Environmental Fate of Neonicotinoids and Classification of Their Potential Risks to Hypogean, Epygean, and Surface Water Ecosystems in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sublethal neonicotinoid insecticide exposure reduces solitary bee reproductive success

1. Pollinating insects provide crucial and economically important ecosystem services to crops and wild plants, but pollinators, particularly bees, are globally declining as a result of various driving factors, including the prevalent use of pesticides for crop protection. Sublethal pesticide exposure negatively impacts numerous pollinator lifehistory traits, but its influence on reproductive success remains largely unknown. Such information is pivotal, however, to our understanding of the long-term effects on population dynamics. 2 We investigated the influence of field-realistic trace residues of the routinely used neonicotinoid insecticides thiamethoxam and clothianidin in nectar substitutes on the entire life-time fitness performance of the red mason bee Osmia bicornis. 3 We show that chronic, dietary neonicotinoid exposure has severe detrimental effects on solitary bee reproductive output. Neonicotinoids did not affect adult bee mortality; however, monitoring of fully controlled experimental populations revealed that sublethal exposure resulted in almost 50% reduced total offspring production and a significantly male-biased offspring sex ratio. 4 Our data add to the accumulating evidence indicating that sublethal neonicotinoid effects on non-Apis pollinators are expressed most strongly in a rather complex, fitness-related context. Consequently, to fully mitigate long-term impacts on pollinator population dynamics, present pesticide risk assessments need to be expanded to include whole life-cycle fitness estimates, as demonstrated in the present study using O. bicornis as a model.

Impact of chronic neonicotinoid exposure on honeybee colony performance and queen supersedure

BACKGROUND: Honeybees provide economically and ecologically vital pollination services to crops and wild plants. During the last decade elevated colony losses have been documented in Europe and North America. Despite growing consensus on the involvement of multiple causal factors, the underlying interactions impacting on honeybee health and colony failure are not fully resolved. Parasites and pathogens are among the main candidates, but sublethal exposure to widespread agricultural pesticides may also affect bees. METHODOLOGY/PRINCIPAL FINDINGS: To investigate effects of sublethal dietary neonicotinoid exposure on honeybee colony performance, a fully crossed experimental design was implemented using 24 colonies, including sister-queens from two different strains, and experimental in-hive pollen feeding with or without environmentally relevant concentrations of thiamethoxam and clothianidin. Honeybee colonies chronically exposed to both neonicotinoids over two brood cycles exhibited decreased performance in the short-term resulting in declining numbers of adult bees (-28%) and brood (-13%), as well as a reduction in honey production (-29%) and pollen collections (-19%), but colonies recovered in the medium-term and overwintered successfully. However, significantly decelerated growth of neonicotinoid-exposed colonies during the following spring was associated with queen failure, revealing previously undocumented long-term impacts of neonicotinoids: queen supersedure was observed for 60% of the neonicotinoid-exposed colonies within a one year period, but not for control colonies. Linked to this, neonicotinoid exposure was significantly associated with a reduced propensity to swarm during the next spring. Both short-term and long-term effects of neonicotinoids on colony performance were significantly influenced by the honeybees' genetic background. CONCLUSIONS/SIGNIFICANCE: Sublethal neonicotinoid exposure did not provoke increased winter losses. Yet, significant detrimental short and long-term impacts on colony performance and queen fate suggest that neonicotinoids may contribute to colony weakening in a complex manner. Further, we highlight the importance of the genetic basis of neonicotinoid susceptibility in honeybees which can vary substantially.

