Comparison of acute toxicity of two neonicotinoid insecticides, imidacloprid and clothianidin, to five cladoceran species

Abstract

The acute toxicity (48-hr) of old (imidacloprid) and new (clothianidin) neonicotinoid insecticides to five cladoceran species and species sensitivity distribution (SSD) for cladocerans and other aquatic organisms to these insecticides are compared here. The sensitivities to both insecticides were in the following descending order: Ceriodaphnia>Daphnia>Moina. Differences in the 5% hazardous concentration (HC5) threshold between the two species taxa to each compound indicated that clothianidin was 4 times less toxic than imidacloprid only to cladocerans.

Introduction

Freshwater ecosystems are threatened by pollution of chemical toxicants,¹⁾ including agricultural pesticides, which are used around the world to assure agricultural quality and productivity. Most pesticides exert negative impacts on non-target organisms in natural ecosystems.^2–4) Thus, the development and use of pesticides with low ecological impact on non-target organisms is imperative for the protection of biodiversity and the ecosystem. Pesticide risk assessment at the community level based on long-term monitoring is essential to accomplish those purposes.^5–7) However, regulatory protocols establish the initial evaluation of the short-term effects of a chemical on individual-level toxicity tests (i.e., zooplankton, fish, and aquatic algae) before the impact of pesticides on communities is estimated.^8,9) Among these test organisms, zooplankton species, particularly cladocerans, are important because they transfer energy and materials from primary producers to higher trophic feeders, such as fish.¹⁰⁾ In addition, cladocerans are highly sensitive to toxic chemicals and changes in the aquatic environment.^11,12)

Among the pesticides most commonly used in agriculture are neonicotinoid insecticides. They are recognized as an important chemical class of systemic insecticides, since they are very effective on target pests (particularly sucking insects) and generally safe for standard test species, such as Daphnia spp., fish, and vertebrates.^13,14) Neonicotinoids are registered globally in more than 120 countries.¹⁵⁾ However, large variations in effective concentrations (2 to 3 orders of magnitude) to non-target aquatic organisms between outdoor and laboratory test conditions have been reported for thiacloprid and imidacloprid.^16–18) Contrary to the expectations, outdoor impact is much higher than suggested by acute toxicity tests. The explanation lies in the time-dependent toxicity of these neonicotinoids and other systemic insecticides, such as fipronil,⁷⁾ which is due to irreversible binding to their respective neuronal receptors.¹⁹⁾

Clothianidin is one of the latest neonicotinoid compounds developed (though it is a metabolite of thiomethoxam), and information on its ecological risks to aquatic organisms is still lacking. Following the OECD protocol, we examined the acute toxicity (48-hr immobilization tests) of clothianidin to cladoceran species, taking into consideration their body size as an important variable influencing the survival and sensitivity of individuals.^18,20)

We then compared the clothianidin species sensitivity distribution (SSD) curves between the cladocerans tested as well as the SSDs for other species of aquatic vertebrates and invertebrates (except cladocerans). Finally, the SSDs for clothianidin were compared to those obtained for older neonicotinoid insecticides, such as imidacloprid,¹⁸⁾ which have the same mode of action (see Nagai and Yokoyama²¹⁾ for a detailed concept of SSD).

Materials and Methods

1. Test design (acute toxicity immobilization tests, 48-hr EC₅₀)

To compare the differences in the susceptibility of cladocerans to imidacloprid¹⁸⁾ and clothianidin, a similar experimental design to that reported by Hayasaka et al.¹⁸⁾ was followed. Test cladocerans chosen in this study were the same as those used in that study (Ceriodaphnia dubia, C. reticulata, Daphnia magna, D. pulex, and Moina macrocopa). These species were obtained from the National Institute for Environmental Studies (NIES), Tsukuba, Japan, and their stock cultures have been in this institute over three decades (see Hayasaka et al.¹⁸⁾ for details on the methodology for culturing these five cladocerans).

To evaluate the realistic acute toxic impact, we used wettable powder of clothianidin to make the insecticidal solutions. Commercial clothianidin [Dantotsu^® Flowable, clothianidin/water, and surfactant (20 : 80, v/v)] was obtained from Kyoyu Agri Co., Ltd., Kanagawa, Japan. Data for the acute toxicity tests of imidacloprid to these cladocerans have already been reported;¹⁸⁾ they were also done using wettable powder [Admire^® Flowable, imidacloprid/water, and surfactant (20 : 80, v/v)]. The acute toxicity tests of clothianidin were performed following the OECD guideline no. 202²²⁾ for 48-hr acute immobilization tests under static conditions. Female neonates (<24-hr old) from the second or later broods were used in all tests. The number of tests for each species/treatment to estimate EC₅₀ of clothianidin is 5 to 7. The concentration ratio between successive solutions in all the tests was 2.0. For the controls, only dechlorinated tap water was used. Each species was tested separately. Five neonates, consisting of four replicates of each concentration, were introduced into each beaker containing 20 mL of the test solutions (see Hayasaka et al.¹⁸⁾ for details of the methodology for the bioassays). Test individuals were not fed during the test period. In parallel with the bioassays, to evaluate the relationship between the acute concentration of clothianidin to test cladocerans and their body sizes, we measured the body lengths of 30–50 female neonates of each species randomly selected. Body lengths, from the crown of the head to the base of the tail spine,^18,23) were measured using a dissecting microscope (Leica DFC490, Leica, Wetzlar, Germany).

