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The toxicological effects of thiamethoxam on Gammarus kischineffensis (Schellenberg 1937) (Crustacea: Amphipoda)
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Available online at www.sciencedirect.com
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The toxicological effects of thiamethoxam on Gammarus kischineffensis (Schellenberg 1937) (Crustacea: Amphipoda) a,∗ ˘ Pelin Ugurlu , Erhan Ünlü b , Elif I˙pek Satar b a b
Dicle University Science and Technology Research and Application Center, 21280 Diyarbakir, Turkey Department of Biology, Section of Hydrobiology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey
a r t i c l e
i n f o
a b s t r a c t
Article history:
Neonicotinoids are a new group of insecticides, and little is known about their toxicity to
Received 9 September 2014
nontarget freshwater organisms an potential effects on freshwater ecosystems. The aim of
Received in revised form
this study is to establish the acute toxicity and histopathological effects of thiamethoxam-
22 January 2015
based pesticide on the gill tissue of Gammarus kischineffensis. In this study G. kischineffensis
Accepted 23 January 2015
samples were exposed to 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100 mg/l
Available online 31 January 2015
of commercial grade thiamethoxam for 96 h. The 24, 48, 72 and 96 h LC50 values were determined as 75.619, 23.505, 8.048 and 3.751 mg/l respectively. In histopathological study the
Keywords:
individuals were exposed to 0.004, 0.04 and 0.4 mg/l thiamethoxam concentrations for 14
Thiamethoxam
days. The results showed that the most common changes at all doses of thiamethoxam
Gammarus kischineffensis
were vacuolization and hemostatic infiltration in the gill tissue of G. kischineffensis. © 2015 Elsevier B.V. All rights reserved.
Acute toxicity Histopathology LC50 value
1.
Introduction
Ecological or environmental risk assessment (ERA) is defined as the procedure by which the likely or actual adverse effects of pollutants and other anthropogenic activities on ecosystems and their components are estimated with a known degree of certainty using scientific methodologies (Depledge and Fossi, 1994). Ecotoxicological risk assessment of contaminants mainly is based on results of standard toxicity tests employing a limited number of laboratory-cultured species (OECD, 1997). The environment is continuously loaded with foreign organic chemicals (xenobiotics) released by urban communities and industries (Van der Oost et al., 2003). Pesticides
∗
used in agriculture are chemical materials or biological agents (virus, bacteria), which are produced to prevent growth of pest insect, animal and plant or to destroy, to repulse these pests or to reduce the number of these pests (EPA, 2007). Even they are useful in agriculture, their extensive and senseless use disturbs environment and, especially, organisms which have sensitive missions in ecosystem. Neonicotinoids or chloronicotinyls are a relatively new group of insecticides that have highly selective toxicity to insects (Beketov and Liess, 2007). Neonicotinoids exhibit high selectivity for nicotinic acetylcholine receptors (nAChRs) in insects, which contributes to their selective toxicity toward insects over vertebrates (Costa et al., 2009). Thaimethoxam is second-generation neonicotinoid and it has been marketed since 1998 under the trademarks Actara® for foliar
Corresponding author. Tel.: +90 412 248 83 20/8457; fax: +90 412 248 83 20. ˘ E-mail addresses: [email protected], [email protected] (P. Ugurlu).
http://dx.doi.org/10.1016/j.etap.2015.01.013 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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application and Cruiser® for seed treatment (Maienfisch et al., 2001). It exhibits outstanding biological properties on a wide number of pest insects with a preference to sucking insects such as aphids, whiteflies, and rice hoppers (Maienfisch et al., 1999). Thiamethoxam degrades in the aquatic/sediment environment with a half-life of 24–44 days under anaerobic conditions and with a half-life of 8–16 days under aerobic conditions. Crustaceans are frequently used as bioindicators and biomonitors in various aquatic systems. One reason is that they are a very successful group of animals, distributed in a number of different habitats including marine, terrestrial and freshwater environments. They are thus interesting candidates for comparative investigations. Some of the special features of crustaceans, particularly of reproduction strategies, may be highly important for the interpretation of data from bioindicator studies using these organisms, and for the development of ecotoxicological endpoints (Rinderhagen et al., 2000). Amphipods of Gammarus sp. are commonly used in freshwater risk assessment as an indicator species (Rinderhagen et al., 2000). They are often found in high densities in headstreams where they are an important reserve of food for macro-invertebrates, i.e. fish, bird and amphibian species (MacNeil et al., 2002) and play a major role in the leaf litter breakdown process (Forrow and Maltby, 2000) and consequently in the entire food web. Moreover, the use of these species is logistically interesting because they can be sampled throughout the year and easily identified, manipulated and maintained in the laboratory or used for in situ bioassays (Xuereb et al., 2009). Gammarus kischineffensis (Schellenberg1937) (Crustacea: Amphipoda) is an amphipod species which is common and nutritious food source for a lot of fish ˘ in clean freshwaters of Europe (Özbek and Ustaoglu, 2006). Toxicity tests are conducted to measure the effects of one or more pollutants on one or more species of organisms (Reish and Oshida, 1987). One of the commonly used measures of toxicity is the LC50 , i.e. the lethal median concentration that causes mortality in 50% of test organisms. A chemical with an LC50 , value of 5 g/l is very highly toxic compared to one with 1000 mg/l which is practically non-toxic (Stephan, 1977). The knowledge gained from concentration-response studies in animals is used to set standards for human exposure and the amount of chemical residue that is allowed in the environment. Histopathological studies have been conducted to help establish causal relationships between contaminant exposure and various biological responses (Antunes-Kenyon and Kennedy, 2007). As an indicator of exposure to contaminants, histology represents a useful tool to assess the degree of pollution, particularly for sub-lethal and chronic effects (Bernet et al., 1999). Histopathological investigations have proved to be a sensitive tool to detect direct effects of chemical compounds within target organs of aquatic organisms in laboratory experiments. Pesticides cause histopathological and biochemical changes in fish and other aquatic organisms. These changes are useful for detecting the effect of pollutants in various tissues and organs. The assessment of the ecotoxicological risks caused by pesticides to ecosystems is based on data on the toxicity and effects of pesticide preparations to non-target organisms.
