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

Chuleemas Boonthai Iwai1, Atcharaporn Somparn1 and Barry Noller2 *1Department of Plant Science and Agriculture Resource, Land Resources and Environment Section, Faculty of Agriculture, Khon Kaen University, 2The University of Queensland, Centre for Mined Land Rehabilitation (CMLR), 1Thailand 2Australia* 

#### **1. Introduction**

266 Pesticides in the Modern World - Risks and Benefits

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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).

Using Zooplankton*, Moina Micrura* Kurz Evaluated

contamination in Thailand aquatic ecosystem.

**2. Materials and methods 2.1 Test organism culture** 

generation was 2 weeks before testing.

**2.2 Test chemicals** 

250µg/L respectively.

**2.3 Experiment design 2.3.1 Acute toxicity test** 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 269

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

*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 0C 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, as well as for experiments, was tap water at the Faculty of Agriculture, Khon Kaen 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

The pesticides test were selected from the common pesticides used in paddy filed of Thailand. Five selected pesticides were malathion 58% w/v (CAS: 121-75-5), chlorpyrifos 40% w/v (CAS:2921-88-2), carbofuran 3 % GR (CAS:1563-66-2), neem extract 40% w/v (CAS:1141-17-6) and glyphosate 36% w/v (CAS:1071-83-6) (Table 1.). Stock solutions were prepared by dissolving the pesticide directly in distill water immediately before to each experiment. Stock solution were added to each of three replication test beakers ( 50 ml total volume) to obtain nominal exposure concentration. Rang in nominal aqueous exposure concentration of chlorpyrifos,malathaion, glyphosate, carbofuran and neem extract on *M. micrura*, were arranged in geometic series between 0.5 -0.0005, 1-50, 500-2500, 3-15 and 50-

Preliminary acute toxicity tests were conducted in order to calculate malathion, chlorpyrifos, carbofuran, neem extract and gyphosate LC50 data. All experiments were performed according to the US.EPA document OPPTS 850.1010 (1996) for determining 48 h LC50 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

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.
