140 ISJ 14: 140-148, 2017 ISSN 1824-307X RESEARCH REPORT Lethal and sub-lethal effects of cypermethrin and glyphosate on the freshwater’s copepod, Acanthocyclops robustus AM Houssou1,2, EJ Daguégué2, E Montchowui1,2 1Laboratory of Research in Aquaculture and Aquatics Biology and Ecology, School of Aquaculture of Vallée, National University of Agriculture, Porto-Novo, BP 43 Kétou, Republic of Bénin 2Laboratory of Hydrobiology and Aquaculture/Faculty of Agricultural Sciences / University of Abomey-Calavi, BP 526 Cotonou, Republic of Bénin Accepted April 18, 2017 Abstract The study aims to evaluate the acute and chronic toxicity of cypermethrin and glyphosate to a freshwater’s copepod, Arcanthocyclops robustus. The acute sensibility was assessed by estimating lethal concentrations. Then the chronic exposure allowed to assess the effects of low concentrations (0.2489 ppb and 0.4978 ppb respectively 10 % and 20 % of LC50 at 48 h of cypermethrin and 1.3 ppm and 2.6 ppm respectively for glyphosate) on the species. The estimated lethal concentrations at 1%, 50 % and 99 % were 2.353 ppb, 4.755 ppb and 9.610 ppb in 24 h, respectively 0.567 ppb, 2.489 ppb and 10.929 ppb in 48 h for cypermethrin. Regarding glyphosate, the lethal concentrations 1 %, 50 % and 99% were 5 ppm, 19 ppm and 73 ppm in 24 h, respectively 8 ppm, 13 ppm and 21 ppm in 48-h. hatching was affected by 20 % LC50 of cypermethrin; only 20 % of females have hatched their eggs against 60 % in the control treatment. Females and nauplii survival was affected by both pesticides. A. robustus is then more sensitive to cypermethrin compared to glyphosate. Low concentration like 0.4978 ppb of cypermethrin could affect it population and then all the ecosystem biodiversity. Key Words: Acanthocyclops robustus; chronic sensibility; cypermethrin; glyphosate; lethal dose Introduction The use of chemical pesticides has greatly expanded in Africa, as everywhere in the world, to fight against both the endemic diseases vectors and crops pests (Lévèque and Paugy, 2006). It results chronic contamination of all ecosystem (aquatic, terrestrial or atmospheric) (Druart, 2011). Only 0.1 % of sprayed pesticides reach their target, the rest is distributed in ecosystems and contaminate the land, water and air. The atmospheric fraction also finally regains, like atmospheric fallout, soil and surface waters during rainfall (Haraguchi et al., 1994). Soil leaching and soil-water dispersion of pesticides therefore lead to contamination of aquatic environments (Kao, 2002; Lalancette, 2012). It results various effects, on ecosystem balance and lead to the extinction of some species (Adégbidi, 2000; Fadoegnon and Midingoyi, 2006; Pesce et al., 2008; Houssou et al., 2015, 2016). ___________________________________________________________________________ Corresponding author: Arsène Mathieu Houssou Laboratory of Research in Aquaculture and Aquatics Biology and Ecology School of Aquaculture of Vallée National University of Agriculture BP 43 Kétou, Republic of Bénin E-mail: arsnehous@yahoo.fr In Bénin republic, aquatic ecosystems are contaminated with agricultural pesticides especially in the cotton basin (Agbohessi et al., 2011), in the Ouémé River (Yèhouénou et al., 2006a and b). Even, cypermethrin and glyphosate are two of the most used pesticides in food crops production in Bénin (preliminary personal investigation: unpublished). The increasing of food crops production in the Ouémé River basin causes a consequent increase of the use of cypermethrin and glyphosate, then the increasing of contamination of the ecosystem. Freshwater’s copepods are an important part of the planktonic biodiversity. They are part of the first organisms that may present a quickly observable responses facing chemical pollution. Due to numerous factors affecting the responses of these organisms to pollutant, specie in two different environments can present wide range of responses to the same substance. Then, to efficiently monitor the status of ecosystems, it is important to have biological models for different environment and know their responses facing