The effects of two widely used pesticides in paddy fields, 2, 4-D, dimethylamine and endosulfan, on growth, photosynthetic rate and the photosynthetic pigments of Chaetoceros sp. and Nannochloropsis sp. were assessed in this study. The 48 hour 50% inhibitory concentration (IC50) value for 2, 4-D dimethylamine on Chaetoceros sp. and Nannochloropsis sp. were 142.2 mg l−1 and 211.8 mg l−1, respectively, and the 48 hour IC50 value for endosulfan on Chaetoceros sp. and Nannochloropsis sp. were 21.9 μ g l−1 and 45.8 μ g l−1, respectively. Endosulfan was much more toxic to the microalgae than 2, 4-D dimethylamine. Both pesticides reduced photosynthetic pigments and oxygen production rate of the microalgae. After 48 hours exposure to the pesticides, the microalgae were transferred to a pesticide-free medium to investigate the post-exposure effect. 2, 4-D dimethylamine and endosulfan did not cause irreversible damage on the microalgae. However, there was a prolonged lag phase and the maximum specific growth rate (μ) of the microalgae was significantly retarded.

Introduction

Pesticides play an important role in today!s agriculture. Increased use of pesticides in the field has elicited concerns on the pesticides! effect on the non-targeted organisms. The effect of pesticides on the primary producers in the aquatic ecosystem is critical. Changes in the quantity and quality of the primary producers would seriously impact the aquatic ecosystem. Phytoplankton is the primary producer in the coastal environment. All chemicals that affect the growth of the primary producers are crucial for the safety and health of the aquatic ecosystem. Phytoplankton can be used to investigate the impacts of chemical pollutants on the ecosystem because of their rapid responses to environmental changes and relatively short generation time. Chaetoceros and Nannochloropsis are common microalgae found in tropical river estuaries (Ahmad et al., 2002; Lacerda et al., 2004; Lassen et al., 2004). Chaetoceros is marine diatom belonging to the bacillariophycea, and it is the largest microalgal genus in the marine environment. These microalgae can be found in a broad range of salinity ranging from 10 to 34 ppt (Fujii et al., 1995). Nannochloropsis spp. are marine unicellular microalgae belonging to eustigmatophyceae (Sandnes et al., 2005). These microalgae are small green algae characterized by the spherical-shape of their cell. Both Chaetoceros spp. and Nannochloropsis spp. are important live feed for shrimp, fish larvae and bivalve due to their high content of polyunsaturated fatty acids (Coutteau and Sorgeloss, 1992).

2, 4-D, dimethylamine is a chlorinated phenoxy compound. It is a selective systemic post emergence herbicide. 2, 4-D, dimethylamine mimics the plant growth hormone, auxin and causes uncontrolled and disorganized growth of the targeted weeds that leads to mortality (Steven and Sunner, 1991). Based on the toxicity of this chlorinated phenoxy herbicide, the World Health Organization (WHO) classified 2, 4-D, dimethylamine as a Class II pesticide. Endosulfan is an organochlorine insecticide that is widely used to control agricultural insects and mites. Endosulfan starts showing its effect on many aquatic organisms even at a concentration as low as 0.5 μ g l−1 (Hii et al., 2007). This insecticide was classified by WHO as a Class IB pesticide. 2, 4-D, dimethylamine and endosulfan are moderately persistent in the environment. The hydrolysis half-life for 2, 4-D, dimethylamine and endosulfan in the aquatic environment is about 10 days and 3–7 days, respectively (Tu et al., 2001; Mackay et al., 2006).

Rice fields are one of the most extensive agricultural land uses in Asia. Most of the rice fields in South East Asia are situated in low land and coastal area. Hence, pesticides applied in the rice fields will eventually end up in the estuaries. 2, 4-D, dimethylamine and endosulfan are pesticides commonly used in the rice fields for controlling weeds and insects. This means that primary producers in the estuaries, such as Chaetoceros sp. and Nannochloropsis sp. will eventually be exposed to these pesticides. Hence, this study was undertaken to assess the effects of the pesticides on the microalgae. The data reported in this study will provide information on the potential effects of these pesticides on the primary productivity of estuary environments and on microalgae in general.

