2-Pyridinecarboxylic acid, 4-amino-3,6-dichloro-

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8 August 2001 to 10 August 2001

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)

Deviations:

no

GLP compliance:

yes

Application method:

oral

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

TEST SOLUTION PREPARATION
For definitive testing, a 6.0 mg a.i./mL primary stock was prepared by adding 0.0635 g of test material to a 10-mL volumetric flask and bringing the flask to volume with a 500 g/L (w/v) sucrose solution. A 50 % dilution series was prepared starting with the primary stock to prepare stocks of 3.0, 1.5 and 0.75 mg a.i./mL. Each stock was prepared with a 500 g/L sucrose solution.

TEST SOLUTION ADMINISTRATION
The bees for the definitive test were starved for approximately 1.75 hours prior to the introduction of pre-weighed feeders (1000-μL pipette tips) containing approximately 200 μL of the appropriate sucrose solution control, or test material-dosed sucrose solutions. Consumption of 100 % of the dosing solutions by the 10 bees in each cage would result in final test dosages of (0.0), 6.0, 15, 30, 60, and 120 μg a.i./bee.
The test was initiated after the bees were given 6 hours to consume the food solutions presented to them. All control and treatment feeders were then removed and the bees were offered ad libitum a 500 g/L sucrose solution until test termination. The feeders were re-weighed to determine the actual amount of test material ingested per replicate.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: honey bee
- Disease free: yes
- Kept according to standard practices: yes

The bees for the definitive test were collected the morning of the definitive test from a single, disease-free colony. The bees were maintained in a clean holding cage at a temperature of approximately 25 °C and at approximately 50 to 80 % relative humidity.

During holding/acclimation and testing, bees were provided ad libitum a 500 g/L (w/v) sucrose solution. The feeding solutions were prepared as needed. The feeding device consisted of a tightly rolled piece of filter paper which was kept saturated with the sucrose solution. One feeder was placed into each cage.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

48 h

Test temperature:

24.8 - 25.6 °C

Humidity:

53 - 67 % (relative)

Photoperiod and lighting:

The chamber was kept dark except when room lighting was used during observation periods.

Details on test conditions:

TEST SYSTEM
Tests were conducted in plastic, screened bee cages (approximately 14 cm wide x 20 cm long x 10 cm high). Prior to test initiation, the chambers were cleaned according to SOP of the test laboratory. The bees were impartially placed into the test chambers by allowing the bees to enter the test chamber at will from the holding chamber via a plastic connecting tube. When at least 10 bees had entered the test chamber it was sealed and a new test chamber was connected to the holding chamber. This process was repeated until all test chambers held at least 10 bees. Extra bees were removed until each chamber contained 10 bees. Treatments were then assigned to each cage. The control and all treatment levels for the definitive test were prepared in triplicate for a total of 30 bees per nominal treatment.

EFFECT PARAMETERS MEASURED
Mortality and behavioural observations were performed at 4, 24, and 48 hours (± 1 hour) after the treated diet had been removed from the cages.

VEHICLE CONTROL PERFORMED: yes

PRELIMINARY TEST
A preliminary test was conducted with nominal test material dosages of 0.10, 1.0, 10, and 100 μg a.i./bee. After the feeding period the control bees had consumed 100 % of their food. Food consumption in the treatments ranged from 74 to 100 % with the highest consumption rates at the lowest test material concentrations. Final dosages considering percent of food eaten ranged from 0.10 to 86 μg a.i./bee. Mortality after 48 hours was 0 % in the treatments and in the control. Based on the preliminary results, the definitive nominal test dosages were set. A multi-concentration definitive test was chosen to demonstrate palatability of the test material and to test at the maximum dose possible.

Nominal and measured concentrations:

Control, 6.0, 15, 30, 60 and 120 µg a.i./bee (nominal)
Calculated dosages ranged from 5.8 to 120 µg a.i./bee

Reference substance (positive control):

yes

Remarks:

dimethoate (0.020, 0.20, and 0.40 μg/bee assuming 100 % consumption of the treated diet)

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 120 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

Consumption of the treated diets ranged from 25 to 100 %. Calculated test material dosages ranged from 5.8 to 120 μg a.i./bee.
Mortality of Apis mellifera 48 hours after consumption of the test material ranged from 0 to 10 % with no dose-response. Overall control mortality was 3 %.

Results with reference substance (positive control):

Consumption of the reference toxicant diets ranged from 7 to 100 %. Consumption of the control solutions was 100 %. Calculated dimethoate dosages ranged from 0.020 to 0.42 μg/bee.
Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments. Overall control mortality was 7 %. The 24-hour estimated LD50 for dimethoate was 0.083 μg/bee. This value was outside of the published range (0.10 - 0.35 μg/bee) for the toxicity of dimethoate to honey bees, however, the value is consistent with historical dimethoate LD50 values obtained at the test laboratory (0.047 - 0.12 μg/bee).