different type of pollutants. Acanthocyclops robustus is one of the most abundant copepod species in the Ouémé River basin (Houssou et al., unpublished). Nowadays, there 141 is no data on the sensitivity of plankton species to the different pollutants in Bénin aquatic environments. The aim of this study is to evaluate the response of A. robustus exposed to cypermethrin and glyphosate, used in food crops production in Bénin. Material and Methods Test organisms: culture and rearing A plankton sample was taken in Ouémé River at Bonou (6°54'30.7"N and 2°26'58.0"E) by using plankton net of 50 µm mesh size. This sample was rushed to the laboratory in ambient conditions in absence of fixative. It was then cultured for 2 weeks in the presence of organic fertilizer (chicken droppings) at 0.6 g/L (Agadjihouèdé et al., 2011). The species were then identified under photonic microscope and ovigerous females of A. robustus were isolated. A total of 10 females have been placed in a culture of phytoplankton (Scenedesmus spp.) and rotifers (Brachionus plicatilis). After hatching, the nauplii were kept in the culture until adulthood. The culture solution was renewed every two weeks. At the time of this study, culture has gone through four generations. Chemicals Cypermethrin is a high active synthetic pyrethroid insecticide. It has light yellow appearance with acidity (as H2SO4) of 0.3 % (w/w) maximum. It toxicity classification is II (Moderatly hazardous). Cypermethrin is lowly soluble in water (4 to 10 µg/L). In this study, cypermethrin is obtained in it supplied formulation (Cyperforce®) containing Cypermethrin 10 % EC (emulsifiable concentrate). Glyphosate is a non-selective systemic herbicide. It is a weak organic acid. Its molecular weight is 169.07 and its solubility in water of 12 g/L at 25 °C. Glyphosate is used in this study as supplied formulation (Kumark® (480 g/L). Different concentrations of both pesticides were dissolved in water to have the needed concentrations of active ingredient. The structural formula of cypermethrin and glyphosate are following (see below). Acute toxicity test Test design and handling The experimental design for each pesticide is composed of twenty-eight (28) glass cup (petri box) (Six concentrations and one control with four replications each) and seven (07) pillboxes. A pillbox containing the test solution of each treatment was used for the measurement of physico-chemical parameters. Seven adults of A. robustus were placed in each glass cup immediately after distribution of the test solutions (28 individuals per treatment and a total of 168 per test). An initial test was performed to determine the concentration range to be used in the definitive test (data not presented). The definitive nominal concentrations used are: 0 (control), 1, 2, 4, 6, 8 and 10 ppb for cypermethrin and 0 (control), 2, 4, 8, 12, 16 and 20 ppm for glyphosate. Each test lasted 48 h. The test solutions were not renewed and the copepods were not fed (USEPA, 2002). The loss of active ingredients concentration in the 48-h was considered insignificant, view cypermethrin is non-volatile (vapor pressure = 3.1x10-9 mmHg at 20 °C; Henry's constant = 4.21x10-7 atm.m3/mol) (SAgE pesticide, 2015). Also its half-life time in the water is 14 days. Glyphosate also is non-volatile (vapor pressure = 1.84x10-7 mmHg at 45 °C; Henry's constant = 4.08x10-19 atm.m3/mol). The photoperiod was 16/8-h (light/darkness). Temperature (26.49 ± 0.25° C), pH (7.11 ± 0.11) and dissolved oxygen (3.91 ± 0.19 mg/L) were measured daily in the different treatments. The individual’s mobility and mortality were monitored at 1-h , 24-h and 48-h of exposure. Copepod was declared dead with lack of movement after a mild stimulus. Chronic tests Test design and handling Hatching, survival of females and nauplii of A. robustus were studied in this part of the study. A total of 30 glass cups (petri box) of 50 ml each (three treatments with 10 replications each) was used for each pesticide. A pillbox (120 ml) was provided for each treatment allowing the measurement of physico-chemical parameters. Two sub-lethal