Using Zooplankton, Moina Micrura Kurz to Evaluate the Ecotoxicology of Pesticides Used in Paddy Fields of Thailand

Thailand is an agricultural country where agriculture is a very important part of the economy. Thailand expanded exports of agricultural products and also imports fertilizers and pesticides intensively. Pesticides are used widely in agriculture and trade of agricultural products to increase agricultural yield and to protect plant from diseases, weeds and insect damage (Department of Agricultural, 2010). Since pesticides were first imported into Thailand under the “Green Revolution Policy” as part of the 1st National Economic and Social Development Plan in 1966, the total amount of imported pesticides has dramatically increased year by year. Most pesticides used in the country are imported (Department of Pollution Control, 2005), and the quantities of imported agricultural pesticides have increased 3 times from 1994 to 2005, reaching more than 80 thousand tonnes in 2004. Pesticides are applied in the highest quantity in vegetable and fruit farming, where market pressure for appearance is higher. In 2000, organophosphates contributed the majority of imported pesticides followed by carbonates and organochlorines; most were herbicides, followed by insecticides, disease control agents and plant growth regulators (Department of Pollution Control, 2002). The result from increasing pesticides uses has resulted in significant increased crop contamination and human health hazard (Office of Epidemiological, 2009). The risk of pesticide contamination in fruits and vegetables in Thai market often occurs. Rice is the major crop and food source for most Asian countries including Thailand. Rice production from paddy fields faces variety of pests that require a range of pesticides and herbicides to manage the presence of insects and weeds, as well as fungal and bacterial pathogens. Indeed, losses of the total world rice crop due to insects have been estimated to occur at a rate of 28% per annum, which is four times greater than the average for other world cereal crops. More than 90% of the global end-user market in pesticides for rice production is applied in Asia (Abdullah, 1995). In Thailand, pesticides play an important part and widely use on rice production because its benefits in pest control and increased rice production. Therefore, pesticide contamination in draining water from paddy field has been one of non-point source pollution in aquatic ecosystem (Sanchez et al., 2006).