Microarray analysis of cytochrome P450 mediated insecticide resistance in Drosophila

Insecticide resistance in laboratory selected Drosophila strains has been associated with upregulation of a range of different cytochrome P450s, however in recent field isolates of D. melanogaster resistance to DDT and other compounds is conferred by one P450 gene, Cyp6g1. Using microarray analysis of all Drosophila P450 genes, here we show that different P450 genes such as Cyp12d1 and Cyp6a8 can also be selected using DDT in the laboratory. We also show, however, that a homolog of Cyp6g1 is over-expressed in a field resistant strain of D. simulans. In order to determine why Cyp6g1 is so widely selected in the field we examine the pattern of cross-resistance of both resistant strains and transgenic flies over-expressing Cyp6g1 alone. We show that all three DDT selected P450s can confer resistance to the neonicotinoid imidacloprid but that Cyp6a8 confers no cross-resistance to malathion. Transgenic flies over-expressing Cyp6g1 also show cross-resistance to other neonicotinoids such as acetamiprid and nitenpyram. We suggest that the broad level of cross-resistance shown by Cyp6g1 may have facilitated its selection as a resistance gene in natural Drosophila populations. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Cytotoxic Effects of Thiamethoxam in the Midgut and Malpighian Tubules of Africanized Apis mellifera (Hymenoptera: Apidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análises morfológica e histoquímicas do ventrículo e do cérebro de operárias recém emergidas de Apis Mellifera L. (Hymenoptera, Apidae) tratadas com Thiamethoxam

Apis mellifera honeybees are social insects of economic importance, by providing honeybee products, and by the pollination of natural areas of vegetation or agricultural areas. The constant use of pesticides, including the thiamethoxam, which is an insecticide belonging to the class of neonicotinoids with neurotoxic action, is subjecting pollinators to situations of severe stress, which has been evidenced by the decrease in the density of honeybees in many parts of the world. By these considerations, the present study aimed to assess the acute toxicity of thiamethoxam for newly emerged workers of A. mellifera, and to investigate the effect of sublethal doses of this insecticide on the survival time and its cytotoxicity to the brain and midgut to the honeybees. It was established the value of the lethal concentration 50 (LC50) equal to 4.28 ng thiamethoxam/μL of food and from it, the sublethal concentrations of 0.428 ng thiamethoxam/μL (CL50/10) and 0.0428 ng thiamethoxam/μL (CL50/100), which were used in bioassays of intoxication of the honeybees. After the bioassays the bees were dissected and the brain and midguts were collected to analyze possible morphological (staining with Hematoxylin-Eosin) and histochemical alterations (Xylidine Ponceau technique, and Feulgen and PAS reactions) caused by exposure to thiamethoxam, and to calculate the lethal time (LT50) for the workers. The duration of the bioassays was 8 days after beginning of feeding. The results obtained showed that the thiamethoxam is toxic to newly-emerged workers of A. mellifera, causing changes in survival time of individuals. This study also shown that the thiamethoxam causes morphological and histochemical alterations on the midgut and brain of workers. These alterations may be reflected in physiological and behavioral changes that can modify the operation of the colony

Potential of the chemical dinotefuran in the control of Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) semi-engorged female ticks

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação bioquímica e imunohistoquímica dos efeitos subletais do tiametoxam sobre Apis mellifera (Hymenoptera, Apidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of pesticides on honey bees (Apis mellifera L.): study of a specific route of exposure and evaluation of biochemical-physiological changes in the assessment of the pesticides toxicity

In this study, some important aspects of the relationship between honey bees (Apis mellifera L.) and pesticides have been investigated. In the first part of the research, the effects of the exposure of honey bees to neonicotinoids and fipronil contaminated dusts were analyzed. In fact, considerable amounts of these pesticides, employed for maize seed dressing treatments, may be dispersed during the sowing operations, thus representing a way of intoxication for honey bees. In particular, a specific way of exposure to this pesticides formulation, the indirect contact, was taken into account. To this aim, we conducted different experimentations, in laboratory, in semi-field and in open field conditions in order to assess the effects on mortality, foraging behaviour, colony development and capacity of orientation. The real dispersal of contaminated dusts was previously assessed in specific filed trials. In the second part, the impact of various pesticides (chemical and biological) on honey bee biochemical-physiological changes, was evaluated. Different ways and durations of exposure to the tested products were also employed. Three experimentations were performed, combining Bt spores and deltamethrin, Bt spores and fipronil, difenoconazole and deltamethrin. Several important enzymes (GST, ALP, SOD, CAT, G6PDH, GAPDH) were selected in order to test the pesticides induced variations in their activity. In particular, these enzymes are involved in different pathways of detoxification, oxidative stress defence and energetic metabolism. The results showed a significant effect on mortality of neonicotinoids and fipronil contaminated dusts, both in laboratory and in semi-field trials. However, no effects were evidenced in honey bees orientation capacity. The analysis of different biochemical indicators highlighted some interesting physiological variations that can be linked to the pesticide exposure. We therefore stress the attention on the possibility of using such a methodology as a novel toxicity endpoint in environmental risk assessment.