The pH and dissolved oxygen (DO) were measured at the beginning and end of the tests in the controls. The initial pH values were 7.65±0.24, and they increased slightly after 48 hr (7.85±0.04). The values of DO at the start and the end were 8.19±0.40 and 7.65±0.31, respectively.

2. Data analysis

From the results of the 48-hr acute immobilization tests, the corresponding acute EC₅₀ values were determined by the Probit method using the program EcoTox-Statics ver. 2.5 (http://www.intio.or.jp/jset/ecotox.htm). The relationship between toxicant sensitivity (48-hr EC₅₀ values) and body lengths was analyzed by Pearson’s correlation coefficient. Differences in the susceptibility between test cladocerans and aquatic organisms other than cladocerans to clothianidin were compared using the 5% hazardous concentration (HC5) values of both species groups, which have been used in the United States and Europe for deriving threshold concentrations that protect most species in a community.^21,24) The HC5 is calculated from the species sensitivity distribution (SSD) of each compound.¹⁸⁾ The acute toxicity data (LC₅₀ and EC₅₀) of clothianidin obtained for other aquatic organisms comprise 3 crustaceans, Procambarus clarkii,²⁵⁾ Hyalella azteca, and Paratya compressa improvisa,²⁶⁾ 3 fish, Cyprinus carpio, Lepomis macrochirus, and Oncorhynchus mykiss,¹⁴⁾ and 2 insects, Cheumatopsyche brevilineatus²⁷⁾ and Chironomus riparius.²⁸⁾ Acute toxicity data for imidacloprid to other aquatic vertebrates and invertebrates, including amphibians, crustaceans, fish, insects, and worms, were obtained from the ECOTOX database (http://cfpub.epa.gov/ecotox/) as reported in Hayasaka et al.¹⁸⁾

Then, based on the degree of discrepancies in the HC5 values between the two species groups, we evaluated the differences in acute toxicity between the old (imidacloprid)¹⁸⁾ and new (clothianidin) neonicotinoid insecticides to aquatic organisms.

Results and Discussion

1. Inter-species variation of acute toxicity

Since Daphnia magna and D. pulex are able to reproduce by cyclical parthenogenesis and can be easily cultured in the laboratory, they supply genetically identical animals, which minimizes analytical errors caused by genetic variation of the test organisms.²⁹⁾ In this study, the 48-hr EC₅₀ estimate for D. magna exposed to wettable flowable clothianidin was 67,564 µg/L (Table 1), and this value is in the same range of the effective concentration (40,000–73,000 µg/L) to this species reported by Haith³⁰⁾ and Saika.²⁶⁾ The most sensitive species (C. dubia) had a 48-hr EC₅₀ of 1691 µg/L. The toxicity of clothianidin was in the following decreasing order: C. dubia>C. reticulata>D. pulex>M. macrocopa>D. magna. For imidacloprid, the order of decreasing toxicity (from 572 to 45,271 µg/L) was slightly different for the most tolerant species: C. dubia>C. reticulata>D. pulex>D. magna>M. macrocopa (Table 1).

Table 1. Acute toxicity (48-hr EC₅₀ in µg/L) and 95% confidence intervals (CI) of imidacloprid and clothianidin for five test cladocerans

Species	48-hr EC50 (95% CI)
	Imidacloprida)	Clothianidin
Ceriodaphnia dubia	571.62 (289.63–841.19)	1691.3 (1077.1–19,844)
Ceriodaphnia reticulata	5552.9 (4213.3–7387.8)	29,474 (21,076–49,968)
Daphnia magna	43,265 (34,302–53,592)	67,564 (48,762–98,441)
Daphnia pulex	36,872 (28,399–48,106)	31,448 (20,881–46,463)
Moina macrocopa	45,271 (34,378–62,218)	61,106 (42,582–106,290)

^a) Data from Hayasaka et al.¹⁸⁾

The inter-species variations of acute toxicity values are small, generally less than a factor of 2 to 3 (maximum 8).³¹⁾ However, variations in the EC₅₀ values for imidacloprid and clothianidin across the 5 cladocerans tested are between a factor of 80 and 40, respectively, whereas, except for C. dubia, the values for the two insecticides are between a factor of 8 and 2, respectively (Table 1). Even Moina macrocopa, which is widely distributed in ponds and rice fields of Japan and is very sensitive to organophosphate insecticides,³²⁾ appears to be more tolerant to neonicotinoid insecticides (Table 1). However, high sensitivity of Ceriodaphnia spp. to other toxicants, such as mercury, zinc, and carbamate insecticides, has also been reported.^23,33,34) On the other hand, Wu et al.²⁹⁾ have indicated that the test organisms and protocols accepted by regulating authorities, such as OECD, do not always reflect local taxa or site-specific conditions, whereas Hose and van den Brink³⁵⁾ have reported that the sensitivity of organisms to toxicants is independent of their geographic origin. Among the cladocerans used in this study, neither C. dubia nor D. magna are indigenous species in Japan. In this context, C. reticulata, which plays an important role in energy flow in aquatic ecosystems and environments through the microbial loop,²³⁾ can be a more suitable ecotoxicological test species for the freshwater environments of Japan.