Introduction
Thailand is an agricultural country where agriculture is a very important part of the economy. Thailand expanded exports of agricultural products and also imports fertilizers and pesticides intensively. Pesticides are used widely in agriculture and trade of agricultural products to increase agricultural yield and to protect plant from diseases, weeds and insect damage (Department of Agricultural, 2010). Since pesticides were first imported into Thailand under the "Green Revolution Policy" as part of the 1st National Economic and Social Development Plan in 1966, the total amount of imported pesticides has dramatically increased year by year. Most pesticides used in the country are imported (Department of Pollution Control, 2005), and the quantities of imported agricultural pesticides have increased 3 times from 1994 to 2005, reaching more than 80 thousand tonnes in 2004. Pesticides are applied in the highest quantity in vegetable and fruit farming, where market pressure for appearance is higher. In 2000, organophosphates contributed the majority of imported pesticides followed by carbonates and organochlorines; most were herbicides, followed by insecticides, disease control agents and plant growth regulators (Department of Pollution Control, 2002). The result from increasing pesticides uses has resulted in significant increased crop contamination and human health hazard (Office of Epidemiological, 2009). The risk of pesticide contamination in fruits and vegetables in Thai market often occurs. Rice is the major crop and food source for most Asian countries including Thailand. Rice production from paddy fields faces variety of pests that require a range of pesticides and herbicides to manage the presence of insects and weeds, as well as fungal and bacterial pathogens. Indeed, losses of the total world rice crop due to insects have been estimated to occur at a rate of 28% per annum, which is four times greater than the average for other world cereal crops. More than 90% of the global end-user market in pesticides for rice production is applied in Asia (Abdullah, 1995). In Thailand, pesticides play an important part and widely use on rice production because its benefits in pest control and increased rice production. Therefore, pesticide contamination in draining water from paddy field has been one of non-point source pollution in aquatic ecosystem (Sanchez et al., 2006). This is attributed to be relatively large amounts pesticides applied in paddy field, in addition to common practice of draining the paddy water in draining canals (Tejada, 1995). Around 95 % of freshwater in Thailand is withdrawn to irrigate the more than 5 million hectares of irrigated agriculture. Waste water from this activity may pose significant environmental hazards for aquatic ecosystem in particularly aquatic biota. Furthermore, this contaminate affect wildlife species ether by direct exposure or through bioaccumulation in food web. Pesticide contamination sites associated with paddy field activities may pose significant environmental hazards for terrestrial and aquatic ecosystems. They are important sources of agro-sourced pollution and may result in ecotoxicological effects, particularly following transfer of irrigation waters following use. Ecotoxicological effects occur at all trophic levels, from the molecular to the ecosystem level and effects may be observed via biomonitoring with both individual organisms and the ecosystem function and structure. Pesticide monitoring is traditionally based on evaluations of individual pesticides identified through chemical analyses. A variety of techniques may permit an examination of actual pesticides, herbicides and their metabolites that are present (Iwai et al., 2007). These techniques are based on sampling approaches that use concentration following collection or during collection. Although these techniques still are not able to show the direct response that ecotoxicity gives, they do give an indication of what is inducing the response of the organism. However, chemical analyses obviously do not reveal complex interaction phenomena and polar degradation products are often missed. In contrast to the use of chemical analyses, the ecotoxicity bioassay approach integrates the biological effects of all compounds present and factors such as bioavailability, synergism, or antagonism are reflected directly in the bioassay results. Ecotoxicological assessment of pesticides in paddy field are therefore expected to give a more comprehensive indication of environmental effects. The use of ecotoxicological assessment to evaluate the impact of pesticide residues in the paddy field is strongly recommended in order to have a more direct and integrated estimate of environmental impact. In fact, biological response to a complex mixture of chemicals integrates different factors such as pH and solubility, antagonism or synergism, and the bioavailability of substances. Pesticides contamination associated with paddy field has been increased a big concern in Thailand. For risk assessment study on the impact of pesticides on aquatic environments that surrounding area, information about effect of pesticides on local species were limited, especially the ecotoxicological data on aquatic organism in Thailand, and it unknown, whether ecological effects test guideline developed elsewhere in the world (US. EPA, ATSM etc) may be use in Thailand. Countries located in the tropical zone rely, mostly, on data from temperate countries about ecotoxicity data. However, this data may be not suitable for tropical countries. Due to the difference organisms species, temperature, rainfall, and agriculture practices that might greatly influence pesticides behavior (Abdullah et al., 1997) and toxicity of pesticides on organisms. Considering the climate adaption of tropical species, assessment of effects of pesticide use on local ecosystem should be performed with local species since their sensitive to toxicants may differ considerably form temperate organism (Domingues et al., 2007). Differential response of organism representing diverse physiological capabilities and niches in aquatic system can help focus field studies where nontarget effect due to off -site movement of pesticides are suspected. Therefore, Thailand need ecological effects test guideline, this guideline typically derived data on toxicological response of local organism to environmental contaminant. The toxicity test is procedure that involves the exposure of organism to complex environmental sample under controled condition to determine if adverse effects have occurred (Edmondson, 1959).
The objective of this study selected the fresh water cladoceran Moina micrura Kurz order Cladocera, family Moinidea. In Thailand, this zooplankton is very common in pond, muddy pool and paddy field and it can be mass culture by some local fish farmer as a high quality fish food. M. micrura is an ideal animal for ecological relevance, wide occurrence, short life cycle, genetic uniformity, relative ease of culture in the laboratory and more sensitive to toxicants (Wang, 1994;Wongrat, 2001). The present study was determine the acute and chronic toxicity of pesticides on M. micrura. The result would be useful as an input to developing a biomonitoring tool and using local species test for evaluation pesticide contamination in Thailand aquatic ecosystem.