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Crustaceans are among the group of non-target aquatic organisms. The present paper is a contribution to the assessment of toxicity and effects of athiamethoxam-based pesticide to G. kischineffensis. The aim of this study is to establish the acute toxic and histopathological effects of thiamethoxam-based pesticide on G. kischineffensis.
2.
Materials and methods
2.1.
Collection and maintenance of animals
G. kischineffensis (Schellenberg 1937) samples were collected with a long-handled sieves and dip nets in an unpolluted slow-running stream under stones and leaves from Tigris River in Diyarbakır, Turkey. In the laboratory undamaged and active individuals were placed in 40 × 35 × 40 cm glass aquariums containing 50:50 mixture field-sampled freshwater and dechlorinated tap water. Each aquarium was aerated with air stones. Approximately 50% of each aquarium’s water was substituted by dechlorinated tap water daily. Both during culturing and experiments, animals were maintained in a controlled room at 18 ± 1 ◦ C under an artificial light regime (13 h light:12 h dark). Animals were fed with decomposed willow (Salix sp.) leaves collected from sampling site and conditioned in field-sampled water for at least 2 weeks prior to use (Cold and Forbes, 2004). The animals were acclimated to the laboratory conditions for 2 weeks before the experiments.
2.2.
The experimental design of acute toxicity test
Prior to experiments, all glass materials used in experiments were prepared according to APHA (1998). In the experiments 2 L glass jars were used. The jars were filled with 1 L dechlorinated tap water. 20 individuals in each of three replicates were used per concentration. No plastic materials were used in the experiments. The test design was static renewal (APHA, 1998). About 50% of the water in the all aquaria were substituted at 24 h intervals to maintain water quality and to prevent degradation of the pesticide. After replenishment, in order to maintain the concentration, 50% of pesticide concentration was added to the test solutions. Commercial grade thiamethoxam (Actara® 240 SC) mixture was purchased from Gani Pesticides Marketing Ltd. Company. The stock thiamethoxam solution was prepared from commercial total pesticide mixture by dissolving this mixture pesticide in distilled water. It was taken into account that there was 240 g thiamethoxam in 1 L of pesticide mixture while test solutions were being prepared. The test concentrations was obtained without any dilution with water. After range finding tests, the test concentrations were determined as 0.0 (control), 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100 mg/l (Song et al., 1997; Barbee and Stout, 2009). Behavioral changes, immobility and mortality were recorded after 24, 48, 72 and 96 h of exposure. In order to determine the LC50 value of commercial grade thiamethoxam, probit analysis in SPSS statistical software was used.
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Table 1 – The % mortality of the individuals exposed to increasing grades of thimethoxam.
Table 2 – LC50 values and 95% confidence limits of commercial grade thiamethoxam for G. kischineffensis.
Concentration (mg/l)
Hours
2.5 5 7.5 10 15 20 25 30 35 40 45 50 60 70 80 90 100 500 1000
Mortality (%) 24 h
48 h
72 h
0 7 7 7 11 13 15 18 30 20 30 33 38 43 50 58 63 83 100
17 20 27 28 46 51 53 62 65 70 73 88 95 95 100 100 100 100 100
30 41 53 63 68 73 78 78 78 85 100 100 100 100 100 100 100 100 100
96 35 53 58 76 79 84 82 93 95 93 100 100 100 100 100 100 100 100 100
2.3. Preparation of tissue samples for histopathological study After finding the LC50 value of commercial grade of thimethoxam, the sublethal concentrations of thiamethoxam were determined according to LC50 value. 1/10, 1/100 and 1/1000 of LC50 value were used as sublethal concentrations of thiamethoxam. 30 animals were used for each concentration and pesticide free (control) group. The exposure period was 14 days. Three replicates were used for each concentration. For determining the histopathological alterations, at 7th and 14th days 5 samples were taken randomly from exposure and control groups. The organisms were sacrified by dissecting them from 2nd and 7th thoracic segments. Then the samples were fixed in formalin solution for 24 h and prepared according to the n-butyl alcohol technic given by Stiles (1934). Sections of 5 m were prepared from paraffin blocks by using a rotary microtome. These sections were then stained with hematoxylin–eosin, examined under a light microscope and the histopathological alterations were photographed.
3.
Results
3.1.
