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Toxicity to terrestrial arthropods

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Reference
Endpoint:
toxicity to terrestrial arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline study used but comparable to national guidelines/standards
Qualifier:
no guideline followed
Principles of method if other than guideline:
Laboratory bioassays were performed to characterize the acute (24 h and 48 h) contact toxicity of oxalic acid to Varroa destructor (Anderson & Trueman) and their honey bee hosts (Apis mellifera L.) in a laboratory bioassay.
GLP compliance:
no
Application method:
contact
Specific details on test material used for the study:
- Name of test material (as cited in study report): Oxalid acid dihydrate (> 99% purity) (The Science Company, Denver, CO) (CAS no. 6153-56-6)
Analytical monitoring:
no
Details on sampling:
No data
Vehicle:
yes
Details on preparation and application of test substrate:
- Method of test material application: [single topical dose]
- Body part: abdomen
- Volume of test solution applied: 10.0, 8.0, 4.0, 2.0, 1.0 or 0.5 ul of a 200 mg/ml stock solution
- Controls: acetone only and a dry control in which bees were anesthetized with CO2 but were not treated with acetone
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution): determined by a preliminary range-finding bioassay
- Evaporation of vehicle before use:no data
Test organisms (species):
Apis mellifera
Animal group:
Hymenoptera (honeybees)
Details on test organisms:
TEST ORGANISM
- Common name: V. destructor (ectoparasitic mite of the bee)
A. mellifera (bee)
- Source: Mites were harvested from adult bees
Bees from C.F. Koehnen & Sons Inc. (Glenn, CA) were shipped to the University of Nebraska
- Age at test initiation (mean and range, SD): Two-to 7-d-old bees obtained from brood frames kept in an incubator
- Date of collection: shipping date of bees is March 2005, definitive bioassay was conducted in September 2005

ACCLIMATION
- Acclimation period: the boxes with bees used for the harvest of mite were stored at 15.6°C in complete darkness until needed for experimentation (no >2 d).
- Feeding: A pint jar of sugar water (1:1 by volume) was attached to each box with bees used for the harvest of mites
Ten bees were placed in each of 32 Benton mailing cages with queen candy
Study type:
laboratory study
Limit test:
no
Total exposure duration:
72 h
Remarks:
24 h exposure for mites; 24, 48 and 72 h exposure for honey bees
Post exposure observation period:
No data
Test temperature:
26°C (mites)
21.7±0.4°C (honey bees)
Humidity:
90% (mites)
46.3±1.5% (honey bees)
Photoperiod and lighting:
mites were exposed in a dark incubator
Details on test conditions:
Glass-Vial Residual Bioassays for V. destructor.
Techniques described by Plapp and Vinson (1977) and Macedo et al. (2002) were used for conducting glass-vial residual bioassays. Serial dilutions of oxalic acid dihydrate (>99% purity) (The Science Company,Denver, CO) (CAS no. 6153-56-6) in acetone were prepared, and a preliminary range-finding bioassay was conducted to determine at least three concentrations that provided V. destructor mortalities >0 and <100%. Seven concentrations of oxalic acid in acetone were prepared for the definitive bioassay (1.0, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 mg/ml). One-half milliliter of each solution was pipetted into four 20-m1 glass scintillation vials for each treatment, including an acetone control. The eight resulting oxalic acid concentrations that were tested in the definitive bioassay were 500,150,50, 15,5,1.5,0.5, and 0.0 pg oxalic acid per vial. All vials (32 total) were rolled on their sides under a fume hood to evaporate the acetone while evenly coating the vials with oxalic acid. The vials were promptly removed after the acetone had evaporated (4-5 min). Ten mites were gently brushed into each vial, the cap was screwed on tightly, and the vials were placed in a dark incubator for 24 h (26°C and 90% RH). One vial of 10 mites constituted a replication and four vials (40 mites) were used for each of the eight concentrations. Mite mortality was scored 24 h later by examining mites under a light microscope. Mites were considered dead if they did not respond to probing with asmall paint brush.