Test organism culture
The Moina micrura obtained from Fisheries Research Institute, Khon Khaen (Khon Khaen, Thailand) and have been maintained in cultured under control laboratory conditions in Ecotoxicology and Environmental Sciences Laboratory, Faculty of Agriculture, Khon Kaen University, Thailand. The culture was incubated at 25± 2 0 C with 16:8 h light:dark photoperiod. M. Micrura were cultured using moderately hardwater and fed on singlecelled green alga, Chlorella vulagaris from axenic culture. The medium, used for zooplankton, a s w e l l a s f o r e x p e r i m e n t s , w a s t a p w a t e r a t t h e F a c u l t y o f A g r i c u l t u r e , K h o n K a e n University, Thailand. Water was filtered by using 0.45µm polymembrane filter. Dissolved oxygen concentration was between 5-7 mg/L and pH was 7-8. The culturing period for one generation was 2 weeks before testing.

Experiment design 2.3.1 Acute toxicity test
Preliminary acute toxicity tests were conducted in order to calculate malathion, chlorpyrifos, carbofuran, neem extract and gyphosate LC 50 data. All experiments were performed according to the US.EPA document OPPTS 850. 1010 (1996) for determining 48 h LC 50 values for M. micrura. Three replication of 10 neonates (<24 h) per treatment and control laboratory well -wate were used. The neonates were exposed in a 150 ml glass beaker containing 50 ml for each test concentration and control were static bioassay under laboratory. Test organisms were not fed during the testing period. Observation motality was made at 24 and 48 h, and results recorded. For water quality, temperature, pH, conductivity and dissolved oxygen were measured according to APHA (1992).

Pesticide
Group of Table 1. Chemical formulation of pesticides tested with M. micrura .

Chronic toxicity test
Chronic toxicity of pesticides to M. micrura followed the procedure recommend by US.EPA document 6004-91/002 (1994). Based on acute toxicity result, M. micrura were exposure to control and concentration test malathion concentration of 0.05, 0.25 and 0.50 µg/L, chlorpyrifos concentration of 0.00005, 0.00025 and 0.00045 µg/L, carbofuran concentration of 0.25,1.00 and 2.50 µg/L, neem extract concentration of 15, 40 and 65 µg/L and glyphosate concentration of 50, 250 and 325 µg/L. In the chronic tests, three replication of 10 neonates ( < 24 h )per treatment and control laboratory well -water were used. The neonates were exposed in a 50 ml glass beaker containing 30 ml for each test concentration and control. Test organism were fed with a concentrated suspension of the green algae, Chlorella sp. Test solution and food were renewed completely every day. The measurement of water quality at the beginning and end of the test on control and treatments. The number of offspring was noted each day used to evaluate the effect of pesticide on reproduction of test organism.

Statistic analysis
The values of lethal concentration 24 and 48 h LC 50 and 95 % confidence limit were caculated by appropriate statistical method intervals by probit analysis.Data from chronic www.intechopen.com test were analyzed using ANOVA with SPSS version 12 statistical software to detected variation significances (P<0.05 ) between treatment group and control.