The acute toxicity test
In the control group, there was no death for 96 h. The death rate increased with the increasing concentration. 100% mortality was observed at 1000 mg/l after 24 h. Every individuals at 500 mg/l died after 48 h. At 25, 30 and 35 mg/l concentrations the mortality was 78% after 72 h. The % mortality of the individuals exposed to increasing grades of thimethoxam was given in Table 1. The water quality variables (the physicochemical properties) calculated across the acute and chronic tests are as follows: pH 7.94 ± 0.505, DO 7.5 ± 0.38 mg/l, total chlor 42.6 mg/l, total hardness 287 ± 2.35 mg/l CaCO3 , Mg
24 48 72 96
LC50 values (mg/l) 75.619 23.505 8.048 3.751
95% confidence limits (mg/l) 66.678–88.504 18.843–27.731 1.319–12.744 3.506–8.332
36 mg/l, conductivity 7.94 Mmho/cm, NO3 -N 2.1 mg/l, NO2 -N 0.002 mg/l, temperature 18 ± 1 ◦ C. After probit analyses, 24, 48, 72 and 96 h LC50 values and 95% confidence limits of commercial grade thiamethoxam for G. kischineffensis were given in Table 2.
3.2.
The histopathological study
Gammaridean amphipods have been characterized by the possession of single coxal gills (epipods) attached to thoracopods 3–8, the gills of most amphipods are thin, flat, oval structures attached to the peraeopods, often by a short stalk (Steele and Steele, 1991). Histologically, all the walls of the coxal gills were composed of a single layer of epithelial cells covered with a thin layer of cuticle, opposing walls of the gill were frequently held together by attachments of the pillar parts to the ones from the opposite site to keep them separated and to maintain a large sinusoid-like hemocoel divided into several compartments for the bloodstream (Fig. 1) (Takeuchi et al., 2003). In crustaceans, gills are the main organs involved in ionic regulation. It constitutes a site of passive and active ionic exchanges between the external and internal media (Issartel et al., 2010). No histopathological changes were observed in the gill of the control group relative to experiment group. The structural details of the gill of control G. kischineffensis are shown in Fig. 1. Histopathological results indicated that gill was the primary target tissue affected by thiamethoxam. The most common
Fig. 1 – The histological structure of gills of control G. kischineffensis samples; epithelium (a), hemocoel (b), cuticula layer (c), pillar cell (d).
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Fig. 2 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 7 days; vacuolization (a).
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Fig. 4 – The histopathological alterations in gills of G. kischineffensis exposed to 0.4 mg/l concentration commercial grade thiamethoxam for 7 days; hemostatic infiltration (a).
changes at all doses of thiamethoxam were vacuolization and hemostatic infiltration. At 7th day of exposure to 0.004, 0.04 and 0.4 mg/l thiamethoxam, the gills of experimental samples showed vacuolization in many areas and collapse of the pillar cells (Figs. 2–4). At 14th day of exposure to 0.004 and 0.04 mg/l thiamethoxam, necrosis and hemostatic infiltration was noticed (Figs. 5 and 6). In the group exposed to 0.4 mg/l thiamethoxam at 14th day, since more than 10% of the G. kischineffensis individuals were dead, results of this experimental group was not included in the test results.
Fig. 5 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 14 days; hemostatic infiltration (a), vacuolization (b).
Fig. 3 – The histopathological alterations in gills of G. kischineffensis exposed to 0.04 mg/l concentration commercial grade thiamethoxam for 7 days; hemostatic infiltration (a).
4.
Discussion
4.1.
The acute toxicity test
According to 96 h LC50 value (3.751 mg/l), the commercial grade thiamethoxam is highly toxic to G. kcshineffensis (Kannan, 1997). Acute toxicity tests provide a measure of the toxicity of compounds to a given species under specific environmental situations (water chemistry, pH, temperature, etc.) (Alam and
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Fig. 6 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 14 days; necrosis (a).
Maughan, 1993). They also reflect the severe and rapid damage caused by sudden exposure to lethal concentrations of contaminants (Cengiz and Ünlü, 1999). Accidental spillage of pesticides or the release of large amounts of pesticides in agricultural runoff would lead to conditions that are comparable to the results of the acute tests. Neonicotinoids are nonvolatile compounds with high water solubility, which makes them potentially mobile in the environment but also might limit their persistence and ameliorate their long-term impact on non-target organisms (Barbee and Stout, 2009). They are the most commonly used group of insecticides worldwide because of their supposed selectivity (Tomizawa and Casida, 2003; Ware and Whitacre, 2004; Guzsvany et al., 2006), and therefore should have low toxicity to warm-blooded animals. Neonicotinoids have low mammalian toxicity. The reason is that the binding sites which are present in insects’ nAChRs are absent in mammals (Iwaya and Kagabu, 1998). Although they are considered harmless for warm-blooded animals, neonicotinoids have high water solubility and potentially may contaminate surface water following rainfall events (EPA, 2003). Since neonicotinoids are used more and more, both in agriculture and for home use, the chance of their polluting water is present despite the low application rates. This pollution can be by means of accidental spilling, spray drift, and runoff, especially if the site of application is irrigated or natural rain occurs within two days of application (Overmyer et al., 2005). Neonicotinoids are thus most toxic to insects, and this also goes for aquatic insects; more so than to other aquatic invertebrates (Overmyer et al., 2005), crustaceans and fish (Tiˇsler et al., 2009). Neonicotinoids are, in general, more environmentally safe than other insecticides because they have small (yet variable) toxicity to crustaceans (Barbee and Stout, 2009). Therefore, applications of neonicotinoid insecticides are expected not to affect non-target crustaceans in planktonic and benthic communities. But in recent studies it has been shown that
neonicotinoids can be harmful to aquatic organisms. In a study carried out by Stark and Banks (2001), it was reported that the 48 h LC50 value of thiamethoxam for Daphnia pulex was 41 mg/l. In another study, 96 h LC50 value of thiamethoxam for crayfish, Procambarus clarkii was reported as 967 g/l (Barbee and Stout, 2009). It is seen that thiamethoxam is more toxic to P. clarkii than G. kischineffensis. Antunes-Kenyon and Kennedy (2007) found that the 96 h LC50 value of thiamethoxam for rainbow trout, Salmo gairdneri was higher than 100 mg/l and for Lepomis macrochirus was higher than 114 mg/l. In general thiamethoxam moderately less toxic than other neonicotiniods. In a study about the toxicity of first generation neonicotinoid imidacloprid, it was determined that the 48 h LC50 value of imidacloprid to Daphnia pulex was 10 mg/l (Song et al., 1997). Cox (2001) has reported that imidacloprid’s 96 h LC50 value for the marine shrimp, Mysidopsis bahia is 37 g/l. In a study carried out by Kidd and James (1991), it was found that 96 h LC50 value of imidacloprid for S. gairdneri was 211 mg/l. These findings showed that neonicotinoids are toxic to aquatic organisms especially crustaceans.