Material and methods

Microalgae culture

Nannochloropsis sp. and Chaetoceros sp. were isolated from the coastal environment of Peninsular Malaysia. The microalgae were maintained in sterilized Guillard F/2 medium (Smith et al., 1993). 300 ml of the sterilized culture medium was transferred aseptically into a sterilized 500 ml Erlenmeyer flask (Pyrex). A 4-day-old microalgae culture was used as inoculum in this study. This was made to ensure that the microalgae were inoculated in the logarithmic growth phase. The microalgae were inoculated at an initial cell density of 4 × 106 cell ml−1. The flasks were then incubated at 25 ± 1°C, pH 8.0 and illumination of 60 ± 2 μ mol photon m−2 s−1for 12 h followed by 12 h darkness (i.e. a 12:12 hours dark:light cycle).

Chemicals and stock solution

Commercial endosulfan; CH endosulfan (Purity 33%, 64–67% α endosulfan; 29–32% β endosulfan) and 2, 4-D, dimethylamine; CH amine 60 (Purity 60%) were used in this study. The pesticide stock solutions were prepared by dissolving the commercial pesticides into culture medium. The test solutions were then prepared from the stock solution by progressive dilution in the culture medium.

Experimental set-up

Static toxicity bioassays were conducted according to the APHA standard method with minor modifications (APHA, 1998). Screening tests were conducted to narrow down the ranges of pesticides concentration for the 48-h EC50 bioassays. In order to assess the 48-h EC50 for the microalgae, Chaetoceros sp. was exposed to 50 mg l−1, 100 mg l−1, 150 mg l−1, 200 mg l−1 and 300 mg l−1 of 2, 4-D, dimethylamine, and to 20 μ g l−1, 40 μ g l−1, 60 μ g l−1, 80 μ g l−1 and 100 μ g l−1 of endosulfan. Nannochloropsis sp. was exposed to 200 mg l−1, 400 mg l−1, 600 mg l−1, 800 mg l−1 and 1000 mg l−1 of 2, 4-D, dimethylamine and 20 μ g l−1, 40 μ g l−1, 60 μ g l−1, 80 μ g l−1 and 100 μ g l−1 of endosulfan. The experiments were conducted with three replicates and a blank with only the culture medium (with no pesticides) was served as control. Population density and photosynthetic pigment of the microalgae were assessed in the experiment. Oxygen produced from the photosynthesis was also monitored continuously for 48 hours by using WTW OXITOP respiratory meter. Endosulfan and 2, 4-D, dimethylamine concentrations at 0 h and 48 h were determined by using APHA method (1998). Exposure concentrations in the experiments were maintained within 10% from the initial (nominal) concentration.

After 48 hours exposure, the microalgae exposed to the highest concentration of the pesticides were harvested by centrifugation at 5000 rpm for five minutes. The supernatant was discarded and new culture medium was added to re-suspend the microalgae. The procedures were repeated three times to discard residual pesticides from the microalgae. The harvested microalgae were then adjusted and re-inoculated into pesticide-free culture medium at 4 × 106 cell ml−1. The microalgal cultures were incubated under the pre-described conditions. Growth and population of the microalgae were assessed for another 96 hours.