Reported statistics and error estimates:

Due to the results of the test, statistical analysis of the test material treatment data was not performed. The Probit method was performed using SAS to define the 24-hour LD50 value for dimethoate.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 48-hour LD50 was estimated to be in excess of 120 µg a.i./bee.

Executive summary:

The toxicity of the test material to honeybees was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 216.

During the study bees were fed test material, in diet (sucrose solution), at doses of 0 (sucrose solution control), 5.8, 6.4, 13, 15, 16, 23, 30, 31, 33,. 49, 110, and 120 µg a.i./bee. The test was initiated after the bees were given 6 hours to consume the food solutions presented to them. The bees were incubated for 48 hours in a temperature-controlled chamber at 25 ± 2 °C. Mortality and behavioural observations were performed at 4, 24, and 48 hours (± 1 hour) after the treated diet had been removed from the cages.

A toxic standard test, using dimethoate, was conducted concurrently with the test material definitive test as a routine check on the response of the animals to a known toxicant. Treatments consisted of a control and nominal dimethoate dosages of 0.020, 0.20, and 0.40 μg/bee assuming 100 % consumption of the treated diet. Each treatment was replicated three times and each replicate cage contained 10 bees.

Mortality of Apis mellifera 48 hours after consumption of the test material ranged from 0 to 10 % with no dose-response. Overall control mortality was 3 %. Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments. Overall control mortality was 7 %. The 24-hour estimated LD50 for dimethoate was 0.083 μg/bee which was within historical dimethoate LD50 values obtained at the test laboratory (0.047 - 0.12 μg/bee).

Therefore, under the conditions of the study, the 48-hour LD50 was estimated to be in excess of 120 µg a.i./bee.

Reference 2

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2001 to 16 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 141-1 (Honey Bee Acute Contact Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Deviations:

no

GLP compliance:

yes

Application method:

contact

Specific details on test material used for the study:

Purity: 94.5%

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

TEST SOLUTION PREPARATION
For the definitive test, a 100 mg a.i./mL primary stock was prepared by adding 1.0582 g of test material to a 10-mL glass volumetric flask and bringing the flask to volume with acetone. The solution was cloudy with a light brown tint.

TEST SOLUTION APPLICATION
A 1 μL drop of the appropriate test or control solution was applied to the dorsal side of the thorax of each bee. Prior to dosing the bees in a cage, the entire cage was placed into a CO₂ atmosphere to anaesthetise the bees. After the bees were anaesthetised, the cage was opened and the appropriate test or control solution was administered. Any extra bees were removed at this time in order to have only ten bees in each cage during the observation period. The cage was then closed.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: honey bee
- Disease free: yes
- Kept according to standard practices: yes

The bees for the definitive test were collected the morning of the definitive test from a single, disease-free colony. The bees were maintained in a clean holding cage at a temperature of approximately 25 °C and at approximately 50 to 80 % relative humidity.

During holding/acclimation and testing, bees were provided ad libitum a 500 g/L (w/v) sucrose solution. The feeding solutions were prepared as needed. The feeding device consisted of a tightly rolled piece of filter paper which was kept saturated with the sucrose solution. One feeder was placed into each cage.

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24.8 - 25.2 °C

Humidity:

55 - 70 % (relative)

Photoperiod and lighting:

The chamber was kept dark except when room lighting was used during observation periods.

Details on test conditions:

TEST SYSTEM
Tests were performed in plastic, screened bee cages (approximately 14-cm wide x 20-cm long x 10-cm high). Prior to test initiation, the chambers were cleaned according to the test laboratory’s SOP. The bees were impartially placed into the test chambers by allowing the bees to enter the test chamber at will from the holding chamber via a plastic connecting tube. When at least 10 bees had entered the test chamber it was sealed and a new test chamber was connected to the holding chamber. This process was repeated until all test chambers held at least 10 bees. The controls and the definitive test material treatment were prepared in triplicate for a total of 30 bees per treatment.

EFFECT PARAMETERS MEASURED
Mortality and behavioural observations were performed at 4, 24, and 48 hours (± 1 hour) after the treatment had been administered.

VEHICLE CONTROL PERFORMED: yes

PRELIMINARY TEST
A preliminary test was conducted and utilised test material doses of 0.1, 1.0, 10, and 100 μg ai/bee. An acetone application (vehicle) control and a water application control were also included in the test design. Each control and test material treatment consisted of one replicate with 10 bees per replicate. After 48 hours, mortality was 0 % at all treatment levels tested. Control mortality was also 0 %. These results were used to define the definitive test as a limit test at 100 μg active ingredient (a.i.)/bee.