4.2.
The histopathological study
The studies about histopathological effects of thiamethoxam on organisms are very rare in the literature. Since thiamethoxam is a synthetic compound, it can cause histopathological alterations in the tissues of living organisms and there are some studies showing that thiamethoxam generated histopathological changes in some organisms (Green et al., 2005; Antunes-Kenyon and Kennedy, 2001). There are few studies about gill histology of Gammaridean species. Especially, light microscopy studies about histopathological alterations caused by toxic compounds are very rare in the literature. The study which was carried out in order to investigate toxic effect of cadmium on Gammarus fossarum was one of the rare studies about histopathological effects of toxic compounds on gills of Gammaridean species (Issartel et al., 2010). In the study researchers investigated cellular and molecular osmoregulatory responses to cadmium exposure in G. fossarum and it was reported that 15 g/l cadmium exposure for 3 days induced particularly abnormal nucleus pigmentation and the absence of pillars; this in turn was linked to a 71% decrease of the gill thickness compared to controls, resulting in the decrease or absence of the hemolymph canals. The researchers determined that a few aneurisms in hyperplasic gills, the significant gill collapse and cell hyperplasia, an increased volume of the gill. In that study at 7th day, the gills of exposed samples showed an increased volume of the gill with a total disappearance of pillars, resulting in an aneurism. In our study we indentified that thiamethoxam caused collapse of pillar cells and as a result of this, atrophy of hemocoel laguna. There have been a lot of studies carried out about the histopathological effects of toxic compounds on the gill tissues of crustacean species. Li et al. (2007) investigated the effects of water-borne copper on gills and hepatopancreas of a freshwater prawn Macrobrachium rosenbergii (Crustacea: Decapoda). They determined swelling of the gill lamellae and epithelial thickening in the gills of individuals exposed to 0.01 mg/l copper concentration. In the
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gills of individuals exposed to 0.05 mg/l copper concentration, hemostatic infiltration in hemocoel laguna and fusion of filaments were recorded. Bhavan and Geraldine (2000) examined histopathological effects of sub-lethal concentration of an organochlorine pesticide endosulfan on the gill tissue of M. malcolmsonii. Researchers reported hemostatic infiltration in hemocoel laguna, edema in gill lamellae, lifting of lamellar epithelium and fusion of lamellae in the gills of individuals exposed to 10.6 ng/l endosulfan. At the highest concentration of endosulfan (32.0 ng/l), swelling of the gill lamellae, thickening and fusion of gill lamellae and necrosis were identified. Our results are similar to these findings. In the gill tissue of individuals exposed to 0.04 and 0.004 mg/l thiamethoxam, we identified necrosis and hemostatic infiltration. Our findings have been showed that even thiamethoxam is an neurotoxic compound, it can cause histopathological changes in the tissues of Amphipods, especially in the gill tissues.
5.
Conclusion
Results have revealed that thiamethoxam is highly toxic to G. kischineffensis. This chemical has rapid and acute effects on the individuals of G. kischineffensis. Accidental leakage of this chemical, even at very low concentrations, can cause lethal consequences for this species. All the histopathological observation indicated that exposure to lethal concentrations of thiamethoxam caused destructive effect in the tissues of G. kischineffensis. Tissue alterations, such as those observed in this study and findings from previous studies, may result in severe functional problems, ultimately leading to the death of organism.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments We are thankful to The Coordination of Scientific Research Projects of Dicle University (DUBAP) for partial support of this work under grant 08-FF-16.
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Stiles, K.A., 1934. Normal butyl alcohol technic for animal tissues with special reference to insects. Stain Technol. 9, 97–100. Takeuchi, I., Matsumasa, M., Kikuchi, S., 2003. Gill ultrastructure and salinity tolerance of Caprella spp. (Crustacea: Amphipoda: Caprellidea) inhabiting the Sargassum community. Fish Sci. 69, 966–973. Tiˇsler, T., Jemec, A., Mozetic, B., Trebˇse, P., 2009. Hazard identification of imidacloprid to aquatic environment. Chemosphere 76, 907–914. Tomizawa, M., Casida, J.E., 2003. Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annu. Rev. Entomo. l48, 339–364. U.S. Environmental Protection and Agency, 2003. Pesticide Fact Sheet, Thiacloprid. Office of Prevention, Pesticides and Toxic Substances, Washington, DC. US Environmental Protection Agency, 2007. What is a pesticide? http://www.epa.gov/kidshometour/pest.htm (accessed 15.12.08). Van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57–149. Ware, G.W., Whitacre, D.M., 2004. An Introduction to Insecticides. Extracted From The Pesticide Book, 6th edition. MeisterPro Information Resources A Division of Meister Media Worldwide, Willoughby, OH, Available at: http://ipmworld.umn.edu/chapters/ware.htm (accessed: 22.01.08). Xuereb, B., Lefèvre, E., Garric, J., Geffard, O., 2009. Acetylcholinesterase activity in Gammarus fossarum (Crustacea Amphipoda): linking AChE inhibition and behavioural alteration. Aquat. Toxicol. 94, 114–122.
