Project
title / Assessing the impact of mixtures of pyrethroids and fungicides on honeybees
/ DEFRA
project code / PN0945

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Assessing the impact of mixtures of pyrethroids and fungicides on honeybees
DEFRA project code / PN0945
Contractor organisation and location / Central Science Laboratory
Sand Hutton
York YO41 1LZ
Total DEFRA project costs / £ 55,718
Project start date / 01/04/01 / Project end date / 31/03/04
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 2

Project
title / Assessing the impact of mixtures of pyrethroids and fungicides on honeybees
/ DEFRA
project code / PN0945

There has been considerable concern over the last few years, including consideration by the Environmental Panel, over the potential for synergism between pyrethroids and EBI fungicides applied to oilseed rape. Incidents reported through the Wildlife Incident Scheme have, according to field data, included mixtures of pyrethroids with fungicides which are not synergistic under laboratory conditions, i.e. chlorothalonil and alphacypermethrin. Interpretation and regulation may be difficult if it is unclear whether synergism or change in repellency is responsible for the incidents. The repellent nature of pyrethroids are important in limiting the exposure of honeybees to this highly toxic group of insecticides. Therefore it is important that this is investigated to ensure increased exposure of honeybees to pyrethroids is not occurring by reducing the repellent nature of the insecticide. This study aimed to determine if the mixing of a range of fungicides, particularly EBIs, with pyrethroids 1. altered the repellent properties of the pyrethroid and 2. increased the toxicity of the mixture and thus increased the risk to honeybees when applied to flowering crops.

The spread of varroa throughout Europe has resulted in an increasing need to resort to synthetic pyrethroid varroacides for its control in honeybee colonies. The use of these synthetic pyrethroid varroacides raises a possibility of interactions between these and fungicides transported to the colony after foraging on treated crops. Therefore this study aimed to determine whether the toxicity of the two pyrethroids used as varroacides, tau-fluvalinate (Apistan) and flumethrin (Bayvarol) was increased with concurrent exposure to a range of fungicides which may be used on flowering crops.

Repellency

A laboratory repellency test was developed in which the bees could access food only by crossing treated paper. Both the pyrethroids, alpha-cypermethrin (Contest) and lambda-cyhalothrin (Hallmark), showed significant repellency although the scale of the repellency differed. Alpha-cypermethrin (Contest) showed 80% repellency whereas lambda-cyhalothrin (Hallmark) showed only 40%. For both pyrethroids three fungicides significantly decreased the repellency of each of the pyrethroids, but they were not the same fungicides. Chlorothalonil (Bravo), difenoconazole (Plover) and tebuconazole (Folicur) significantly decreased the repellency of alphacypermethrin (Contest). Prochloraz (Sportak), flusilazole (Sanction) and propiconazole (Tilt) significantly decreased the repellency of lambda-cyhalothrin (Hallmark). The greatest decline in repellency was observed with alpha-cypermethrin and chlorothalonil (Bravo) where repellency was significantly reduced (p<0.001) from a mean of 80% to a mean of 6%.

Toxicity

The increases in toxicity of the pyrethroid pesticides a-cypermethrin and l-cyhalothrin and varroacides tau-fluvalinate and flumethrin were assessed both with the active ingredients and formulations. The greatest increase in toxicity of the active ingredients was observed for prochloraz in combination with fluvalinate (53 fold) with prochloraz resulting in increases of at least 10 fold with all pyrethroids, and flusilazole increasing the toxicity of fluvalinate and flumethrin at least 19 fold.

The LD50 for the formulated alpha-cypermethrin and l-cyhalothrin and their combination with formulated fungicides were also calculated. The maximum increase in toxicity (decrease in LD50) observed was a 6.7 fold increased in the toxicity of l-cyhalothrin in the presence of prochloraz. Six of the eight fungicides increased the toxicity of l-cyhalothrin and three increased the toxicity of alpha-cypermethrin. The maximum increase observed in combination with alpha-cypermethrin was 2.2 fold with prochloraz.

Chlorothalonil + alphacypermethrin

In 2002/03 a semi-field trial was undertaken with Bravo (chlorothalonil) and Contest (alphacypermethrin) which was identified as decreasing the repellency shown by the pyrethroid in laboratory studies. Mortality levels after pyrethroid and mixture applications were higher than controls and the mixture application resulted in increasing mortality over days 5-7. Lower foraging activity was observed immediately after application of all the pesticides and was continued throughout the day for Contest but not after application of the Bravo/Contest mixture or after application of Bravo alone. Activity was also lower at the first observation on the day after spraying for the Contest treated tunnels but not for the other treatments. Interestingly foraging activity in both the Contest and Contest + Bravo treated colonies was apparently elevated on days 3-7 (% day –1) compared with the Bravo treated tunnels. The data suggest that the mixture has some repellent properties but is a shorter-term and lower level effect than the pyrethroid alone.