concentrations (10 % and 20 % LC50 in 48 h) were tested in addition to the zero- concentration (control). After distribution of the test solutions in the experimental design, one ovigerous female of A. robustus was immediately placed in each cup. The feeding was ad-libitum with concentrated freshwater’s rotifer (B. plicatilis) and phytoplanktons. The test solution was renewed in all tests every 96-h. Cypermethrin and glyphosate being non-volatile with half-life time in water more than 96-h, the test solution renewal time made insignificant the loss of active mater. The test lasted 10 days and the hatching was controlled daily. Female and nauplii mortality have also been monitored daily. The photoperiod was 16/8-h (light/darkness). The temperature (24.73 ± 0.06°C), pH (6.67 ± 0.06) and dissolved oxygen (3.57 ± 0.12 mg/L) were measured daily in the different treatments. 142 Table 1 Cumulative mortality of Acanthocyclops robustus (n = 28 each concentration) exposed to cypermethrin and glyphosate Data analysis The copepod survival values were used to estimate the lethal concentrations (LC1-10-20-30-40-50-60- 70-80-90-99) at 24-h and 48-h. Lethal concentrations were calculated using probit method in the computing program PoloPlus v.1.0. The chi-square test was used to assess difference in response other exposure concentration. The hatching rate, the death rate of nauplii and females were calculated of each treatment. The One way analysis of variance (ANOVA) was used to assess variability of data other treatment (Statistica v.7 Software was used). Results Acute toxicity Cumulative numbers of dead individual of A. robustus to increase concentration of cypermethrin and glyphosate during the exposure time (24 and 48-h) are presented in Table 1. There is significant increased number of dead individual with increasing concentration for both pesticides. Respectively two deaths were recorded in the control treatment (zero concentration) at 24-h and 48-h for cypermethrin exposure and four in the two periods for glyphosate exposure. In cypermethrin exposure, there was 100 % mortality from 8 ppb to 10 ppb (24-h) and from 6 ppb to 10 ppb (48-h). Respective to the glyphosate exposure, a maximum of 53.57% (24-h) and 100% (48-h) was respectively observed at 16 ppm and 20 ppm. The estimated lethal concentrations of cypermethrin and glyphosate to 1 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % and 99 % of A. robustus population are respectively presented in Tables 2 and 3. In case of cypermethrin, the median lethal concentration (LC50) at 24-h (4.755 ppb) was high than that of 48-h (2.489 ppb). Regarding glyphosate, the LC50 was 19 ppm (24-h) and 13 ppm (48-h). Sub-lethal effects The percentage of females that hatched their eggs according to different treatments is presented in Table 4 respectively for cypermethrin and glyphosate. Hatching was very low in females exposed to 20 % LC50 of cypermethrin. Only 20 % of females have hatched their eggs against 60 % in the control and 10 % LC50 cypermethrin treatment. In the case of glyphosate contamination, the maximum hatching rate was 70 % in the control treatment. The lowest hatching was observed in the 20 % LC50 treatment. Table 2 Lethal concentration (LC1 to 99) and 95 % confidence limits of cypermethrin to Acanthocyclops robustus Point Concentration (ppb) 24h 48h Doses 95%CL 95%CL LC1 2.353 0.667 - 3.314 0.567 0.156 - 1.001 LC10 3.228 1.853 - 3.964 1.102 0.466 - 1.632 LC20 3.687 1.932- 4.464 1.457 0.734 - 2.015 LC30 4.058 2.415 - 4.778 1.783 1.015 - 2.355 LC40 4.405 2.912 - 5.080 2.119 1.332 - 2.705 LC50 4.755 3.451 - 5.409 2.489 1.706 - 3.099 LC60 5.134 4.048 - 5.817 2.924 2.160 - 3.59 LC70 5.573 4.705 - 6.417 3.474 2.726 - 4.290 LC80 6.134 5.389 - 7.495 4.251 3.454 - 5.472 LC90 7.006 6.145 - 9.839 5.623 4.526 - 8.126 LC99 9.610 7.745 - 20.352 10.929 7.698 - 23.230 Chi-square 10.96 Degrees of freedom: 2 p = 0.004 Glyphosate Cypermethrin Concentration (ppm) No of mortality Concentration (ppb) No of mortality 24-h 48-h 24-h 48-h 0 4 4 0 2 2 2 3 5 1 5 9 4 5 6 2 10 13 8 7 8 4 12 17 12 11 14 6 20 28 16 15 23 8 28 28 