Available online at www.sciencedirect.com
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The toxicological effects of thiamethoxam on Gammarus kischineffensis (Schellenberg 1937) (Crustacea: Amphipoda) a,∗ ˘ Pelin Ugurlu , Erhan Ünlü b , Elif I˙pek Satar b a b
Dicle University Science and Technology Research and Application Center, 21280 Diyarbakir, Turkey Department of Biology, Section of Hydrobiology, Faculty of Science, University of Dicle, 21280 Diyarbakir, Turkey
a r t i c l e
i n f o
a b s t r a c t
Article history:
Neonicotinoids are a new group of insecticides, and little is known about their toxicity to
Received 9 September 2014
nontarget freshwater organisms an potential effects on freshwater ecosystems. The aim of
Received in revised form
this study is to establish the acute toxicity and histopathological effects of thiamethoxam-
22 January 2015
based pesticide on the gill tissue of Gammarus kischineffensis. In this study G. kischineffensis
Accepted 23 January 2015
samples were exposed to 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100 mg/l
Available online 31 January 2015
of commercial grade thiamethoxam for 96 h. The 24, 48, 72 and 96 h LC50 values were determined as 75.619, 23.505, 8.048 and 3.751 mg/l respectively. In histopathological study the
Keywords:
individuals were exposed to 0.004, 0.04 and 0.4 mg/l thiamethoxam concentrations for 14
Thiamethoxam
days. The results showed that the most common changes at all doses of thiamethoxam
Gammarus kischineffensis
were vacuolization and hemostatic infiltration in the gill tissue of G. kischineffensis. © 2015 Elsevier B.V. All rights reserved.
Acute toxicity Histopathology LC50 value
1.
Introduction
Ecological or environmental risk assessment (ERA) is defined as the procedure by which the likely or actual adverse effects of pollutants and other anthropogenic activities on ecosystems and their components are estimated with a known degree of certainty using scientific methodologies (Depledge and Fossi, 1994). Ecotoxicological risk assessment of contaminants mainly is based on results of standard toxicity tests employing a limited number of laboratory-cultured species (OECD, 1997). The environment is continuously loaded with foreign organic chemicals (xenobiotics) released by urban communities and industries (Van der Oost et al., 2003). Pesticides
∗
used in agriculture are chemical materials or biological agents (virus, bacteria), which are produced to prevent growth of pest insect, animal and plant or to destroy, to repulse these pests or to reduce the number of these pests (EPA, 2007). Even they are useful in agriculture, their extensive and senseless use disturbs environment and, especially, organisms which have sensitive missions in ecosystem. Neonicotinoids or chloronicotinyls are a relatively new group of insecticides that have highly selective toxicity to insects (Beketov and Liess, 2007). Neonicotinoids exhibit high selectivity for nicotinic acetylcholine receptors (nAChRs) in insects, which contributes to their selective toxicity toward insects over vertebrates (Costa et al., 2009). Thaimethoxam is second-generation neonicotinoid and it has been marketed since 1998 under the trademarks Actara® for foliar
Corresponding author. Tel.: +90 412 248 83 20/8457; fax: +90 412 248 83 20. ˘ E-mail addresses: [email protected], [email protected] (P. Ugurlu).
http://dx.doi.org/10.1016/j.etap.2015.01.013 1382-6689/© 2015 Elsevier B.V. All rights reserved.
