Comparison of Diazinon Toxicity to Temperate and Tropical Freshwater Daphnia Species

+e presence of pesticides in water bodies presents unique challenges to the ecosystem and all the life forms. Biological methods have been widely used to examine the toxic effects of various toxicants including pesticides. +e present study aims at determining the adverse effects of diazinon, a nonsystemic organophosphate insecticide, on two cladoceran species including the temperate Daphnia magna (D. magna) and the tropicalDaphnia lumholtzi (D. lumholtzi).+e 48 h LC50 values demonstrated higher toxicity of diazinon forD. lumholtzi at a concentration of 3.41 μg·L compared toD.magna at a concentration of 4.63 μg·L. After 14 days of exposure to diazinon, the survival capacity as well as the reproduction potential of the two cladoceran species clearly reduced and their rate of population increase (RPI) decreased at concentrations >0.1μg·L. +e present study indicated that the tropical cladoceran (D. lumholtzi) was more sensitive than the temperate D. magna. +erefore, it could be used as an indicator for toxicity assessment in tropical environments. +e presence of diazinon in water bodies can be associated with significant risk to aquatic organisms.


Introduction
In recent decades, most of the developed countries are shifting toward a chemical-free agriculture, also known as ''organic'' agriculture/farming, or in other words, decreasing the use of pesticides [1,2]. is has resulted in substantial improvements in agronomic practices guided by stringent legislations and in the quality of natural water bodies, that is, less risk to the aquatic lives [1,2].Most of the developing countries in the tropical regions are increasing their use of pesticides and fertilizers as they become wealthier [3].As a result, the concentration of pesticides in tropical environments has constantly increased, causing a decline in species diversity.Although pesticides have certain beneficial effects for agricultural crops, their use can cause a wide range of toxic effects on different nontarget organisms [4].
Diazinon (O,O-diethyl-O-[2-isopropyl-6-methyl-4pyrimidinyl] phosphorothioate) is a nonsystemic organophosphate insecticide.It was commonly used for insert control in agricultural crops [4,5].e toxicity of diazinon results from inhibiting the enzyme acetylcholinesterase (AChE), leading to the accumulation of neurotransmitters and altered signal transmission in chemical synapses [6].In the domestic and international markets, there are more than 500 registered products that contain diazinon as the active substance [4].Due to its widespread use, diazinon is frequently found in freshwater ecosystems [7].In a recent study, Montuori et al. [7] reported that diazinon is prevalent in aquatic systems all over Europe, with the highest concentration recorded to be 785 ng•L −1 in the Ebro River, Spain.Moreover, high concentrations of diazinon up to 1.5 μg•L −1 have been found in urban waterways in California [8].In recent decades, diazinon has been widely used in tropical regions [9].However, pesticide laws and regulations have not been implemented adequately in these regions despite its heavy applications.e toxic effects of diazinon are available in the literature for the temperate cladoceran D. magna.However, the toxic effects of diazinon on tropical zooplanktons have not been well documented and studied in the literature.
Diazinon generates high acute toxicity to a wide variety of aquatic organisms, leading to a wide range of sublethal biochemical effects, damage to specific target organs and tissues, and adverse ecological impacts.e toxicity of diazinon has extensively studied in fish [4,[9][10][11] and crustaceans [6,12,13], and it has been reported to be moderately toxic to early life stages of zebrafish [4].e 96 h LC 50 values ranging from 0.32 to 1.53 mg•L −1 for larval and from 2.2 to 10.3 mg•L −1 for adult of several fish have already been recorded [10].Yen et al. [11] reported that diazinon lowers larval swimming activity and inhibits AChE activity in zebrafish, as well as an increase in the Hsp70 content.In cladocerans, diazinon at the concentration from 0.18 to 0.30 μg•L −1 caused adverse effects on the survival of D. magna [6,14].
ese diazinon concentrations have been reported to cause a decrease in mean total young per female, mean brood size, and rate of population increase (RPI) and development of D. magna.Only the concentration above 0.25 μg•L −1 caused a delay in the time to first reproduction [14].Toxicokinetic and toxicodynamic models of diazinon and its by-product 2-isopropyl-6-methyl-4-pyrimidinol in crustacean species were investigated by Kretschmann et al. [15].Results suggested that the oxidative dearylation of diazinon to pyrimidinol is a crucial cellular detoxification step which is catalyzed by the enzyme P450 [15].However, most ecotoxicological studies use the temperate cladoceran D. magna as an ecotoxicological model; the toxicity of insecticide on tropical zooplankton species has not been examined to the same extent [16].
In this study, an acute 48 h assay and a chronic 14-day assay were performed to study the effects of diazinon on two cladoceran species.
e toxicity of diazinon to early life stages of the temperate species D. magna was tested, and the toxicity on D. magna was compared to that on the tropical species D. lumholtzi.e results will provide baseline information to establish the benchmark for organophosphate insecticides in tropical waters.