Within the same cladoceran genus, body size is positively related to acute toxicity, i.e., smaller species are more sensitive to toxic stress than the larger ones for obvious reasons,³⁶⁾ whereas clear relationships between zooplankton body size and sensitivities to toxicants are not found across different species.^37,38) In this study, significant relationships between the EC₅₀ values of each species for both neonicotinoid insecticides and their body lengths were not found (imidacloprid, r²=0.585, p=0.132; clothianidin, r²=0.593, p=0.129) (Fig. 1). On the other hand, the acute toxicity of phenyl-pyrazole fipronil was significantly correlated with body length.¹⁸⁾ Since the magnitude of species sensitivity may be highly chemical-dependent, tendencies found for one chemical may not be adequately reflected in others.

Fig. 1. Correlations between the 48-hr EC₅₀ values of imidacloprid (solid line) and clothianidin (dotted line) to five test cladocerans and their body length. Open and solid symbols are for imidacloprid and clothianidin, respectively (○, Ceriodaphnia dubia; □, Ceriodaphnia reticulata; -, Daphnia magna; △, Daphnia pulex; ◇, Moina macrocopa). Results of imidacloprid as per Hayasaka et al.¹⁸⁾

2. Differences in SSD between cladocerans and other aquatic organisms

SSDs have been used to determine hazardous concentrations for the protection of ecosystems and are frequently used for the quantitative determination of the ecological risks of toxicants. The SSDs of both neonicotinoid insecticides showed a clear difference between the five cladocerans tested here and other aquatic vertebrates and invertebrates (excluding cladocerans) reported in the existing literature (Fig. 2). Although Daphniidae are more sensitive to metal and organophosphate pesticides than other aquatic vertebrates and invertebrates,^39,40) the opposite tendencies are found for neonicotinoid insecticides (Fig. 2). The 5% hazardous concentration (HC5) values of imidacloprid for the tested cladocerans and other aquatic organisms were 513.68 µg/L and 0.67 µg/L, respectively. The HC5 values for clothianidin to the two taxa groups were 1929.72 µg/L and 0.34 µg/L, respectively, suggesting that freshwater aquatic taxa other than cladocerans were about 2,000 times more sensitive to the neonicotinoid insecticides. The toxicity patterns of clothianidin for aquatic organisms, except cladocerans, might be the same as those of imidacloprid due to their identical mode of action. However, HC5 values for fipronil are similar for cladocerans (0.88 µg/L) and other aquatic species (0.10 µg/L).¹⁸⁾ Except for Daphnia, high toxicity of imidacloprid to chironomidae larvae, dragonfly nymphs, crustaceans including ostracods, amphipods, and crayfish, has been reported by many researchers.^41–44)

Fig. 2. Species sensitivity distribution (SSD) of five test cladocerans (solid line) and other aquatic organisms except cladocerans (dotted line) reported in the existing literature for (a) imidacloprid and (b) clothianidin. Symbols of each test cladoceran species as in Fig. 1. Other aquatic organisms tested for sensitivity to imidacloprid include amphibians, crustaceans, fish, insects, and worms (see Hayasaka et al.¹⁸⁾ for detailed information on imidacloprid). Those tested for sensitivity to clothianidin include crustaceans, fish, and insects.

Since the neonicotinoid chemical class may be safe only to cladocerans, the evaluation of the ecotoxicological risks of the neonicotinoids based on acute toxicity to cladocerans can systematically underestimate the risk of these pesticides to all other aquatic species and communities. Indeed, other scientists have indicated the need for establishing a different risk assessment to this and other classes of systemic insecticides.³⁰⁾ Sarma et al.³⁴⁾ have also indicated the need to employ a range of cladoceran species for ecotoxicological tests given that no single species was consistently sensitive to stress from pesticides. In any case, the community effects of pesticides should not be linked to the lethal effects alone because sublethal responses of individual species and indirect effects on other species in the community also play a role.⁶⁾

Acknowledgments

We thank Akiko Miyamoto for her kind assistance with the acute toxicity tests. We are indebted to Fusae Oyama and Nozomi Fujiwara for providing the culture organisms and their food. The authors wish to thank Megumi Nakagawa for valuable technical advice. The paper benefited from the constructive comments of two anonymous reviewers. This research has been partly supported by a grant from the Ministry of the Environment, Japan.

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