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application and Cruiser® for seed treatment (Maienfisch et al., 2001). It exhibits outstanding biological properties on a wide number of pest insects with a preference to sucking insects such as aphids, whiteflies, and rice hoppers (Maienfisch et al., 1999). Thiamethoxam degrades in the aquatic/sediment environment with a half-life of 24–44 days under anaerobic conditions and with a half-life of 8–16 days under aerobic conditions. Crustaceans are frequently used as bioindicators and biomonitors in various aquatic systems. One reason is that they are a very successful group of animals, distributed in a number of different habitats including marine, terrestrial and freshwater environments. They are thus interesting candidates for comparative investigations. Some of the special features of crustaceans, particularly of reproduction strategies, may be highly important for the interpretation of data from bioindicator studies using these organisms, and for the development of ecotoxicological endpoints (Rinderhagen et al., 2000). Amphipods of Gammarus sp. are commonly used in freshwater risk assessment as an indicator species (Rinderhagen et al., 2000). They are often found in high densities in headstreams where they are an important reserve of food for macro-invertebrates, i.e. fish, bird and amphibian species (MacNeil et al., 2002) and play a major role in the leaf litter breakdown process (Forrow and Maltby, 2000) and consequently in the entire food web. Moreover, the use of these species is logistically interesting because they can be sampled throughout the year and easily identified, manipulated and maintained in the laboratory or used for in situ bioassays (Xuereb et al., 2009). Gammarus kischineffensis (Schellenberg1937) (Crustacea: Amphipoda) is an amphipod species which is common and nutritious food source for a lot of fish ˘ in clean freshwaters of Europe (Özbek and Ustaoglu, 2006). Toxicity tests are conducted to measure the effects of one or more pollutants on one or more species of organisms (Reish and Oshida, 1987). One of the commonly used measures of toxicity is the LC50 , i.e. the lethal median concentration that causes mortality in 50% of test organisms. A chemical with an LC50 , value of 5 g/l is very highly toxic compared to one with 1000 mg/l which is practically non-toxic (Stephan, 1977). The knowledge gained from concentration-response studies in animals is used to set standards for human exposure and the amount of chemical residue that is allowed in the environment. Histopathological studies have been conducted to help establish causal relationships between contaminant exposure and various biological responses (Antunes-Kenyon and Kennedy, 2007). As an indicator of exposure to contaminants, histology represents a useful tool to assess the degree of pollution, particularly for sub-lethal and chronic effects (Bernet et al., 1999). Histopathological investigations have proved to be a sensitive tool to detect direct effects of chemical compounds within target organs of aquatic organisms in laboratory experiments. Pesticides cause histopathological and biochemical changes in fish and other aquatic organisms. These changes are useful for detecting the effect of pollutants in various tissues and organs. The assessment of the ecotoxicological risks caused by pesticides to ecosystems is based on data on the toxicity and effects of pesticide preparations to non-target organisms.
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Crustaceans are among the group of non-target aquatic organisms. The present paper is a contribution to the assessment of toxicity and effects of athiamethoxam-based pesticide to G. kischineffensis. The aim of this study is to establish the acute toxic and histopathological effects of thiamethoxam-based pesticide on G. kischineffensis.
2.
Materials and methods
2.1.
Collection and maintenance of animals
G. kischineffensis (Schellenberg 1937) samples were collected with a long-handled sieves and dip nets in an unpolluted slow-running stream under stones and leaves from Tigris River in Diyarbakır, Turkey. In the laboratory undamaged and active individuals were placed in 40 × 35 × 40 cm glass aquariums containing 50:50 mixture field-sampled freshwater and dechlorinated tap water. Each aquarium was aerated with air stones. Approximately 50% of each aquarium’s water was substituted by dechlorinated tap water daily. Both during culturing and experiments, animals were maintained in a controlled room at 18 ± 1 ◦ C under an artificial light regime (13 h light:12 h dark). Animals were fed with decomposed willow (Salix sp.) leaves collected from sampling site and conditioned in field-sampled water for at least 2 weeks prior to use (Cold and Forbes, 2004). The animals were acclimated to the laboratory conditions for 2 weeks before the experiments.
2.2.
The experimental design of acute toxicity test
Prior to experiments, all glass materials used in experiments were prepared according to APHA (1998). In the experiments 2 L glass jars were used. The jars were filled with 1 L dechlorinated tap water. 20 individuals in each of three replicates were used per concentration. No plastic materials were used in the experiments. The test design was static renewal (APHA, 1998). About 50% of the water in the all aquaria were substituted at 24 h intervals to maintain water quality and to prevent degradation of the pesticide. After replenishment, in order to maintain the concentration, 50% of pesticide concentration was added to the test solutions. Commercial grade thiamethoxam (Actara® 240 SC) mixture was purchased from Gani Pesticides Marketing Ltd. Company. The stock thiamethoxam solution was prepared from commercial total pesticide mixture by dissolving this mixture pesticide in distilled water. It was taken into account that there was 240 g thiamethoxam in 1 L of pesticide mixture while test solutions were being prepared. The test concentrations was obtained without any dilution with water. After range finding tests, the test concentrations were determined as 0.0 (control), 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 and 100 mg/l (Song et al., 1997; Barbee and Stout, 2009). Behavioral changes, immobility and mortality were recorded after 24, 48, 72 and 96 h of exposure. In order to determine the LC50 value of commercial grade thiamethoxam, probit analysis in SPSS statistical software was used.
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Table 1 – The % mortality of the individuals exposed to increasing grades of thimethoxam.
Table 2 – LC50 values and 95% confidence limits of commercial grade thiamethoxam for G. kischineffensis.
Concentration (mg/l)
Hours
2.5 5 7.5 10 15 20 25 30 35 40 45 50 60 70 80 90 100 500 1000
Mortality (%) 24 h
48 h
72 h
0 7 7 7 11 13 15 18 30 20 30 33 38 43 50 58 63 83 100
17 20 27 28 46 51 53 62 65 70 73 88 95 95 100 100 100 100 100
30 41 53 63 68 73 78 78 78 85 100 100 100 100 100 100 100 100 100
96 35 53 58 76 79 84 82 93 95 93 100 100 100 100 100 100 100 100 100
2.3. Preparation of tissue samples for histopathological study After finding the LC50 value of commercial grade of thimethoxam, the sublethal concentrations of thiamethoxam were determined according to LC50 value. 1/10, 1/100 and 1/1000 of LC50 value were used as sublethal concentrations of thiamethoxam. 30 animals were used for each concentration and pesticide free (control) group. The exposure period was 14 days. Three replicates were used for each concentration. For determining the histopathological alterations, at 7th and 14th days 5 samples were taken randomly from exposure and control groups. The organisms were sacrified by dissecting them from 2nd and 7th thoracic segments. Then the samples were fixed in formalin solution for 24 h and prepared according to the n-butyl alcohol technic given by Stiles (1934). Sections of 5 m were prepared from paraffin blocks by using a rotary microtome. These sections were then stained with hematoxylin–eosin, examined under a light microscope and the histopathological alterations were photographed.
