Chlorothalonil

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to bees: acute oral

Remarks:

and toxicity to bees: acute contact

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jul 1999 to 30 Jul 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

Version / remarks:

Sep 1998

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Version / remarks:

Sep 1998

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.3020 (Honey Bee Acute Contact Toxicity)

GLP compliance:

yes

Application method:

other: contact and oral

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Tetrahydrofuran

Details on preparation and application of test substrate:

Due to the low solubility of the test substance in the organic solvents tested the top doses were made by preparing a solution containing 96.1 mg a.i./mL test substance. This solution was then further diluted to prepare the top dose (range-finding) or 50.6 mg a.i./mL (main test). All solutions were made in tetrahydrofuran and were used within 2 hours of preparation. One set of control bees were treated with tetrahydrofuran. The other set of control bees were undosed.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Source: Test bees for the tests in this study were adult worker bees (Apis mellifera L.) taken from colony 12 for the range-finding test and colony 76 for the main test owned and maintained by the testing facility for use in such tests.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

In the oral test, a single dose was given and mortality was assessed at 1 and 4 hours after dosing.

Test temperature:

25 ± 1°C

Humidity:

65 + 5% relative humidity)

Photoperiod and lighting:

Not specified

Details on test conditions:

TEST SYSTEM
- Collection of worker bees: Worker bees were collected from the hive by using a small amount of smoke, gently shaking them from the combs and transferring them (40-50 per cage) into cylindrical mesh cages.
- Holding: In the laboratory the mesh cages were placed into the incubator until needed for the test (bees used in the oral tests were starved for 1.5 to 2 hours before being used for the tests). Immediately prior to treatment each group of bees in its mesh cage was anaesthetised with carbon dioxide gas by placing the cage into a 2 litre beaker filled with CO2.
- Replication: A range of dose levels and a dosed and undosed control were used for each test, with three replicates of 10 bees per dose level.

EFFECT PARAMETRES MEASURED:
- Contact test: Mortality and sub-lethal effects were assessed at intervals of 1, 4, 24 and 48 hours after dosing.
- Oral test: Mortality and sub-lethal effects effects were assessed at 1 and 4 hours after offering doses. The glass test feeders, containing any unconsumed portions of the doses, were then removed. Fresh 50% w/v sucrose solution was then supplied in feeders within the cages and further assessments made at 24 and 48 hours after removal of the glass test feeders.
- Sub-lethal effects determination: Sub-lethal effects were assessed according to pre-determined categories: Knocked down (i.e. alive but immobile), Stumbling (i.e. moving but in a poorly co-ordinated manner)

CONTACT TEST
- Test substance: Due to the low solubility of the test substance in the organic solvents tested the top doses were made by preparing a solution containing 96.1 mg ai/mL test substance. This solution was then further diluted to prepare the top dose (range-finding) or 50.6 mg ai/mL test substance (main test). All solutions were made in tetrahydrofuran and were used within 2 hours of preparation. One set of control bees were treated with tetrahydrofuran. The other set of control bees were undosed.

- Toxic reference: On the day of the test a stock solution of 3.2 mg a.i./mL (range-finding test) or 3.1 mg a.i./mL (main test) dimethoate in deionised water containing 1g/L Triton X100 (as surfactant) was prepared and a series of dilutions made. All solutions were made in deionised water containing 1g/L Triton X100. Control bees were treated with µg/L Triton XI 00 in deionised water. Doses were used within 2 hours of preparation.

- Test method: The bees were anaesthetised with carbon dioxide immediately before dosing and were gently tipped out onto filter paper and counted into the petri dish cage (drones were discarded). Each bee was dosed on the thorax with two (test substance) or one (dimethoate) 1 µL drops of a given pesticide concentration or two 1 µL drops of tetrahydrofuran (for controls with test substance) or one 1 µL drop of water containing 1g/L Triton XI00 (for controls with toxic reference) using an Anachem EDP Plus micropipette. The lid was placed on the cage, the bees were allowed to recover and kept in the incubator with a continuous supply of 50% w/v aqueous sucrose solution as food.

ORAL TEST
- Test substance: Thus 0.5 mL of each contact test substance solution was diluted to 10 mL with 50% w/v aqueous sucrose solution and each group of 10 bees were offered 0.25 mL test substance in a glass feeder. The maximum dose in the main run oral test was 2.53 mg ai/mL test substance which equates to 63.3 µg ai/bee for groups of 10 bees which consumed the entire 250 µL dose. Doses were offered within 2 hours of preparation. One set of control bees were given 50% w/v aqueous sucrose solution containing 0.5 mL tetrahydrofuran, i.e. a final concentration of 5% tetrahydrofuran. Although the final solvent concentration in the test substance doses was greater than the 1% recommended in the OECD guidelines, this level of solvent was required to ensure the test substance remained in solution and had no apparent adverse effects on control bees. The other set of control bees were supplied with untreated 50% w/v sucrose.

