**2.3 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 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

Using Zooplankton*, Moina Micrura* Kurz Evaluated

al., 2006; Nelson et al.,1997,1998; Ankley et al., 1991).

**3. Results and discussion** 

toxicity test with *M. micrura*.

Hoeven and Gerrisen, 1997).

(Goktepe and Plhak, 2002,2003)**.** 

respectively.

48 h.

**Acute toxicity** 

Ecotoxicology of Pesticides Used in Paddy Field in Thailand 271

test were analyzed using ANOVA with SPSS version 12 statistical software to detected

Table 2. show the estimated 48-h LC50 for pesticides, with were calculated from standard

**Malathion.:** Our 48-h *M. Micrura* LC50 of 10.44 µg/L was compareable to the 48-h LC50 to other Cladocera species. This results shown 48-h- LC50 *M. Micrura* were nealy repoted the 5- 10 µg/L for *M. marcocopa* by Wang et al. (1994) and the 8 and 13 µg/L that reported by Khan et al. (1993) and Siefirt (1987) for *D. Magna.* Differnce in LC50 value were observed wih *Cerodahnia dubia* have been reported between 1.14 -3.35 µg/L(Hernadez et al.,2004; Maul et

**Chlorpyrifos:** Of the five pesticides tested in this study, chlorpyrifos was the most toxic to *M. Micrura*. The 48-h LC50 value for *M. micrura* was 0.08 µg/L. Other values in literature were higher between 0.13-3.7 µg/L using *D. ambigue, D. Magnaand and D. Duplex* (Caceres et al.,2007; Van Wijngaarden et al., 1993; Barata et al.,2004; Kersting et al.,1997; Van der

**Carbofuran:** The 48-h LC50 for *M. micrura* obtain in this study 6.96 µg/L is compareable to the 48-h LC50Of 2.69 µg/L obtain with *C. Dubia* (Nerberg et al.,1997) for carbofuran in *D. magna* were higher than concentration tested (6.96 µg/L). In comparison, Poirer (1990); DBR (2000) and Dopsikova (2003) found an acute 48-h LC50 were 86.1, 38.6 and 18.7 µg/L

**Neem extract:** The present study found that the 48-h LC50 for neem extract in *M. micrura* was 196.3 µg/L. The acute toxicity data for *D.magna* with 48-h LC50 were 570- 1,250 µg/L (John , 2001; Stark ,2001; Scott and Kaushik, 2001), and were <6000 – 380,000 µg/L for *D. Duplex* 

**Glyphosate:** Glyphosate was the lowest toxic (LC50 was 3042 µg/L) to *M. micrura*. Other values in reports were higher between 1150 – 107,000 µg/L and 30000 µg/L for *C. Dubia and D. Magnaa,* respectively*.* The LC50 value of pesticides showed that toxicity of chlorpyrifos > carbofuran > malathion > neem extract > glyphosate. *M micrura* were susceptible to pesticides from µg/L to mg/L, with chlorpyrifos was the most toxic (LC50 =

**Pesticides 48 h-L50 ( µg/L)**

Table 2. Acute toxicity (Medium lethal concentration [LC50 ]) of pesticides on *M. micrura* at

0.08 µg/L) and glyphosate was the lowest toxic (LC50 = 3042 µg/L) to *M. micrura*.

Malathion 10.44 ( 9.10 -11.85) Chlorpyrifos 0.08 ( 0.03 - 0.20 ) Carbofuran 6.96 ( 5.97 – 7.63) Neem extract 196.3 ( 161.5 -263.9) Glyphosate 3043 ( 1974 – 1778)

variation significances (P<0.05 ) between treatment group and control.


48 h, and results recorded. For water quality, temperature, pH, conductivity and dissolved oxygen were measured according to APHA (1992).

Source: Extoxnet (1996 ); Chemical Book (2007); Compendium of Pesticide common name (2008 a, 2008 b, 2008 c)

Table 1. Chemical formulation of pesticides tested with *M. micrura* .

#### **2.3.2 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.

#### **2.4 Statistic analysis**

The values of lethal concentration 24 and 48 h LC50 and 95 % confidence limit were caculated by appropriate statistical method intervals by probit analysis.Data from chronic test were analyzed using ANOVA with SPSS version 12 statistical software to detected variation significances (P<0.05 ) between treatment group and control.