3.
Results
3.1.
The acute toxicity test
In the control group, there was no death for 96 h. The death rate increased with the increasing concentration. 100% mortality was observed at 1000 mg/l after 24 h. Every individuals at 500 mg/l died after 48 h. At 25, 30 and 35 mg/l concentrations the mortality was 78% after 72 h. The % mortality of the individuals exposed to increasing grades of thimethoxam was given in Table 1. The water quality variables (the physicochemical properties) calculated across the acute and chronic tests are as follows: pH 7.94 ± 0.505, DO 7.5 ± 0.38 mg/l, total chlor 42.6 mg/l, total hardness 287 ± 2.35 mg/l CaCO3 , Mg
24 48 72 96
LC50 values (mg/l) 75.619 23.505 8.048 3.751
95% confidence limits (mg/l) 66.678–88.504 18.843–27.731 1.319–12.744 3.506–8.332
36 mg/l, conductivity 7.94 Mmho/cm, NO3 -N 2.1 mg/l, NO2 -N 0.002 mg/l, temperature 18 ± 1 ◦ C. After probit analyses, 24, 48, 72 and 96 h LC50 values and 95% confidence limits of commercial grade thiamethoxam for G. kischineffensis were given in Table 2.
3.2.
The histopathological study
Gammaridean amphipods have been characterized by the possession of single coxal gills (epipods) attached to thoracopods 3–8, the gills of most amphipods are thin, flat, oval structures attached to the peraeopods, often by a short stalk (Steele and Steele, 1991). Histologically, all the walls of the coxal gills were composed of a single layer of epithelial cells covered with a thin layer of cuticle, opposing walls of the gill were frequently held together by attachments of the pillar parts to the ones from the opposite site to keep them separated and to maintain a large sinusoid-like hemocoel divided into several compartments for the bloodstream (Fig. 1) (Takeuchi et al., 2003). In crustaceans, gills are the main organs involved in ionic regulation. It constitutes a site of passive and active ionic exchanges between the external and internal media (Issartel et al., 2010). No histopathological changes were observed in the gill of the control group relative to experiment group. The structural details of the gill of control G. kischineffensis are shown in Fig. 1. Histopathological results indicated that gill was the primary target tissue affected by thiamethoxam. The most common
Fig. 1 – The histological structure of gills of control G. kischineffensis samples; epithelium (a), hemocoel (b), cuticula layer (c), pillar cell (d).
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Fig. 2 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 7 days; vacuolization (a).
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Fig. 4 – The histopathological alterations in gills of G. kischineffensis exposed to 0.4 mg/l concentration commercial grade thiamethoxam for 7 days; hemostatic infiltration (a).
changes at all doses of thiamethoxam were vacuolization and hemostatic infiltration. At 7th day of exposure to 0.004, 0.04 and 0.4 mg/l thiamethoxam, the gills of experimental samples showed vacuolization in many areas and collapse of the pillar cells (Figs. 2–4). At 14th day of exposure to 0.004 and 0.04 mg/l thiamethoxam, necrosis and hemostatic infiltration was noticed (Figs. 5 and 6). In the group exposed to 0.4 mg/l thiamethoxam at 14th day, since more than 10% of the G. kischineffensis individuals were dead, results of this experimental group was not included in the test results.
Fig. 5 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 14 days; hemostatic infiltration (a), vacuolization (b).
Fig. 3 – The histopathological alterations in gills of G. kischineffensis exposed to 0.04 mg/l concentration commercial grade thiamethoxam for 7 days; hemostatic infiltration (a).
4.
Discussion
4.1.
The acute toxicity test
According to 96 h LC50 value (3.751 mg/l), the commercial grade thiamethoxam is highly toxic to G. kcshineffensis (Kannan, 1997). Acute toxicity tests provide a measure of the toxicity of compounds to a given species under specific environmental situations (water chemistry, pH, temperature, etc.) (Alam and
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Fig. 6 – The histopathological alterations in gills of G. kischineffensis exposed to 0.004 mg/l concentration commercial grade thiamethoxam for 14 days; necrosis (a).