Nominal and measured concentrations:

100 μg active ingredient (a.i.)/bee

Reference substance (positive control):

yes

Remarks:

dimethoate (0.020, 0.20, and 0.40 μg/bee)

Key result

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

Mortality in the 100 μg formulation/bee treatment was 0 % at the conclusion of the 48-hour test. Control and vehicle control mortality was 0 %. There were no observations of sublethal effects during the test.

Results with reference substance (positive control):

Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments. Control and vehicle control mortality was 3 and 0 %, respectively. The 24-hour estimated LD50 for dimethoate was 0.063 μg/bee. This value was outside of the published range of LD50 values (0.10 - 0.35 μg/bee) for the toxicity of dimethoate to honey bees, however, the value is consistent with historical dimethoate LD50 values obtained at the test laboratory (0.019 - 0.16 μg/bee).

Reported statistics and error estimates:

Due to the results of the test, no statistical analyses were performed on the test material treatment data. The Probit method was performed using SAS to define the LD50 values for dimethoate.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the estimated 4-, 24- and 48-hour LD50 values were >100 μg ai./bee, respectively.

Executive summary:

The toxicity of the test material to honeybees was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 141-1 and OECD 214.

During the study, bees were treated with 1 µL of the test (100 µg a.i.) or control (acetone) solutions to the dorsal side of the thorax of each bee. A reference substance, dimethoate was included as toxic control.

Mortality in the 100 μg formulation/bee treatment was 0 % at the conclusion of the 48-hour test. Control and vehicle control mortality was 0 %. There were no observations of sublethal effects during the test. Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments.

Therefore, under the conditions of the study, the estimated 48-hour LD50 value was >100 μg a.i./bee.

Description of key information

ACUTE CONTACT TOXICITY
48 h LD50 = >100 µg a.i./bee (honey bee), EPA OPP 141-1,  OECD 214, Auferheide (2004)
ACUTE ORAL TOXICITY
48 h LD50 = > 120 µg a.i./bee (homey bee), OECD 216, Auferheide (2001) 

Additional information

Two studies, investigating the toxicity of the test material to the terrestrial arthropod, Apis mellifera, are available. The first was an acute contact toxicity test and the second an acute oral toxicity test. Both studies were conducted under GLP conditions and in accordance with standardised guidelines. Both studies were assigned a reliability score of 1 in line with the criteria of Klimisch et al. (1997).

In the first study, reported by Auferheide (2004) the toxicity of the test material to honeybees was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines EPA OPP 141-1 and OECD 214.

During the study, bees were treated with 1 µL of the test (100 µg a.i.) or control (acetone) solutions to the dorsal side of the thorax of each bee. A reference substance, dimethoate was included as toxic control.

Mortality in the 100 μg formulation/bee treatment was 0 % at the conclusion of the 48-hour test. Control and vehicle control mortality was 0 %. There were no observations of sublethal effects during the test. Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments.

Therefore, under the conditions of the study, the estimated 48-hour LD50 value was >100 μg a.i./bee.

In the second study, reported by Auferheide (2001) the toxicity of the test material to honeybees was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 216.

During the study bees were fed test material, in diet (sucrose solution), at doses of 0 (sucrose solution control), 5.8, 6.4, 13, 15, 16, 23, 30, 31, 33,. 49, 110, and 120 µg a.i./bee. The test was initiated after the bees were given 6 hours to consume the food solutions presented to them. The bees were incubated for 48 hours in a temperature-controlled chamber at 25 ± 2 °C. Mortality and behavioural observations were performed at 4, 24, and 48 hours (± 1 hour) after the treated diet had been removed from the cages.

A toxic standard test, using dimethoate, was conducted concurrently with the test material definitive test as a routine check on the response of the animals to a known toxicant. Treatments consisted of a control and nominal dimethoate dosages of 0.020, 0.20, and 0.40 μg/bee assuming 100 % consumption of the treated diet. Each treatment was replicated three times and each replicate cage contained 10 bees.

Mortality of Apis mellifera 48 hours after consumption of the test material ranged from 0 to 10 % with no dose-response. Overall control mortality was 3 %. Mortality in the reference toxicant test ranged from 0 to 100 % in the individual dimethoate treatments. Overall control mortality was 7 %. The 24-hour estimated LD50 for dimethoate was 0.083 μg/bee which was within historical dimethoate LD50 values obtained at the test laboratory (0.047 - 0.12 μg/bee).

Therefore, under the conditions of the study, the 48-hour LD50 was estimated to be in excess of 120 µg a.i./bee.