Topical Application of oxalic acid to Adult Bees.
Serial dilutions of oxalic acid dihydrate (>99% purity) (The Science Company) (CAS no. 6153-56-6) in acetone were prepared, and a preliminary range-finding bioassay was conducted to determine at least three doses that provided honey bee mortalities >0 and <100%. Two-to 7-d-old bees from C.F. Koehnen & Sons Inc. (Glenn, CA) were shipped to the University of Nebraska in March 2005 and were used for the range-finding bioassay. The results from the range-finding bioassay were used to establish the definitive bioassay dosage levels.
The definitive bioassay was conducted in September 2005 by using 2- to 7-d-old bees that were obtained from brood frames kept in an incubator. Ten bees were placed in each of 32 Benton mailing cages with queen candy. One cage of 10 worker bees constituted a replication and four cages (40 bees) were tested for each treatment. Each cage was randomly assigned to one of the eight treatments, and all 10 bees within a cage received the same treatment. A 200 mg/ml solution of oxalic acid in acetone was prepared. Honey bees were dosed with 10.0, 8.0, 4.0, 2.0, 1.0, or 0.5 µl of the 200 mg/ml stock solution. These doses correspond to 2,000, 1,600, 800, 400, 200, and 100 µg OA per bee, respectively. After bees had been anesthetized with CO2 the doses were applied to individual bee abdomens by using a Hamilton microsyringe and repeating dispenser (Hamilton Company, Reno, NV). A 10-}µl control (acetone only) was included in the bioassay along with a dry control in which bees were anesthetized with CO2 but were not treated with acetone. Bees were held at 21.7 ± 0.4°C and 46.3 ± 1.5% RH in darkness for 72 h except for brief periods when water was administered to the cages. Bees were provided water twice daily throughout the experiment by brushing the cage screens with several drops of water.
Mortality was evaluated 24, 48, and 72 h after treatment.
Nominal and measured concentrations:
nominal
Reference substance (positive control):
not specified
Key result
Duration:
24 h
Dose descriptor:
LD10
Effect conc.:
564.05 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
LD50
Effect conc.:
1 575.85 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
other: LD90
Effect conc.:
4 402.6 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LD10
Effect conc.:
176.68 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
LD50
Effect conc.:
372.01 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
48 h
Dose descriptor:
other: LD90
Effect conc.:
783.27 µg per animal
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
LD10
Effect conc.:
1.4 other: ug/20 mL (fotr mites)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
LD50
Effect conc.:
5.12 other: ug/20 mL (mites)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
24 h
Dose descriptor:
other: LD90
Effect conc.:
18.69 other: ug/20 mL (mites)
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality at end of exposure period:

V. destructor Bioassays.
The natural mortality for the definitive bioassay was 9.7 ± 3.4% after
24 h.

Honey Bee Bioassays.
Doses of oxalic acid <100,ug per bee did not cause significant mortality after 48 h in the range-finding bioassay. Furthermore, it was impossible to calculate the 24- and 48-h LDm values for honey bees tested in the range-finding bioassay because significant mortality did not occur until at least 72 b posttreatment. Aliquots of a 200 mg/ ml solution of OA in acetone were used to dose honey bees in both the range-finding and definitive bioassays, because solutions >200 mg/ ml clogged the microsyringe, making it impossible to deliver accurate doses of oxalic acid.
Only the 10-d acetone control group was used for Probit analysis of the definitive bioassay data, because the 10-µl acetone control had slightly more mortality than the dry control group. The natural mortality for the definitive bioassay was 4.0 ± 2.5% after 48 h. We were unable to calculate a confidence interval for the 72-h LDS, because all bees in the 2,000,1,600, 800, and 400 ttg of oxalic acid per bee treatments died after 72 h. The estimated 72-h LDm for honey bees was 194.89 Ag per bee, based on the 100 and 200 pg of oxalic acid per bee treatments.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
Results were analyzed by Probit analysis (Finney 1971) by using the POLO-PC statistical software (LeOra Software 1991), and natural
mortality was taken into account. The concentrations used for Probit analysis of mite bioassay data were expressed as micrograms per vial. The doses used for Probit analysis of honey bee bioassay data were expressed as micrograms per bee.
Validity criteria fulfilled:
yes
Conclusions:
The 24 h LD 50 was 1575.85 ug/bee, the 48 h LD50 was 372.01 ug/bee.
Executive summary:

Laboratory bioassays were performed to characterize the acute contact toxicity of oxalix acid to the mite Varroa destructor and their honey bee hosts (Apis mellifera L.).

Mites (Varroa destructor) and bees (Apis mellifera) were exposed to oxalic acid during 24 to 48 hours according to no specific guideline. A 24 h LD 50 of 1575.85 ug/bee and of 5.12 ug/20 ml (mites) was determined. The 48 h LD50 was 372.01 ug/bee.

Description of key information

A 24 h LD 50 of 1575.85 ug/bee is reported.

Key value for chemical safety assessment

Additional information