Maughan, 1993). They also reflect the severe and rapid damage caused by sudden exposure to lethal concentrations of contaminants (Cengiz and Ünlü, 1999). Accidental spillage of pesticides or the release of large amounts of pesticides in agricultural runoff would lead to conditions that are comparable to the results of the acute tests. Neonicotinoids are nonvolatile compounds with high water solubility, which makes them potentially mobile in the environment but also might limit their persistence and ameliorate their long-term impact on non-target organisms (Barbee and Stout, 2009). They are the most commonly used group of insecticides worldwide because of their supposed selectivity (Tomizawa and Casida, 2003; Ware and Whitacre, 2004; Guzsvany et al., 2006), and therefore should have low toxicity to warm-blooded animals. Neonicotinoids have low mammalian toxicity. The reason is that the binding sites which are present in insects’ nAChRs are absent in mammals (Iwaya and Kagabu, 1998). Although they are considered harmless for warm-blooded animals, neonicotinoids have high water solubility and potentially may contaminate surface water following rainfall events (EPA, 2003). Since neonicotinoids are used more and more, both in agriculture and for home use, the chance of their polluting water is present despite the low application rates. This pollution can be by means of accidental spilling, spray drift, and runoff, especially if the site of application is irrigated or natural rain occurs within two days of application (Overmyer et al., 2005). Neonicotinoids are thus most toxic to insects, and this also goes for aquatic insects; more so than to other aquatic invertebrates (Overmyer et al., 2005), crustaceans and fish (Tiˇsler et al., 2009). Neonicotinoids are, in general, more environmentally safe than other insecticides because they have small (yet variable) toxicity to crustaceans (Barbee and Stout, 2009). Therefore, applications of neonicotinoid insecticides are expected not to affect non-target crustaceans in planktonic and benthic communities. But in recent studies it has been shown that
neonicotinoids can be harmful to aquatic organisms. In a study carried out by Stark and Banks (2001), it was reported that the 48 h LC50 value of thiamethoxam for Daphnia pulex was 41 mg/l. In another study, 96 h LC50 value of thiamethoxam for crayfish, Procambarus clarkii was reported as 967 g/l (Barbee and Stout, 2009). It is seen that thiamethoxam is more toxic to P. clarkii than G. kischineffensis. Antunes-Kenyon and Kennedy (2007) found that the 96 h LC50 value of thiamethoxam for rainbow trout, Salmo gairdneri was higher than 100 mg/l and for Lepomis macrochirus was higher than 114 mg/l. In general thiamethoxam moderately less toxic than other neonicotiniods. In a study about the toxicity of first generation neonicotinoid imidacloprid, it was determined that the 48 h LC50 value of imidacloprid to Daphnia pulex was 10 mg/l (Song et al., 1997). Cox (2001) has reported that imidacloprid’s 96 h LC50 value for the marine shrimp, Mysidopsis bahia is 37 g/l. In a study carried out by Kidd and James (1991), it was found that 96 h LC50 value of imidacloprid for S. gairdneri was 211 mg/l. These findings showed that neonicotinoids are toxic to aquatic organisms especially crustaceans.
4.2.
The histopathological study
The studies about histopathological effects of thiamethoxam on organisms are very rare in the literature. Since thiamethoxam is a synthetic compound, it can cause histopathological alterations in the tissues of living organisms and there are some studies showing that thiamethoxam generated histopathological changes in some organisms (Green et al., 2005; Antunes-Kenyon and Kennedy, 2001). There are few studies about gill histology of Gammaridean species. Especially, light microscopy studies about histopathological alterations caused by toxic compounds are very rare in the literature. The study which was carried out in order to investigate toxic effect of cadmium on Gammarus fossarum was one of the rare studies about histopathological effects of toxic compounds on gills of Gammaridean species (Issartel et al., 2010). In the study researchers investigated cellular and molecular osmoregulatory responses to cadmium exposure in G. fossarum and it was reported that 15 g/l cadmium exposure for 3 days induced particularly abnormal nucleus pigmentation and the absence of pillars; this in turn was linked to a 71% decrease of the gill thickness compared to controls, resulting in the decrease or absence of the hemolymph canals. The researchers determined that a few aneurisms in hyperplasic gills, the significant gill collapse and cell hyperplasia, an increased volume of the gill. In that study at 7th day, the gills of exposed samples showed an increased volume of the gill with a total disappearance of pillars, resulting in an aneurism. In our study we indentified that thiamethoxam caused collapse of pillar cells and as a result of this, atrophy of hemocoel laguna. There have been a lot of studies carried out about the histopathological effects of toxic compounds on the gill tissues of crustacean species. Li et al. (2007) investigated the effects of water-borne copper on gills and hepatopancreas of a freshwater prawn Macrobrachium rosenbergii (Crustacea: Decapoda). They determined swelling of the gill lamellae and epithelial thickening in the gills of individuals exposed to 0.01 mg/l copper concentration. In the
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gills of individuals exposed to 0.05 mg/l copper concentration, hemostatic infiltration in hemocoel laguna and fusion of filaments were recorded. Bhavan and Geraldine (2000) examined histopathological effects of sub-lethal concentration of an organochlorine pesticide endosulfan on the gill tissue of M. malcolmsonii. Researchers reported hemostatic infiltration in hemocoel laguna, edema in gill lamellae, lifting of lamellar epithelium and fusion of lamellae in the gills of individuals exposed to 10.6 ng/l endosulfan. At the highest concentration of endosulfan (32.0 ng/l), swelling of the gill lamellae, thickening and fusion of gill lamellae and necrosis were identified. Our results are similar to these findings. In the gill tissue of individuals exposed to 0.04 and 0.004 mg/l thiamethoxam, we identified necrosis and hemostatic infiltration. Our findings have been showed that even thiamethoxam is an neurotoxic compound, it can cause histopathological changes in the tissues of Amphipods, especially in the gill tissues.
5.
Conclusion
Results have revealed that thiamethoxam is highly toxic to G. kischineffensis. This chemical has rapid and acute effects on the individuals of G. kischineffensis. Accidental leakage of this chemical, even at very low concentrations, can cause lethal consequences for this species. All the histopathological observation indicated that exposure to lethal concentrations of thiamethoxam caused destructive effect in the tissues of G. kischineffensis. Tissue alterations, such as those observed in this study and findings from previous studies, may result in severe functional problems, ultimately leading to the death of organism.
Conflict of interest The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments We are thankful to The Coordination of Scientific Research Projects of Dicle University (DUBAP) for partial support of this work under grant 08-FF-16.
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