20 14 28 10 28 28 Chi-Square 15.076 14.203 23.385 17.624 df 22 22 22 22 143 Table 3 Lethal concentrations (LC1 to 99) and corresponding 95 % confidence limits of glyphosate to Acanthocyclops robustus Regarding the females which hatched their eggs, 100 % had died within 10 days of exposure to 20 % LC50 of cypermethrin (Table 4). Only 50 % of females had died under 10 % LC50 while in the control 33,33 % mortality was observed. Respective to the females which have not hatched their eggs, 100 % mortality was observed with 20 % LC50 of cypermethrin against 75 % and 50 % respectively for 10 % LC50 and the control. In case of glyphosate exposure (Table 4) 57.14 % of females which hatched their eggs was died in control treatment. In 10 %LC50 and 20 %LC50 treatment, 66.67 % and 80 % of died was observed respectively. For those which didn’t hatch their eggs in glyphosate exposure, 100 % was died under 10 % LC50 while 66.67 % and 60 % was noted respectively in control and 20 % LC50 treatment. The cumulative mortality of hatched nauplii during the study showed increase sensitivity to the exposure dose of cypermethrin (Fig. 1). The dose of 20 % LC50 in 48-h (0.4978 ppb) was more toxic to the nauplii of A. robustus during the 10 days of exposure. A high mortality rate was also observed in the absence of contaminants (control). Respective to glyphosate (Fig. 2), 10 % LC50 and 20 % LC50 induced more mortality in nauplii. The average number of live nauplii obtained per female after 10 days of exposure is presented on Figures 3 and 4 respectively for cypermethrin and glyphosate. In cypermethrin exposure, the lowest average number of nauplii per female was obtained with 20 % LC50 (9.5 ± 3.5 nauplii/female). This mean number is significantly different from those obtained in the other two treatments (ANOVA 1, Tukey test, p < 0.05). The averages of 21.67 ± 5.8 nauplii/female and 24.17 ± 10.96 nauplii/female were observed in the control and 10 % LC50 respectively. For glyphosate, no significant difference was observed between treatments. 21.5 ± 7.6 nauplii/female was the highest survival nauplii obtained in glyphosate exposure after 10 days. Table 4 Hatching rate and mortality of female of Acanthocyclops robustus under sub-lethal concentrations of cypermethrin and glyphosate. Control 10%LC50 20%LC50 Hatching (%) Cyperméthrin 60 60 20 Glyphosate 70 60 50 Mortality of females after hatching (%) Cyperméthrin 33,33 50 100 Glyphosate 57,14 66,67 80 Mortality of females without hatching (%) Cyperméthrin 50 75 100 Glyphosate 66,67 100 60 Point Concentration (ppm) 24h 48h Doses 95%CL Doses 95%CL LC1 5 1 - 8 8 5 - 10 LC10 9 2 - 12 10 7 - 12 LC20 12 6 - 15 11 8 - 12 LC30 14 9 - 18 12 9 - 13 LC40 16 12 - 22 12 10 - 14 LC50 19 14 - 28 13 11 - 14 LC60 22 17 - 39 14 12 - 15 LC70 26 20 - 55 14 13 - 16 LC80 31 23 - 86 15 14 - 17 LC90 40 27 - 162 17 15 - 20 LC99 73 40 - 751 21 18 - 28 Chi-square 28.08 Degrees of freedom: 2 p = 0.000 144 Fig. 1 Cumulative mortality of hatched nauplii other cypermethrin treatments Discussion The immobilization follow by mortality of A. robustus is higher when the exposure time is long and the concentration of cypermethrin or glyphosate is high. The increasing doses have therefore lead dysfunction of the nervous system causing paralysis. Cypermethrin’s action has focused on interference with the functioning of the sodium channel in the central nervous system, stimulating the nervous discharges repeatedly, causing paralysis (SAgE pesticide, 2015). Christensen et al. (2005) reported that cypermethrin at concentrations greater than 0.1 ppb causes immobilization of crustacean zooplankton in general and in Daphnia magna particularly after respectively 6-h, 24-h, and in 48-h exposure. Lethal doses determined showed that A. robustus is very sensitive to cypermethrin (Cyperforce®). Kuldeep et al. (2014) reported that cypermethrin is highly toxic to aquatic invertebrates. This high toxicity is observed on A. robustus with median lethal concentrations (LC50) 24-h and 48-h respectively 4.755 ppb and 2.489 ppb. In case of glyphosate, 19 ppm and 13 ppm was LC50 to