Test Organisms.
Two cladocerans species were used in the present study: D. lumholtzi was isolated from a fish pond in the north of Vietnam.D. magna was obtained from MicroBioTest Inc. (Belgium).Both daphnids were grown in 1 L beakers filled with COMBO medium [17] and kept at a temperature of 25 ± 1 °C under a light : dark cycle of 12 h : 12 h.e animals were fed with microgreen alga (Chlorella sp.) and a 1 : 1 : 1 mixture of yeast, cerrophyl, and trout chow digestion (YTC) [18].e food and culture medium were renewed every two days.

Acute Toxicity Assay.
e acute immobilization test was performed according to Protocol 202 of the Organization for the Economical Cooperation and Development [19]. is assay was performed to evaluate the sensitivity of the species and to establish the range of concentrations to be used in chronic assays.Briefly, Daphnia neonates (<24 h old) were maintained in 50 mL beakers containing 30 mL COMBO medium spiked with diazinon at a concentration range of 0, 0.5, 1, 2, 5, and 10 µg•L −1 .In each exposure concentration, 15 neonates were exposed per concentration of diazinon and blank control.All test exposures were prepared in triplicate.
e test containers were placed at a controlled temperature of 25 ± 1 °C under a light : dark cycle of 14 h : 10 h during 48 h of incubation.e assessed response for this assay was the immobility or death of the cladocerans.e criterion for test acceptance was a survival rate greater than or equal to 90% in the control group.Finally, the mortality data recorded at the end of the toxicity tests (48 h) were used to determine the median lethal concentration (48 h LC 50 ).

Chronic Toxicity Assay.
Based on the acute toxicity results and environmentally relevant concentrations of diazinon in previous studies [7,8], daphnids were exposed during 14 days to the following sublethal diazinon concentrations: 0 (control), 0.05, 0.1, 0.2, 0.5, and 1.0 µg•L −1 .e reproduction test was conducted according to the standard protocol described in APHA [20] with minor modifications.Briefly, neonates of less than 24 h old were individually incubated in 50 mL beakers containing 20 mL control medium or exposed to diazinon concentrations.Diazinon concentrations and food (a mixture of green algae Chlorella sp., at a density of 5 × 10 6 cells•mL −1 , and YTC) were renewed every two days.e survival, reproduction (fecundity), time for the first reproduction, total number of neonates per female, number of broods, and brood size were recorded daily.e body length of parent daphnids was measured at the end of the experiment.

Statistical Analysis.
e 48 h median lethal concentrations (48 h LC 50 ) were predicted by probit analysis as previously reported by Stephan [21].
e rate of population increase (RPI) was calculated according to the method suggested by Euler-Lotka [22]: e -rx l x m x � 1, where l x � the proportion of surviving to age x, m x � age-specific fecundity, and x � time in days.All the calculations were based on 14day experiments [23].
e differences between treatment groups and controls were determined through one-way analysis of variance (ANOVA).Significant differences (p < 0.05) were distinguished by using Dunnet's test method.All data are presented as median ± SD. e toxic effects of pesticides on aquatic organisms are often investigated using the temperate D. magna under laboratory conditions.However, there is still little understanding about the acute and chronic effects of diazinon on crustaceans, especially to species that originated from tropical regions.e 48 h LC 50 values of diazinon reported in this study were in the range with the 48 h LC 50 values reported for nauplius of copepod Eodiaptomus (48 h LC 50 � 2.8 µg.L −1 ), Mesocyclops (48 h LC 50 � 2.9 µg•L −1 ), and ermocyclops (48 h LC 50 � 4.1 µg•L −1 ) [24].However, the values were lower than the 48 h LC 50 values for adults of Eodiaptomus (48 h LC 50 � 46.8 µg•L −1 ), Mesocyclops (48 h LC 50 � 30.6 µg•L −1 ), and ermocyclops (48 h LC 50 � 40.2 µg•L −1 ) [24], or higher than 48 h LC 50 values for other cladocerans such as Ceriodaphnia dubia (48 h LC 50 � 0.21 µg•L −1 ) [12].Results from acute assays documented higher sensitivity of tropical D. lumholtzi neonates to diazinon when compared to the temperate D. magna.From a practical application perspective, these results clearly showed that the D. lumholtzi may serve as a suitable surrogate for the temperate species D. magna, that is, as a toxicity indicator species, under tropical conditions.