Measurement of growth

Population density of the microalgae in the experiments was determined by using a Neubauer-improved haemocytometer. The microalgae were counted under Eclipse E400 microscope (Nikon). The specific growth rate (μ) of the microalgae was determined by the following equation (Schanz and Zahler, 1981):

formula
where:
  • Nt = Cell population at the end of the logarithm growth phase, cell ml−1

  • N0 = Cell population at the beginning of the logarithm growth phase, cell ml−1

  • t = Time of the growth, day

While, the population doubling time (T2) and generation time (G) was estimated by using the following equations:
formula
Growth inhibition was determined by average reduction in cell number after exposure to the pesticide during the experiments relative to the controls. Growth inhibition of the microalgae was evaluated by comparing the area below the growth curve. The area (A) below the microalgal growth curve was calculated based on the following equation (Wong and Couture, 1986):
formula
where:
  • N0 = Number of cell per ml at time t0

  • N1 = Number of cell per ml at time t1

  • Nn = Number of cell per ml at time tn

  • t1 = Time of first measurement after beginning of test

  • tn = Time of nth measurement after beginning of test

Percentage of growth inhibition (IA) was calculated by using the following equation:
formula
where:
  • Ac = Area below the growth curve of control

  • AT = Area below the growth curve of cell after exposure to pesticide.

Photosynthetic pigments content

Photosynthetic pigments contents of the microalgae were determined by using spectrophotometrical method as described by Parsons et al. (1984). Briefly, the culture was filtered through GFC filter paper (Whatman) under a constant 0.5 atmospheric pressure. The filter paper was then placed in a 15 ml centrifuge tube. 10 ml of 90% acetone (Merck, A.R.) was added into the centrifuge tube. The centrifuge tube was kept overnight in a freezer at −20°C. The supernatant was then decanted into a 1 cm cuvette and the sample was measure by using UV-VIS spectrophotometer (Shimadzu) at 480 nm, 510 nm, 630 nm, 647 nm and 664 nm. The photosynthetic pigments were determined by the following equations.

formula
Chlorophyll concentration (expressed as μ g chlorophyll l−1) was calculated by multiplying the volume of acetone used in the extraction and the volume of sample used for the extraction.

Determination of oxygen production

A respiratory meter (WTW) was used to measure oxygen produced by the microalgae under illumination as described above. To do this, 250 ml of the sample was transferred into a 300 ml bottle. The bottle was capped with the WTW sensor. Sodium hydroxide tablets were placed in the sensor to remove carbon dioxide produced during metabolism. The oxygen partial pressure was recorded by the sensor every 10 minute. Oxygen production was calculated based on the following equation:

formula
Where,
  • MW = Molecular weight

  • R = Gas constant (83.144 mbar mol.k−1)

  • T0 = Reference temperature

  • Tm = Temperature

  • Vt = Volume of bottle

  • V1 = Volume of sample

  • α = Bunsen adsorption coefficient (0.03103)

  • Δ p(02) = Difference of oxygen partial pressure

Statistical analysis

Probit analysis was used to determine the 24-h and 48-h 50% inhibitory concentration (IC50). Data normality and homogeneity were examined by using a homogeneity test. Normal-distributed data were statistically analyzed by one-way analysis of variance (ANOVA). When there was a significant difference, the means were compared by multiple ranges Duncan analysis. Kruskal-Wallis analysis was used to analyze non-parametric data. All the statistical analyses were conducted with 5% significance level (p < 0.05) to establish differences from corresponding controls.

Results

Effect of 2, 4-D, dimethylamine on Chaetoceros sp.

2, 4-D, dimethylamine retarded the growth of Chaetoceros sp. as shown in Figure 1a. The herbicide inhibited Chaetoceros sp. growth as soon as 5 hours after exposure. The effect of 2, 4-D, dimethylamine was pronounced within 24 hours, and the 24-h IC50 and 48-h IC50 of 2, 4-D, dimethylamine on Chaetoceros sp. were 161.5 mg l−1 and 142.2 mg l−1, respectively. Growth inhibition on the microalgae was not significantly different after 24 and 48 hours exposure (p > 0.05). Population doubling time (T2) of Chaetoceros sp. was significantly affected by 2, 4-D, dimethylamine (P < 0.00). When the microalgae were exposed to 300 mg l−12, 4-D, dimethylamine, the population doubling time (T2) of Chaetoceros sp. was extended about three times from 1.33 days to 4.29 days, and the generation time (G) was reduced from 0.75 day−1 in the control to 0.23 day−1. Table 1 lists some of the physiological responses of Chaetoceros sp. after exposure to 2, 4-D, dimethylamine. After exposure to the herbicide, there was a significant reduction in oxygen production rate (p = 0.005) and chlorophyll-a (p = 0.021). Chlorophyll-a content of the microalgae was not linearly correlated to the dosage of the herbicide and there was a slight (non-significant) decrease in the carotenoid content.