- Toxic reference: Thus 0.5 mL of each contact toxic reference solution was diluted to 10 mL with 50% w/v aqueous sucrose. The maximum dose in the main run oral test was 0.030 mg ai/mL dimethoate which would equate to 0.6 µg ai/bee dimethoate for groups of 10 bees which consume the entire 200 µL dose. Doses were offered within 2 hours of preparation. Control bees were given 50% w/v aqueous sucrose solution containing 0.5 mL Triton X100 at the same concentration as in the toxic reference doses (0.05 g/L).

- Test method: The bees were anaesthetised with carbon dioxide immediately before dosing and were gently tipped out onto filter paper and counted into the petri dish cage (drones were discarded). Each group of 10 bees was offered 0.25 mL (test substance) or 0.20 mL (dimethoate) of a given concentration (or controls as above), the dose being measured into a small, pre-weighed, glass feeder within the cage using a variable volume Finnpipette. After 4 hours the glass feeders were removed and weighed and the sucrose feeders filled with approximately 3 mL 50% w/v aqueous sucrose so that bees had continuous access to sucrose for the remainder of the study. The dose consumed was determined by comparison of the weight of the dose remaining in the glass feeders with the weight of a known volume of the test solutions.



RANGE-FINDING STUDY
A range-finding and a main run test, each comprising one contact and one oral test, were performed. The range-finding test was used to determine the dose levels to be used in the main test. As less than 50% mortality was observed in the range-finding test the number of dose levels in the main test was changed so as to repeat the rangefinding test. One contact and one oral test using dimethoate as the toxic reference were run concurrently.

Nominal and measured concentrations:

- Nominal concentrations contact test: 0 (solvent control), 0.101, 1.01, 10.1 and 101 µg a.i./bee
- Nominal concentrations oral test: 0 (solvent control), 0.0633, 0.633, 6.33 and 63.3 µg a.i./bee

Reference substance (positive control):

yes

Remarks:

Dimethoate

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 101 µg per animal

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Contact test

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 63 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Oral test

Details on results:

- Test solutions: The test substance at 100 mg ai/ mL test substance came out of solution rapidly at room temperature. Suspensions in acetone or ethanol were less stable than in DMSO or tetrahydrofuran. At 20 mg ai/mL test substance in acetone and ethanol suspensions were formed but the test substance dissolved in DMSO and in tetrahydrofuran. At 50 mg ai/mL a more stable suspension was formed in tetrahydrofuran and therefore this was selected as the highest concentration that could be used in the tests. Due to rapid precipitation the dispersion test was not conducted over 1 hour. All solutions were mixed well immediately prior to use, thus ensuring that the bees were exposed to the maximum doses.

CONTACT TEST
The results show that the 24-hour and 48-hour LD50 levels are greater than 101 µg ai/bee the test substance. The NOEL is also at least 101 µg ai/bee the test substance at both 24 and 48 hours. Control mortality was less than 10% at 24 or 48 hours. The 48-hour results were similar to those at 24 hours, indicating there were no delayed effects. These results show a toxicity level within the OECD guidelines and similar to those previously reported within the National Bee Unit, indicating that the bees were reacting normally in the present tests.

ORAL TEST
For the test substance, the 24-hour and 48-hour LD50 levels are greater than 63 µg ai/bee based on actual intake data. The NOEL is also at least 63 µg ai/bee test substance at both 24 and 48 hours. Control mortality was less than 10% at 24 or 48 hours. The 48-hour results were similar to those at 24 hours, indicating there were no delayed effects. These results show a toxicity level within the OECD guidelines and similar to those previously reported within the National Bee Unit indicating that the bees were reacting normally in the present tests.

Results with reference substance (positive control):

Mortality of bees exposed to the toxic reference in main tests allowed calculation of a 48-hour LD50 of 0.27 µg dimethoate/bee (95% CL 0.22-0.37 µg ai/bee) for contact dosing and 0.21 µg dimethoate/bee (95% CL 0.18-0.25 µg ai/bee) for oral dosing

Reported statistics and error estimates:

When greater than 50% mortality was observed the mortality data from the tests with the test substance and dimethoate were analysed using the CSL probit programme (Probit 1, version 4).