Ricerca di contaminanti in tessuti animali e in alimenti destinati all'uomo.

La contaminazione chimica rappresenta uno dei rischi principali per la sicurezza alimentare e può arrecare anche gravi danni alla salute umana. Rientrano in questa tesi di dottorato tre famiglie di contaminanti: Micotossine, Metalli e Insetticidi. La ricerca di aflatossina B1 è stata effettuata su 90 confezioni di farina, sia biologici sia convenzionali. La presenza della micotossina è stata rilevata solo nelle farine di mais. Solo un campione di produzione convenzionale ha superato il limite di 2 ppb definito per legge. Il dato di maggior rilievo è stato che il quantitativo di 5 grammi di campionamento si è dimostrato non rappresentativo sul totale della confezione commerciale di farina. Più attendibile si è invece dimostrato un campionamento di 20 grammi. L’aflatossina M1 è stata ricercata in 58 campioni di latte di cui 35 sono risultati positivi. Tuttavia, i livelli riscontrati erano costantemente inferiori al limite previsto per legge. Sono stati sottoposti a estrazione e purificazione, e analizzati con metodica HPLC-FL per la ricerca di Ocratossina A, 114 campioni di bile, 35 campioni di plasma, 40 campioni di rene prelevati da polli in Giordania. Le analisi hanno fornito risultati costantemente negativi. Sono stati analizzati 72 campioni (30 di muscolo, 29 di fegato e 13 di rene) prelevati da 30 bovini nel macello di Irbid (Giordania), di età compresa tra 8 e 30 mesi e provenienti da allevamenti diversi, per la ricerca di 13 elementi essenziali e non essenziali. In questo studio nessun campione supera i livelli massimi stabiliti dalla normativa europea per quanto riguarda gli elementi considerati. Infine, sono stati analizzati 37 campioni di latte ovino e 31 campioni di latte bovino, prelevati in Giordania in diversi allevamenti, per la ricerca di 4 neonicotinoidi (imidacloprid, acetamiprid, thiamethoxam e thiacloprid). I campioni, analizzati con sistema HPLC/MS/MS, sono risultati costantemente negativi ai quattro neonicotinoidi ricercati.

Sublethal effects of a common neonicotinoid pesticide, thiamethoxam, on honey bees: impact on locomotion and thermoregulation

Neonicotinoids have been pointed to as a factor responsible for the increased honey bee colony losses in the last decades. Many studies have investigated the effects of the first marketed neonicotinoid, imidacloprid, while fewer have focused on thiamethoxam. One recent study showed that sublethal doses of thiamethoxam lead to colony failure by decreasing forager homing flight success. We thus decided to investigate the mechanism which caused this phenomenon. Our hypothesis was that this effect was caused by impairment of forager locomotion abilities. Therefore we tested the effects of sublethal acute and chronic exposures to thiamethoxam on forager walking (Chapter 2) and flight (Chapter 3) performances. The acute treatment (1.34 ng/bee) affected walking locomotion firstly triggering hyperactivity (30 min post-treatment) and then impairing motor functioning (60 min post-treatment). 2-day continuous exposures to thiamethoxam (32.5, 45 ppb) elicited fewer effects on walking locomotion, however both exposure modes elicited an increased positive phototaxis. Similarly, in flight experiments, the single dose (1.34 ng/bee) elicited hyperactivity shortly after intoxication (increased flight duration and distance), while longer and continuous exposures (32.5, 45 ppb) impaired forager motor functions (decreased flight duration, distance, velocity). It is known that flight muscles temperature needs to be precisely regulated by bees during flight. Therefore, we further hypothesized that the impaired flight performances of neonicotinoid intoxicated bees were caused also by thermoregulation anomalies. We tested the effects that acute thiamethoxam exposures (0.2, 1, 2 ng/bee) elicit on forager thorax temperature (Chapter 4). Foragers treated with high doses exhibited hyperthermia or hypothermia when respectively exposed to high or low environmental temperatures. In summary, we show that sublethal doses of thiamethoxam affected forager walking and flight locomotion, phototaxis and thermoregulation. We also display the intricate mode of action of thiamethoxam which triggered, at different extents, inverse sublethal effects in relation to time and dose.

