostopoulou M., Kotrikla A., Gatidou G., Thomaidis N.S., Golfinopoulos S., Makri Ch., Babos D., Vagi M., Stasinakis A., Petsas A., Lekkas D.F., (2004). Evaluation of the pollution of the surface waters of Greece from the priority compounds of List II, 76/464/EEC Directive, and other toxic compounds. Env. Int. 30, Li X., Ping X., Xiumei S., Zhenbin W., and Liqiang X., (2005). Toxicity of cypermethrin on growth, pigments, and superoxide dismutase of Scenedesmus obliquus. Environ. Saf., 60, Ma J., Lin F., W. Qin W. and Wang P., (2004). Differential Response of Four Cyanobacterial and Green Algal Species to Triazophos, Fentin Acetate, and Ethephon. Bull. Environ. Contam. Toxicol., 73, Ma J., Wang P., Huang C., Lu N., Qin W. and Wang Y., (2005). Toxicity of organophosophorus insecticides to three cyanobacterial and five green algal species. Bull. Environ. Contam. Toxicol., 75, Nyholm N. and Kallqvist T., (1989). Methods for growth inhibition toxicity tests with freshwater algae. Environ. Toxicol. Chem., 8, OECD, (2004). Guidelines for Testing of Chemicals, Test No 201: Testing of effects on biotic systems, alga, growth inhibition test. Environ. Health and Safety Div., OECD Environ. Directorate, Paris. URL: 28/03/2013). 9. PGNCCMP (2013). Medium Recipes. Medium f/2. Provasoli-Guillard National Center for Culture of Marine Phytoplankton, Bigelow Laboratory for Ocean Sciences. Maine, U.S.A. URL: 03/03/2013). 10. Strickland J.D.H. and Parsons T.R., (1972). A Practical Handbook of Seawater Analysis, 2 nd Ed. Bulletin of Fisheries Research Bord of Canada 167, Ottawa, Canada. 11. Thurberg F.P., Dawson M.A. and Collier R.S., (1973). Effects of Copper and Cadmium on Osmoregulation and Oxygen Consumption in Two Species of Estuarine Crabs. Mar. Biol. (Berl.), 23(3), Tomlin C., (1997). The Pesticide Manual, Crop Production Publications, New York. 13. Vagi M.C., Kostopoulou M.N., Petsas A.S., Lalousi M.E., Rasouli Ch. and Lekkas T.D., (2005). Toxicity of organophosphorus pesticides to the marime alga Tetraselmis suecica. Global Nest Intern. J. 7(2),