Validity criteria fulfilled:

yes

Conclusions:

The 48-h LD50 values were determined to be > 101 and >63 µg/bee for acute contact and oral and contact exposure, respectively.

Executive summary:

GLP-compliant tests were carried out to determine the acute contact and oral toxicity of the test substance to adult honey bees (Apis mellifera L.). The protocol followed the EPPO guidelines (1992) and was in accordance with the draft EPA Ecological Effects Test Guidelines (OPPTS 850.3020 Honey Bee Acute Contact Toxicity) and OECD TG 213 Honeybees, Acute Oral Toxicity Test and 214 Acute Contact Toxicity. All doses and toxicity data for the test substance refer to the test substance as the active ingredient and, for the purpose of this study, have been corrected for analysed content of the test substance. Three batches of bees, in groups of 10 bees, were topically dosed on the thorax with two 1µL drops containing 50.6, 5.06, 0.506 and 0.0506 µg/bee test substance dissolved in tetrahydrofuran. Mortality and sub-lethal effects were assessed at 1, 4, 24 and 48 hours after dosing. Results indicated that the 24-hour and 48-hour contact LD50 of the test substance is greater than 101 µg/bee. The No Observed Effect Level (NOEL) is also at least 101 µg/bee. The 48-hour results were similar to the 24-hour results, indicating there were no delayed effects. Similar groups of bees were also offered the equivalent doses of 63.3, 6.33, 0.633 and 0.0633 µg/bee test substance in 50% w/v aqueous sucrose solution, the technical material having first been dissolved in tetrahydrofuran. At the highest treatment level dose groups consumed 63, 59 and 28 µg/bee test substance. Mortality was assessed at 1 and 4 hours after dosing. Glass test feeders were then removed and further assessments made at 24 and 48 hours after removal of the glass test feeders. Results indicated that the 24-hour and 48-hour oral LD50 of the test substance is greater than 63 µg ai/bee. The NOEL is also at least 63 µg/bee based on actual intake at the highest dose. The 48-hour results were similar to the 24-hour results indicating there were no delayed effects. These results indicate that the test substance is “virtually non-toxic” by contact and at worst “slightly toxic” by ingestion (based on mean maximum dose ingested) under the conditions of these tests (International Commission for Bee Botany, 1985).

Description of key information

The 48-h LD50 values were determined to be > 101 and > 63 µg/bee for acute contact and oral and contact exposure, respectively, OECD TG 213 and 214, Thompson 2000

Additional information

GLP-compliant tests were carried out to determine the acute contact and oral toxicity of the test substance to adult honey bees (Apis mellifera L.). The protocol followed the EPPO guidelines (1992) and was in accordance with the draft EPA Ecological Effects Test Guidelines (OPPTS 850.3020 Honey Bee Acute Contact Toxicity) and OECD TG 213 Honeybees, Acute Oral Toxicity Test and 214 Acute Contact Toxicity. All doses and toxicity data for the test substance refer to the test substance as the active ingredient and, for the purpose of this study, have been corrected for analysed content of the test substance. Three batches of bees, in groups of 10 bees, were topically dosed on the thorax with two 1µL drops containing 50.6, 5.06, 0.506 and 0.0506 µg/bee test substance dissolved in tetrahydrofuran. Mortality and sub-lethal effects were assessed at 1, 4, 24 and 48 hours after dosing. Results indicated that the 24-hour and 48-hour contact LD50 of the test substance is greater than 101 µg/bee. The No Observed Effect Level (NOEL) is also at least 101 µg/bee. The 48-hour results were similar to the 24-hour results, indicating there were no delayed effects. Similar groups of bees were also offered the equivalent doses of 63.3, 6.33, 0.633 and 0.0633 µg/bee test substance in 50% w/v aqueous sucrose solution, the technical material having first been dissolved in tetrahydrofuran. At the highest treatment level dose groups consumed 63, 59 and 28 µg/bee test substance. Mortality was assessed at 1 and 4 hours after dosing. Glass test feeders were then removed and further assessments made at 24 and 48 hours after removal of the glass test feeders. Results indicated that the 24-hour and 48-hour oral LD50 of the test substance is greater than 63 µg ai/bee. The NOEL is also at least 63 µg/bee based on actual intake at the highest dose. The 48-hour results were similar to the 24-hour results indicating there were no delayed effects. These results indicate that the test substance is “virtually non-toxic” by contact and at worst “slightly toxic” by ingestion (based on mean maximum dose ingested) under the conditions of these tests (International Commission for Bee Botany, 1985).