Flusilazole + lambda-cyhalothrin

In 2003/04 a semi-field trial was undertaken with Genie (flusilazole) and Hallmark (l-cyhalothrin) which was identified as increasing the toxicity shown by the pyrethroid in laboratory studies. Immediately after application (day 0 pm) mortality levels the mixture applications were higher than those of the pyrethroid alone which were higher than controls and the mixture application continued to show higher levels of mortality on days 1 and 2. The increased mortality following with the application of the mixture was observed despite far lower levels of foraging activity in these tunnels (mean 22% pre-trial). Application of Hallmark resulted in lower levels of foraging immediately post-foraging but recovered to pre-trial levels during the following assessment and was a mean of 59% pretrial. The flusilazole treated tunnels showed no apparent effects on foraging immediately post-application (mean 91% pre-trial). Activity was similar in all tunnels on the day after spraying. The data suggests that the decreased foraging observed in the mixture treated tunnels was insufficient to prevent higher levels of mortality due to the synergy of the mixture.

Fungicides + varroacides

The effects of exposure to the fungicides on the toxicity of the varroacide formulations Apistan and Bayvarol and the agricultural formulation Mavrik were investigated. The increase in toxicity was expressed qualitatively as the fold increase in toxicity was dependent on the level of mortality shown in the presence of the varroacide alone, which in turn was dependent on the level of contact of the bees with the varroacide strips within the cages. The effect of exposure to fungicides and varroacides showed that synergy may occur at levels below the application rate in the field. However, it must be borne in mind that the exposure to varroacides in this study was extreme with bees confined to cages containing the varroacide strips. However, the studies with active ingredients showed that the highest levels of synergy were observed with the varroacide active ingredients, tau-fluvalinate and flumethrin with the fungicides prochloraz and flusilazole and all the fungicides showed at least a 2 fold increase in toxicity. The fungicides which are of most concern in the formulation studies were tebuconazole, difenoconazole, flusilazole and propiconazole all of which showed high levels of mortality at rates well below the maximum application rate.

The ranking in potential of the fungicides to increase the toxicity of the pyrethroids tested was prochloraz>flusilazole> propiconazole> tebuconazole>carbendazim=difenoconazole > thiophanate-methyl on the basis of active ingredients. It is interesting that thiophanate –methyl has lower potential to increase the toxicity of pyrethroids than carbendazim as it is the precursor. In terms of the pyrethroids the greatest increase in toxicity was shown by fluvalinate>flumethrin >alphacypermethrin>lambda-cyhalothrin. This variation in relative effect on the toxicity of the pyrethroids is probably due to the relative importance of the three main pathways of pyrethroid metabolism – hydrolysis, oxidation and conjugation. Increased levels of synergy are mostly likely to be observed with EBI fungicides for those pyrethroids where oxidation is a major pathway. The relative importance of these pathways varies not only between pyrethroids but also between insect species and may vary with the age of bees due to the development of detoxication pathways with age.

The effects of the observed increase in toxicity and decrease in repellency in the laboratory were combined to assess the effects on the risk posed by applications of mixtures compared with pyrethroids alone . This suggests that the risk posed by application of prochloraz with l-cyhalothrin is 11 fold greater than l-cyhalothrin alone (due to increased toxicity) and the risk posed by application of chlorothalonil and alphacypermethrin is 10 fold greater than alphacypermethjrin alone (due to decreased repellency). The effects of chlorothalonil and cypermethrin and flusilazole and lambda-cyhalothrin were assessed under semi-field conditions. Addition of chlorothalonil to alphacypermethin appeared to decrease the time for which alphacypermethrin was repellent. Addition of flusilazole to lambda-cyhalothrin increased the levels of mortality although it appeared to decrease foraging activity immediately post-application, suggesting no adverse effects on repellency.

The data from both the laboratory and semi-field trials suggest that the addition of fungicide formulations to pyrethroids affect the repellency of the mixtures but this requires confirmation at the field scale. The observation of an effect with a non-EBI fungicide raises the possibility that any tank mix may produce such an effect on repellency.

The increased toxicity of l-cyhalothrin in the presence of prochloraz and propiconazole were slightly lower than those to that previously reported for formulations (9 fold and 16 fold respectively) This is probably due to the lower ratio of fungicide used in this study 1:30 vs 1:50 for prochloraz, 1:8 vs 1:16 for propiconazole . The mechanism of synergism between l-cyhalothin and prochloraz has previously been reported as delayed metabolism, detoxication and excretion of the pyrethroid by inhibition of microsomal oxidation. The level of synergy was also different between the ai and formulated product highlighting the role that co-formulants may play in synergy.