2, 4-D, dimethylamine is a strong inhibitor to Chaetoceros sp. however; short-term exposure (< 48 hours) did not cause irreversible damage to the cell population. When the cells (after exposure to 300 mg l−1 2,4-D, dimethylamine) were harvested and inoculated into a pesticide-free medium, the microalgae demonstrated a prolonged lag-phase of 24 hour (Figure 2a). The maximum specific growth rate of the non-exposed Chaetoceros sp. was significantly higher than the post-exposed cells (p = 0.017). The maximum specific growth rate of the non-exposed cells and pre-exposed cells were 0.7 and 0.37 day−1, respectively.

Effect of endosulfan on Chaetoceros sp.

Chaetoceros sp. was very sensitive to endosulfan. After 48 hours exposure to endosulfan, growth of the microalgae was significantly (p = 0.000) reduced (Figure 1b). Specific growth rate of the microalgae decreased from 5.04 × 10−1 day−1 to 6.12 × 10−2 day−1 at 300 μ g l−1. The 48-h IC50 obtained from the non-linear dose-responses regression curve was 21.9 μ g l−1. Thus, endosulfan is extremely toxic to Chaetoceros sp. There was no significant difference for percentage of inhibition at 24 hours and 48 hours exposures (p > 0.05). Table 2 shows some of the physiological responses of Chaetoceros sp. after exposure to endosulfan. Endosulfan caused strong inhibition on growth, photosynthetic pigments and oxygen production rate of the microalgae. Oxygen production rate of Chaetoceros sp. after exposure to 100 μ g endosulfan l−1 was reduced from 50.6± 3.0 nmol O2 h−1 106 cell−1in the control down to 30.5± 1.3 nmol O2 h−1 106 cell−1. The microalgal growth rate was most sensitive to endosulfan. Chaetoceros sp. did not fully recover from the effect of endosulfan even only a short-term exposure (< 48 hours) (Figure 2b). The maximum specific growth rate of the non-exposed and post-exposed cells was 0.44 and 0.13 day−1, respectively.

Effect of 2, 4-D, dimethylamine on Nannochloropsis sp.

2, 4-D, dimethylamine inhibited growth of Nannochloropsis sp. rapidly (Figure 1c) and there was no significant difference between percentage of cell inhibition after 24 and 48 hours exposure (p > 0.05). The 48-h IC50 of 2, 4-D, dimethylamine on Nannochloropsis sp. was 211.8 mg l−1. After exposure to 1000 mg l−1 2, 4-D, dimethylamine, the specific growth rate of Nannochloropsis sp. decreased almost ten times from 2.78 × 10−1 day−1 in the control down to 1.36 × 10−2 day−1. T2 increased from 2.5 day in the control to 50.92 days, and G decreased from 0.4 day−1 in the control to 0.02 day−1. The values of T2 and G were significantly correlated to the pesticide concentration. Table 2 reveals some of the physiological changes of the microalgae after exposure to 2, 4-D, dimethylamine. Decrease of the photosynthetic pigment was significantly correlated to the level of the herbicide (p = 0.00). The herbicide also affected chlorophyll-a content. After exposure to 1000 mg l−1 of 2, 4-D, dimethylamine for 48 hours, the chlorophyll-a content was reduced by 64%.