Chronic Toxicity.
e effects of sublethal diazinon concentrations on the survival and reproduction of D. magna and D. lumholtzi during 14 days of incubation are shown in Table 1 and Figure 1. e survival of both daphnids decreased with increasing concentrations of diazinon during the 14-day test.Significant decreases in life history responses were observed for D. magna and D. lumholtzi when exposed to diazinon.Both daphnids grew well in the control incubation (the length of D. magna and D. lumholtzi increased up to 4.1 and 2.4 mm, respectively, at the end of the experiment).All individuals in the control survived throughout the experimental period (14 days) and produced numerous offspring (27.4 ± 3.6 juveniles per D. magna and 18.2 ± 2.1 per D. lumholtzi).
Diazinon caused significant effects and dose-dependent increases on the reproduction (number of broods per female) of both daphnids (Table 1).For D. magna, diazinon at the concentration up to 0.1 µg•L −1 or higher resulted in a significant decrease in reproduction (number of broods per female), while for D. lumholtzi, a significant reduction of reproduction was recorded from the lowest concentration tested (0.05 µg•L −1 ).e reproduction of both daphnids was significantly delayed (p < 0.05), from 6.5 days for the control to 9.6 days for D. magna (as the diazinon concentration up to 0.1 µg•L −1 or higher), and from 3.5 days in the control to 5.4 days for D. lumholtzi (as the diazinon concentration up to 0.05 µg•L −1 or higher) (Table 1).e results of both tested species indicated that the number of neonates born per female significantly declined at the diazinon concentration of 0.1 µg•L −1 or higher (Table 1).
e mean body length significantly decreased in those daphnids (as the diazinon concentration up to 0.2 µg•L −1 or higher) within the 14-day test period, from 4.1 to 3.1 mm and from 2.4 to 1.9 mm in D. magna and D. lumholtzi, respectively.
e results of the life-response history of both D. magna and D. lumholtzi species.Exposure to sublethal concentrations of diazinon under laboratory conditions showed that diazinon significantly reduced the number of offspring produced per female and delayed in the age at first brood.e obtained results are in agreement with previous observations which showed a decrease in the mean offspring production and suppression of growth in D. magna or Ceriodaphnia dubia following exposure to diazinon [12,14,25].
e results from the chronic assay showed that D. lumholtzi was a sensitive species to diazinon.Sánchez et al. [25] suggested that crustaceans are closely related to insects, more than other invertebrates.Hence, they are more sensitive to pesticides than other invertebrates.Modra et al. [5] indicated that the toxicity of diazinon is affected by many factors such as the biotransformation ability of the organism itself, water temperature, presence of other pollutants, and other nonidentified environmental variables.On the other hand, by using a toxic kinetic and dynamic model for studying diazinon toxicity, Kretschmann et al. [13] suggested that the sensitivity of test species to diazinon may depend on the detoxification ability of diazinon and diazoxon (a toxic metabolite of diazoxon).
ese authors revealed that, when compared to D. magna, the amphipod crustacean Gammarus pulex is less sensitive to diazinon because the detoxification of diazinon and diazoxon is six times faster which in turn causes less damaging effects.
e authors also suggested that mechanistic-based effect models should be used to explain the actual causes, effects, and the minor differences among different aquatic invertebrates [13].

Effects of Diazinon on Rate of Population Increase.
e effects of diazinon on the RPI are shown in Figure 2. Diazinon concentrations equal and/or greater than 0.1 µg•L −1 significantly reduced the RPI of both daphnids during the 14-day chronic test (p < 0.05).It can be observed that both daphnids exposed to diazinon showed a nearly similar trend during the 14-day chronic test.In natural environments, diazinon is known to cause adverse effects on many zooplankton species including Daphnia, even at low concentrations (from 5.3 to 26.3 nM) [6,15].e RPI of the Daphnia population is an important indicator for prediction population trends.e survival rate, number of offspring per female, and age of fecundity are all crucial for the prediction [26].A reduction in the RPI indicated chronic toxicant stress of pesticides on Daphnia [27].
Our results indicated that both test species were sensitive to diazinon and could be employed to predict the risk of insecticides.
e present results clearly suggest that the growth rates of D. magna and D. lumholtzi in the control treatments were within the range reported in the literature [25,26].In tropical countries, it is necessary to establish the benchmark for organophosphate insecticides.To minimize environmental risk associated with pesticides in tropical ecosystems, we strongly recommend further studies on short-and long-term toxic effects of organophosphate insecticides on different tropical groups of organisms.

Conclusions
e present study confirmed that diazinon poses significant risk to aquatic organisms, namely, nontarget Daphnia species.e population growth of D. magna and D. lumholtzi was adversely affected by diazinon after a chronic exposure period.Compared with D. magna, D. lumholtzi showed even higher sensitivity to diazinon in the acute test.
e results of this study are important for prediction of toxic effects and environmental risk associated with insecticides.Further studies using additional organophosphate insecticides, different tropical test species, and test conditions are needed to assess the possible environmental risk associated with pesticides in tropical aquatic ecosystems.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

3. 1 .
Acute Toxicity.No mortality occurred in the control during the experimental time of acute test.e highest tested concentration of diazinon resulted in 100% mortality of both daphnids.However, diazinon showed higher toxicity to D. lumholtzi.e 48 h LC 50 values of diazinon for D. magna and D. lumholtzi under the tested experimental conditions, at 95% confidence interval, were 4.63 and 3.41 µg•L −1 , respectively.

Figure 2 :
Figure 2: e effect of on the rate of population increase of Daphnia.* Significant difference with the control (p < 0.05).

Table 1 :
Fecundity, survival, and body length of D. magna and D. lumholtzi after exposure to different concentrations of diazinon for 14 days.