Chronic toxicity
Effect of sublethal pesticides concentration on the number of offspring per female of M. micrura is shown in (Table 4). Number of offspring per female of M. micrura was significant reduced (P<0.05) at malathion concentration 0.50 µg/L, chlorpyrifos concentation greater than 0.00025 µg/L, at carbofuran concentration at 2.50 µg/L and at glyphasate concentration 325 µg/L. For neem extract concentration had no effect on the number of offspring per female significantly (P>0.05). Sublethal effects for each pesticide, were founded similar to other reports (Wong et al, 1995;Alberdi et al,1996;US EPA 2006). An estimate of no observed effect concentration (NOEC) and lowest observed concentration (LOEC) were 0.25 and 0.50 µg/L for malathion, 0.00005 and 0.00025 µg/L for chlorpyrifos, 1.00 and 2.50 µg/L for carbofuran, 250 and 325 for glyphosate and LOEC 65 µg/L for neem extract. Cladocerans contribute an important component of aquatic ecosystem especially, for fish food source. If the number of clardocerans were down, it may affect fish and another organisms. The number of offspring per female is one endpoint used to determine the maximum acceptable -toxicant concentration (MATC). The 16 % reproduction impairment have been used as the endpoint for many aquatic ecotoxicology (Biesinger and Chistensen, 1972). Therefore, this studies used 16 % reproduction impairment estimate the chronic values MATCs for pesticides (Table 4). According to the obtained results the calculated values of MATCs and 48-h LC 50 were for estimate application factor (AF) of pesticides on M. micrura (Table 5). This value was used to predict the safe concentration (SC)applies for pollutant prevention in aquatic ecosystem. However, the application factor will vary with type of pesticide and organism (Mounth and Stephan, 1967 Aquatic ecosystems in tropical regions differ from those in temperate regions. The biodiversity in tropical zones is higher than that in temperate zones, which means that in tropic regions there are potentially more species that can be exposed to certain pollutants. However, many countries in the tropics are developing countries, in which pollution control is not carried out due to a lack of funds and other resources. Furthermore environmental quality criteria for some pollutants are often obtained by extrapolating toxicity data derived for a reduced number of species mainly distributed in temperate regions (e.g. Europe or the US) (Kim et al., 2001in Kwok et al., 2007. Kwok et al. (2007) investigated to which extent the sensitivity distributions of temperate species to toxic substances were similar to those of tropical species. They found that the temperate species seemed to be more sensitive to metals than the tropical species (Kwok et al., 2007). However, it should be noted that these differences might be due to the different species composition included in the species sensitivity distributions (SSD). Kwok et al (2007) used mainly fish species, which could be less sensitive to pollutants than the invertebrate species that are predominantly used in the temperate species sensitivity distributions. A better comparison can be made when using similar taxonomic groups for the distribution.
In Thailand ecotoxicological research is quite new and has many limitations. Although ecotoxicological issues arise in this country and there is a need for water quality management and ecological risk assessment tools, there is a lack of ecotoxicological data on aquatic organisms from Thailand. Until now, like other developing countries, they have relied on over sea data to develop ecotoxicological test guidelines. However, these guidelines may be unsuitable for Thailand. The Thai indigenous aquatic organisms might be more or less sensitive to contaminants than their temperate surrogate species (Iwai, 2004;Somparn et al., 2010). Moreover, there are differences in physicochemical and biological characteristics of aquatic habitats between tropical and temperate regions (Kwok et al., 2007). The characteristic of the sediment and water in Thai rivers may differ from those in other countries Somparn et al., 2010), influencing the concentration, availability and accumulation of pollutants and therefore their toxicity. An example of this is given by Jeon et al. (2010). They found that clay and food content in the water influence the toxicity of pollutants on aquatic biota. Tirado et al. (2008) report that the main rivers in Thailand were monitored from 1993 to 1999 for the presence of pesticide residues; most water samples contained insecticide and herbicide residues in levels above advisable limits, whereas less contamination was observed in sediment samples. In river water, organochlorine pesticides were detected in 40.62% of the samples (in concentration ranging from 0.01 to 1.21 μg/L), organophosphate pesticides were detected in 20.62% of samples (in concentration ranging from 0.01 to 5.74 μg/L). The safety limit established by the European Union is 0.1 μg/L for any single pesticide and 0.5 μg/l for the sum of all pesticides detected. Both organochlorine and organophosphate pesticide residues were found above those safety limits. Additional compounds, like carbamate pesticides were detected in 12.39% of samples (in concentration ranging from 0.01 to 13.67 μg/l), triazines were detected in 20.0% of samples (in concentration ranging from 0.01 to 6.63 μg/L), and paraquat was detected in 21.36% of samples (in concentration ranging from 0.14 to 87.0 μg/L) (Chulintorn et al., 2002). An earlier study has also found residues of the pesticides DDT and dieldrin in five Thai rivers (Upper Ping, Lower Ping, Wang, Yom, Nan, Chee), in concentrations above acceptable standard levels (Sombatsiri, 1997). The Division of Agricultural Toxic Substances in the Department of Agriculture (Ministry of Agriculture and Cooperatives) has also monitored the presence of pesticide residues in rivers and canals around agricultural areas in the country. The contamination of pesticides in water and sediments was generally low in water resources used for domestic consumption like ponds and reservoirs that have no connection to agricultural plantations. However, the water resources in certain agricultural areas, like orchid and ornamental plantations, were contaminated with organophosphate and www.intechopen.com carbamate insecticides. From 1999 to 2001, a survey of three major rivers along paddy field areas (Thachin river in Suphanburi and Nakornpathom, the Chao Phraya river in Pathumthani and Nonthaburi, and the Bangpakong river in Chachengsao), found the highest residues of the insecticide endosulfan in the Thachin River, followed by the Chao Phraya and Bangpakong Rivers. In all cases, the levels of pesticide residues were above the safety limit set by the European Union (0.1 μg/L) (Chatsantiprapha, et. al., 2002).
In 2001, groundwater in the lower Central and the lower Northeastern region of Thailand was contaminated with pesticides residues, in many cases in concentration above the safety limit set by the EU (0.1 μg/l). In the lower Central region during the rainy season in 2001, 68% of 15 GRL-TN-03-2008 the total groundwater samples were contaminated with endosulfan and other insecticides, in concentration ranging from 0.02 to 3.2 μg/l, and paraquat, 2,4-D, butachlor, atrazine and metribuzin herbicide residues ranging from 0.02 to 18.9 μg/l. In lower Northeastern region during the dry season in 2001, 71.2% of the total groundwater samples were contaminated with endosulfan and other insecticides, in concentrations from 0.01 to 0.33 μg/l, and atrazine and paraquat herbicide residues at the level of 0.5-4.0 μg/l (Sakultiangtrong, et.al., 2002). In 1993, the Department of Agriculture investigated shallow groundwater wells from Rayong Province. From 160 samples collected from wells, 67% were contaminated with organochlorine and organophosphate pesticides, but in concentration below the safety limits (Pollution Control Department, 2004