Figure 2c compares growth of the post-pesticide exposure cell and non-pesticide exposure cell. There was about a 24-hour lag phase in the post-exposure cells. Although the microalgae were not acutely affected by the herbicide, the maximum specific growth rate was significantly affected (p = 0.00). The maximum specific growth rates for the non-exposed and post-exposed cells were 0.31 and 0.21 day−1, respectively.

Effect of endosulfan on Nannochloropsis sp.

Endosulfan inhibited growth of Nannochloropsis sp. When the concentration of endosulfan approached 100 μ g l−1, the pesticide stunted the growth of the microalgae (Figure 1d). The 48-h IC50 of endosulfan for Nannochloropsis sp. was 45.8 μ g l−1. After exposure to endosulfan at 100 μ g l−1, the specific growth rate of Nannochloropsis sp. was reduced about 10 times from 2.26 × 10−1 day−1 in the control down to 2.58 × 10−2 day−1. Both T2 and G were subsequently affected. Table 2 lists some of the physiological changes of the microalgae after exposure to different concentrations of endosulfan. The effect of endosulfan on the microalgae was rapid, and there was no significant difference in percentage of growth inhibition after exposure for 24 and 48 hours (p > 0.05). Oxygen production rate was affected by the pesticide, and after exposure to 100 μ g l−1 endosulfan for 48 hours, oxygen production rate decreased from 85.0 ± 5.7 nmol O2 106 cell−1 h−1 in the control to 20.7 ± 0.9 nmol O2 106 cell−1 h−1. Carotenoid content of Nannochloropsis sp. was affected by endosulfan as compared to the chlorophyll-a. After 48 hours exposure to 100 μ g l−1 endosulfan, carotenoid content in the microalgae was reduced by 51.5%, while the chlorophyll-a content was only reduced by 34%.

Figure 2d shows growth of the endosulfan post-exposed and non-exposed cells. The maximum specific growth rate for the non-exposed and post-exposed Nannochloropsis sp. was 0.34 and 0.08 day−1, respectively.

Discussion

2, 4-D, dimethylamine and endosulfan are both commonly used in paddy fields. Their use in the paddy fields may eventually lead to exposure of the primary producers in the estuary. The effect of the pesticides on the tropical species Nannochloropsis sp. and Chaetoceros sp. are not well documented. 2, 4-D, dimethylamine and endosulfan posed acute effect on microalgae. The pesticides toxicity was profound even after short exposure, and noticeable within the first 24 hours.

Effect of 2, 4-D, dimethylamine and endosulfan on many aquatic organisms have been reported previously. Okay and Gaines (1996) reported LC50 of 2, 4-D, dimethylamine on Dunaliella tertiolecta and Phaeodactylum tricornutum of 185 mg l−1 and 362 mg l−1, respectively. According to that report, the green algae was more impacted by 2, 4-D, dimethylamine than the diatom, which is possibly due to the cell wall!s readiness for chemical transportation. The present study revealed that IC50 of 2, 4-D, dimethylamine for Chaetoceros sp. (diatom) was lower as compared to Nannochloropsis sp. (green algae). Presumably there were other factors that affect sensitivity of the microalgae besides the cell wall!s readiness for chemical transportation. This herbicide is believed to reduce electron transfer of Hill reaction in photosystem II, and, thus, caused a significant reduction in oxygen production rate in both Nannochloropsis sp. and Chaetoceros sp. instead of a drastic decrease in the photosynthetic pigment of the microalgae.

High concentration of 2, 4-D, dimethylamine in the aquatic environment would inhibit microalgal growth. However, short-term exposure to the herbicide did no lead to cell rupture. The post-exposure Chaetoceros sp. and Nannochloropsis sp. to 2, 4-D, dimethylamine exhibited a prolonged lag phase. Similar observation was reported on Chlorella vulgaris after exposure to isoproturon (Rioboo et al., 2002). 2, 4-D, dimethylamine is generally less toxic as compared to endosulfan. When 2, 4-D, dimethylamine is present at low concentration in the aquatic environment; it did promote microalgae bloom and improved protein and amino acid contents of some microalgae. Zhou et al., 2003 reported that 0.05 μ g l−1 of 2, 4-D, dimethylamine increased the protein and amino acid contents of Chaetoceros muelleri Lemmermann and Phaeodactylum tricornutum, and Kobraei and White (1996) reported that 2 mg l−1 or less of 2, 4-D, dimethylamine would stimulate total community growth of algae. A similar stimulation effect was reported on filamentous green alga, Oedogonium acmandrium as well (Sarma and Tripathi, 1980).