A. robustus at 24-h and 48-h exposure respectively. These results showed that cypermethrin is more toxic to A. robustus than glyphosate, as Golombieski et al. (2008) reported that insecticides are more toxic to aquatic organisms than other pesticides. Chris (2009) also reported that glyphosate is less toxic to fish than other pesticide as: methidathion, beta-cyfluthrin, endosulfan et carbendazine. Summarizing these observations, the sensitivity of aquatic species to a pollutant, is affected by both interspecific differences and the type of pollutant (see Tables 5 and 6). Takahashi et al. (2006) have also showed a large difference in sensitivity between life stages of specie exposed to same pollutant. They demonstrated that adult copepods are higher resistant to Diazinon and Carbaryl than nauplii. In the present paper, this aspect was not evaluated for A. robustus facing cypermethrin and glyphosate. But the high acute resistance observed by Takahashi et al. (2006) in adult copepods to Diazinon and Carbaryl, confirmed the interspecific and pollutant difference effect in sensitivity of aquatic invertebrates. Fig. 2. Cumulative mortality of hatched nauplii other glyphosate treatments. 145 Fig. 3. Mean number of nauplii obtained per female of Acanthocyclops robustus after 10 days of exposure to different doses of cypermethrin. Plots having different letter are significantly different (Turkey, p < 0.05). In aquatic ecosystems where shores are strongly used for agricultural production (as Oueme river), concentrations higher than LC50 estimated for A. robustus (especially for cypermethrin) can easily be exceed in water. It would result a strong change in its population and zooplankton community in general (Relyea, 2005). According to Sarkar et al. (2005), the 96-h LC50 of aquatic invertebrates to cypermethrin are generally between 0.01 and 5 ppb. The observations of this study at 24-h and 48-h of exposure fit into this range and confirm the high sensitivity of the species. Regarding the glyphosate, the lethal dose observed is largely high than that of Roundup® (glyphosate) to the callanoïda Phyllodiaptomus annae (1.06 ppm) (Ashoka-Deepananda et al., 2011). The difference may be due to the difference in used formulation of glyphosate (Kumark® in the present study) also in the genetic difference between the two species of copepod (table 6). However, the department of agriculture of UAS has reported that lethal doses of glyphosate Roundup® to aquatic invertebrates varied between 4 and 37 ppm (Ashoka-Deepananda et al., 2011). The chronic effects tests showed that chronic exposure of A. robustus at sub-lethal doses of cypermethrin and glyphosate affects reproduction and survivorship of nauplii. In fact, chronic exposure to both pesticides caused mortality in ovigerous females before and after hatching (reproduction). Hatching is strongly affected and survivorship of nauplii is also affected. The results confirm those of Hanazato (2001) which showed that pesticides can affect population dynamics of freshwater zooplankton by reducing their survival rates, by affecting the eggs hatching and reducing their richness and specific diversity. Lina et al. (2003) also showed zooplankton population reduction in the presence of cypermethrin. Ratushnyak et al. (2005) showed that cypermethrin at concentrations above 0.002 ppb to 0.2 ppb does not affect the survival of freshwater’s invertebrates after 21 days of exposure. Lutnicka et al. (2014) also showed that a concentration of 0.02 ppb of cypermethrin has no observable toxic effect on reproduction and growth of freshwater organisms. This justified the fact that a concentration of 0.2489 ppb (10% LC50, 48-h), had no significant effect neither on reproduction neither on the survival of A. robustus after 10 days of exposure. Significant effects were then obtained with a concentration of 0.4978 ppb (20 % LC50, 48- h) after 10 days of exposure. Contrary to Ratushnyak et al. (2005), Wendt-Rasch et al. (2003) have previously reported that cypermethrin causes a reduction in zooplankton population at concentrations greater than 0.13 ppb