The results of this study show that not only synergy but also repellency should be taken into account in assessing the risk of tank mixes of pyrethroids with other pesticides. This is particularly important with pyrethroids which are highly toxic and where repellency is key in reducing the risk posed by the pesticide such as alphacypermethrin. However, even when repellency was observed increased mortality occurred in a semi-field study with lambda-cyhalothrin and flusilazole. In this case the results were similar in the laboratory and semi-field studies with a 2 fold increase in toxicity observed in the laboratory and an increase from 20 to 47 dead bees (2.4 fold increase) observed on the day after spray application in the semi-field trial whilst in the laboratory an increase of 2.5 fold was observed with the active ingredients and 2.2 fold with the combined formulations. Repellency has been reported to occur at 1-4 ng/bee for permethrin.

The methodology developed in this study and the results observed suggests that the repellency and increased toxicity of tank mixes should be more widely assessed to determine the increased risk to non-target wildlife. As all combinations cannot be tested routinely a model for screening for potential effects, e.g. development of QSAR, should be considered based on mode of action, e.g. P450 inhibition and pyrethroid toxicity to identify possible issues. As in most cases higher levels of toxicity were observed with active ingredients than formulated products this may be the most appropriate route until the role of co-formulants is more fully understood. Synergy due to inhibition of oxidation is not restricted to the pyrethroid insecticides. A recent report showed that piperonyl butoxide, triflumizole and propiconazole increased the toxicity of the cyano-substituted neonicotinoid insecticides acetamiprid and thiacloprid by up to 1141 fold but had no effect on the toxicity of imidacloprid and no apparent effect under extended laboratory conditions.

CSG 15 (Rev. 6/02) 2

Project
title / Assessing the impact of mixtures of pyrethroids and fungicides on honeybees
/ DEFRA
project code / PN0945
Scientific report (maximum 20 sides A4)
To tab in this section press the tab key and the Control key together
Press the DOWN arrow once to move to the next question.

CSG 15 (Rev. 6/02) 2

Project
title / Assessing the impact of mixtures of pyrethroids and fungicides on honeybees
/ DEFRA
project code / PN0945

1. Introduction

There has been considerable concern over the last few years, including consideration by the Environmental Panel, over the potential for synergism between pyrethroids and fungicides applied to oilseed rape (Thompson, 1996). In response to the concerns of the Environmental Panel there is work within WIIS to identify residues of fungicides in submitted honeybee samples and thus whether synergism is responsible for incidents. However, incidents reported through the Wildlife Incident Scheme have, according to field data, included mixtures of pyrethroids with fungicides which are not synergistic under laboratory conditions, i.e. chlorothalonil and alphacypermethrin. Interpretation and regulation may be difficult if it is unclear whether synergism or change in repellency is responsible for the incidents. The repellent nature of pyrethroids are important in limiting the exposure of honeybees to this highly toxic group of insecticides. Therefore it is important that this is investigated to ensure increased exposure of honeybees to pyrethroids is not occurring by reducing the repellent nature of the insecticide, i.e. the risk assessment based on repellency is valid. This study aimed to determine if the mixing of fungicides with pyrethroids 1. altered the repellent properties of the pyrethroid and 2. increased the toxicity of the mixture and thus increased the risk to honeybees when applied to flowering crops. The study assessed effects in the laboratory to identify combinations to be tested under semi-field conditions, tunnels containing bee colonies and treated Phacelia.

Fungicides are widely used on flowering crops and are generally of low toxicity to honeybees. The spread of varroa throughout Europe has resulted in an increasing need to resort to synthetic pyrethroid varroacides for its control in honeybee colonies. The widescale use of pyrethroid varroacides has resulted in the development of resistance in varroa in many countries (including the UK in 2001) and use of other chemical controls. Pyrethroid varroacides are generally used in the spring and/or autumn in the UK. Treatment involves placing strips containing relatively high levels of the pyrethroid in the brood chamber of the honeybee colony for 6 weeks. The pyrethroid varroacide fluvalinate is regularly detected in the incidents reported to WIIS due to its use as a varroacide in honeybee colonies. The use of these synthetic pyrethroid varroacides raises a possibility of interactions between these and fungicides transported to the colony after foraging on treated crops. The exposure of bees to high levels of fungicides may affect their susceptibility to the toxic effects of the pyrethroid which are normally considered safe. Therefore this study aimed to determine whether the toxicity of the two pyrethroids used as varroacides, tau-fluvalinate (Apistan) and flumethrin (Bayvarol) was increased with concurrent exposure to a range of fungicides which may be used on flowering crops.