As compared to 2, 4-D, dimethylamine, the IC50 of endosulfan for the microalgae were much lower. Presence of the pesticides in the growth medium caused concentration-dependent growth inhibition of the microalgae. The pesticides adversely affected oxygen production rate and photosynthetic pigments of the microalgae. The photosynthetic pigments; chlorophyll-a and carotenoid contents of the microalgae also decreased subsequently after exposure.

Endosulfan is toxic to a wide spectrum of aquatic organisms. As early as 1960s!, endosulfan was reported to cause 86.6% decrease in the productivity of the natural phytoplankton community at 1 mg l−1 after 4 hours exposure (Butler, 1963). DeLorenzo et al. (2002) reported EC50of 427.8 μ g l−1 endosulfan was determined for Pseudokirchneriella subcapitatum. Mohapatra and Mohanty (1992) found endosulfan to be more toxic to the cyanobacterium Anabaena doliolum (10 days LC50 of 2.15 mg l−1) than to the green alga Chlorella vulgaris (10 days LC50 of 41.5 mg l−1). Endosulfan affected membrane components of the cell and caused irreversible damage on the organisms (Srivastava and Misra, 1981). The pesticide also caused severe effect on photosystem II activity in the spheroplasts of Clamydomonas sp. and blocked electron flow at the water oxidation side (Gandhi et al., 1988). Endosulfan also accelerated the formation of active oxygen species in the cell; these active oxygen species undergo deleterious reactions and caused oxidative stress in cellular systems. When Nannochloropsis sp. and Chaetoceros sp. were exposed to endosulfan, they probably suffered from serious cell wall damage and jamming of electron flow at their water oxidation site.

Short-term endosulfan exposure (less than 24 hours) on Nannochloropsis sp. and Chaetoceros sp. caused serious inhibition on the total community growth. Even when the microalgae were transferred (48 hours exposure) into pesticide-free medium, cell growth was seriously retarded. The long-lasting effects of endosulfan were probably due to serious cell wall leakage and blockage of electron transfer in the photosynthetic system. Serious cell wall leakage could also lead to irreversible damage on microalgae.

When the microalgae were removed from the short-term exposure to 2, 4-D, dimethylamine (48 hours exposure), the microalgae showed an extended lag phase. After the extended lag phase, the microalgae were also growing at a lower specific growth rate. The lagged growth rate would cause instability in an aquatic ecosystem. 2, 4-D, dimethylamine and endosulfan concentration detected in the Muda rice field in Malaysia ranged from 0.07 to 2.80 μ g l−1and 0.04 to 2.55 μ g l−1, respectively (Cheah et al., 1998). These pesticides, at their present concentrations may not cause acute effects on microalgae. However long-term exposure to these pesticides may cause instability in the aquatic environment due to more complex unforeseen interactions.

Conclusions

2, 4-D, dimethylamine and endosulfan are both strong inhibitors of growth and physiological functions in Nannochloropsis sp. and Chaetoceros sp. even after short exposure. Both microalgae showed reduced photosynthetic pigments, population and oxygen production rates after exposure to these compounds. When the microalgae were transferred into pesticide-free medium after a 48-h exposure, the post-exposed microalgae showed an extended lag phase in their cell growth and the maximum specific growth rate (μ) of the microalgae was significantly retarded.

Acknowledgements

The authors would like to acknowledge the Ministry of Higher Education, Malaysia and University Malaysia Terengganu for funding the studies.

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