during 11 days of exposure. Also Lina et al. (2003) showed that copepods facing concentrations of cypermethrin greater than 0.03 ppb, presented firstly ovigerous females mortality and secondly an increase egg hatching rate among survivors. This demonstrates the variation of species responses to a single pollutant whether the environments are different. After 21 days of exposure of Daphnia magna to different concentration (0.26 and 0.38 ppm) of glyphosate, no effect was observed on neonates’ survivorship and growth, but the reproduction has affected with increasing concentration (Maycock et al., 2010). On freshwater copepods, concentration Fig. 4 Mean number of nauplii obtained per female of Acanthocyclops robustus after 10 days of exposure to different doses of glyphosate. Plots having different letter are significantly different (Turkey, p < 0.05). 146 Table 5 Comparative toxicity of cypermethrin in different formulation to different planktonic crustacean species Species Cypermethrin formulation Acute toxicity Chronic effects References Exposure time Toxicity LC50 (ppb) Concentration (ppb) Effects Ceriodaphnia dubia (cladoceran) Pestanal® 48-h 0.23 0,0978 Affected growth of neonate Shen et al. (2012) Daphnia magna (cladoceran) Pestanal® 48-h 0.72 Ashauer et al. (2011) Daphnia magna (cladoceran) Cypermethrin (CAS no.52315-07-8) ≥0.1 Significant reduction of the swimming ability. Christensen et al. (2005) Daphnia schoedleri (cladoceran) Pestanal® 48-h 0,6 0.0005, 0.0054 and 0.054 All population parameters were significantly reduced. Reduction in reproduction is observed Martínez- Jerónimo et al. (2013) Diaptomus forbesi (copepod) Pestanal® 48-h 0.03 Saha and Kaviraj (2008) Acartia clause (copepod) Pestanal® 48-h 2.67 Willis and Ling (2004) Pseudocalanus elongates (copepod) 48-h >5 Acanthocyclops robustus (copepod) Cyperforce® 48-h 2.49 0.25 Reduced egg hatching in ovigerous females Reduced nauplii survivorship Present study greater than 0.01 ppm shouted reduce eggs hatchability (Maycock et al., 2010). In the present study, A. robustus exposed to chronic doses 1.3 ppm and 2.6 ppm (respectively 10% and 20% of LC50 in 48-h) of glyphosate has presented any significant effects on hatching rate. Significant effect on female survival after eggs hatching at 10% LC50 exposure (may be due to physical treatments). It is then observed a high resistance of A. robustus to sub-lethal doses of glyphosate. Conclusion A. robustus, a copepod species present in Ouémé River ecosystem, appeared very sensitive to cypermethrin. The median lethal concentration (LC50) is 4.755 ppb in 24 h and 2.489 ppb respectively in 48-h. This specie was less sensitive to glyphosate with respective LC50 of 19 ppm and 13 ppm. Low concentrations rang of 0.2489 ppb and 0.4978 ppb of cypermethrin affected it reproduction by reducing egg hatching. The survival of ovigerous females and nauplii in the early development stages was also affected. Regarding glyphosate, exposure to sub-lethal dose as 2.6 ppm also reduced survival factors of the species. Low levels of cypermethrin in the aquatic environment can therefore significantly affect aquatic biodiversity. Glyphosate in fact is less toxic for aquatic invertebrates. Table 6 Comparative toxicity of glyphosate in different formulation to different planktonic crustacean species Species Glyphosate formulation Acute toxicity Chronic effects References Exposure time Toxicity LC50 (ppm) Concentration (ppm) Effects Daphnia magna (cladoceran) Glyphosate IPA 48-h 31 4.05 Significant reduction in survival Significant reduction of body size Significant decrease of fecundity Cuhra et al. (2013) Simocephalus vetulus (cladoceran) Eskobat® 48-h 21.5 ≥6.4 Age at first reproduction was delayed 2 to 4 days number of neonates/females was significantly reduced Reno et al. (2014) Notodiaptomus conifer (Copepod) Eskobat® 48-h 95.2 80 Significant delayed of the sexual maturity Reno et al. (2014) ≥160 N. conifer could not reach the adult stage Phyllodiaptomu s annae (Copepod) Roundup® 48-h 1.06 Ashoka- Deepanand a et al. 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