Sublethal neonicotinoid insecticide exposure reduces solitary bee reproductive success

1.Pollinating insects provide crucial and economically important ecosystem services to crops and wild plants, but pollinators, particularly bees, are globally declining as a result of various driving factors, including the prevalent use of pesticides for crop protection. Sublethal pesticide exposure negatively impacts numerous pollinator life-history traits, but its influence on reproductive success remains largely unknown. Such information is pivotal, however, to our understanding of the long-term effects on population dynamics. 2.We investigated the influence of field-realistic trace residues of the routinely used neonicotinoid insecticides thiamethoxam and clothianidin in nectar substitutes on the entire life-time fitness performance of the red mason bee Osmia bicornis. 3.We show that chronic, dietary neonicotinoid exposure has severe detrimental effects on solitary bee reproductive output. Neonicotinoids did not affect adult bee mortality; however, monitoring of fully controlled experimental populations revealed that sublethal exposure resulted in almost 50% reduced total offspring production and a significantly male-biased offspring sex ratio. 4.Our data add to the accumulating evidence indicating that sublethal neonicotinoid effects on non-Apis pollinators are expressed most strongly in a rather complex, fitness-related context. Consequently, to fully mitigate long-term impacts on pollinator population dynamics, present pesticide risk assessments need to be expanded to include whole life-cycle fitness estimates, as demonstrated in the present study using O. bicornis as a model.

Impact of Chronic Neonicotinoid Exposure on Honeybee Colony Performance and Queen Supersedure

Background Honeybees provide economically and ecologically vital pollination services to crops and wild plants. During the last decade elevated colony losses have been documented in Europe and North America. Despite growing consensus on the involvement of multiple causal factors, the underlying interactions impacting on honeybee health and colony failure are not fully resolved. Parasites and pathogens are among the main candidates, but sublethal exposure to widespread agricultural pesticides may also affect bees. Methodology/Principal Findings To investigate effects of sublethal dietary neonicotinoid exposure on honeybee colony performance, a fully crossed experimental design was implemented using 24 colonies, including sister-queens from two different strains, and experimental in-hive pollen feeding with or without environmentally relevant concentrations of thiamethoxam and clothianidin. Honeybee colonies chronically exposed to both neonicotinoids over two brood cycles exhibited decreased performance in the short-term resulting in declining numbers of adult bees (−28%) and brood (−13%), as well as a reduction in honey production (−29%) and pollen collections (−19%), but colonies recovered in the medium-term and overwintered successfully. However, significantly decelerated growth of neonicotinoid-exposed colonies during the following spring was associated with queen failure, revealing previously undocumented long-term impacts of neonicotinoids: queen supersedure was observed for 60% of the neonicotinoid-exposed colonies within a one year period, but not for control colonies. Linked to this, neonicotinoid exposure was significantly associated with a reduced propensity to swarm during the next spring. Both short-term and long-term effects of neonicotinoids on colony performance were significantly influenced by the honeybees’ genetic background. Conclusions/Significance Sublethal neonicotinoid exposure did not provoke increased winter losses. Yet, significant detrimental short and long-term impacts on colony performance and queen fate suggest that neonicotinoids may contribute to colony weakening in a complex manner. Further, we highlight the importance of the genetic basis of neonicotinoid susceptibility in honeybees which can vary substantially.

Neonicotinoid insecticides severely affect honey bee queens

Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances.

Low doses of a neonicotinoid insecticide modify pheromone response thresholds of central but not peripheral olfactory neurons in a pest insect

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