Conclusion
The aim of this study was using zooplankton, Moina micrura Kurz. which is an important species in aquatic ecosystem of Thailand to evaluated ecotoxicity of main pesticide used in paddy field (malathion, chlorpyrifos, carbofuran, neem extract (azadirachtin) and glyphosate). The acute toxicity (48-h LC 50 ) of malathion, chlorpyrifos, carbofuran, neem extract and glyphosate on M. micrura were 10.44, 0.08, 6.96, 196.3 and 3043 µg/L, respectively. Chlorpyrifos had highest toxicity followed by carbofuran, malathion, neem extract and glyphosate, respectively. Chronic toxicity test, the effect of pesticides to M. micrura on reproduction was studies by observing the number of offspring per female. Reproduction have significant reduced (P<0.05), with concentration of malathion at 0.50 µg/L, chlorpyrifos greater than 0.0025 µg/L, carbofuran at 2.50 µg/L and the concentration of glyphosate at 325 µg/L affected on reducing the number of offspring per female significantly (P<0.05). The neem extract had no significantly (P>0.05) effect on the number of offspring per female. The maximum acceptable -toxicant concentration (MATCs) of malathion, chlorpyrifos, carbofuran, neem extract and glyphosate were 0.36, 0.0001, 2.41, 172 and 281.9 µg/L, respectively. The result would be useful as an input to developing a biomonitoring tool for evaluation pesticide contamination in Thailand aquatic ecosystem. Effect of experimental condition including duration test organism and end point on observed toxicity of pesticide to M. micrura were evaluated. Relative sensitivities of test varies with pesticide type. Among five pesticides toxicity test, chlorpyrifos had highest acute toxicity on M. micrura followed by carbofuran, malathion, neem extract and gyphosate, respectively. The significant reducing effect on number of offspring per female of M. micrura were observed in the present of malathion, chlorpyrifos carbofuran and glyphosate. For neem extract had no effect on the number of offspring per female. The results indicate that reproductivity parameters are very important interm of pesticide impact on aqutic population such as M. micrura. However, in the natural environment aquatic organism are often exposure to multiple pesticides simultaneously. Therefore under natural condition, there is the potential of pesticides may act in additive or synergistic manner, although the sensitivity of aquatic biota to multiple pesticides cannot be predicted by the individual pesticide sensitivities generate in this study. The results showed M. micrura to be sensitive test organism, Thus its a good bioindicator and useful to developing a biomonitoring tool for evaluation pesticide contamination in Thailand aquatic ecosystem. However, in order to obtain more precise and conclusive toxicology data on application of these pesticide in paddy field and evaluation toxicity of pesticides on organism, similar study using another local freshwater in Thailand.