2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Toxicity to terrestrial arthropods

Administrative data

Endpoint:

toxicity to bees: acute oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study initiated/completed: 15 May 2001 to 18 January 2002.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Bees are not considered for the risk assessment under REACH (NB are not considered to be terrestrial according to ECHA)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPPO 22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

oral

Test material

Test material information

Constituent 1

Reference substance name:

2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

EC Number:

264-980-3

EC Name:

2-chloro-N-[[[4-(trifluoromethoxy)phenyl]amino]carbonyl]benzamide

Cas Number:

64628-44-0

Molecular formula:

C15H10ClF3N2O3

IUPAC Name:

3-(2-chlorobenzoyl)-1-[4-(trifluoromethoxy)phenyl]urea

Test material form:

liquid

Details on test material:

Appearance: liquid / white to light beige
Storage: ambient temperature, dark

Sampling and analysis

Analytical monitoring:

not specified

Details on sampling:

This method is proposed for testing the side-effects of plant protection products on honey bee brood. Most attention was directed to the development of the bee brood because Triflumuron SC 480 is known as an insect growth-regulator. Treatment rate per colony:
Test substance (T1 ) - 41.7 mg a.s.
Test substance (T2) - 7.5 mg a.s.
Test substance (T3) - 2.5 mg a.s.
Toxic standard - 25 mg a.s. (100mg product)

Test substrate

Vehicle:

yes

Remarks:

Water and sugar solution

Details on preparation and application of test substrate:

The test substance solution containing Triflumuron SC 480 was prepared shortly before feeding in the laboratory of the testing facility at the test concentration of 41.7 mg a.s. in 1 L sugar solution in the first run and at the concentrations of 7.5 mg a.s. and 2.5 mg a.s. in 1 L sugar solution in the second run.
For the test substance treatment the specific amount of Triflumuron SC 480 was first dissolved in water and afterwards the water / test substance solution was mixed with a definite amount of a 50 % (w/v) aqueous sucrose solution in order to get the feeding solution. The colonies of the untreated control group were fed with pure sugar solution and the colonies of the toxic standard treatment were fed with sugar solution containing 25 mg a.s Insegar 25 WG in 1 litre A quantity of 1 L of the test solution was offered by placing a feeding container with the test solution into each colony. The feeding was started simultaneously for all colonies and lasted until the solution was
consumed.

Test organisms

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

The honey bee is an important beneficial insect due to its pollination activity in fruit, berry and seed growing. Due to the specific use of honey bees in the crops to be pollinated (migratory beekeeping) they are an irreplaceable productive factor. In addition to this, they contribute to the preservation of a multitude of wild flowering plants because of their high constancy in pollination activity.

Study design

Study type:

field study

Limit test:

no

Total exposure duration:

21 d

Test conditions

Humidity:

Climatic conditions during the 1st run of the test ranged from 54.8 - 89.7

Details on test conditions:

Study design: There were 3 replicates at each treatment level (2.5, 7.5 and 41.7 mg a.s./colony, T3, T2, T1 respectively).

Observations:

Mortality of Adult Honey Bees and Pupae: Bee traps with gauze on bottom and on 50 % of the top were attached to the entrance of the hives in order to register those dead bees and pupae which were carried out of the hives by the bees. The mortality of adult honey bees and pupae in the bee trap at the entrance of the colonies was assessed prior to (evaluations once a day), during (evaluations shortly before feeding), and after feeding (once a day).

The observations of the flight intensity at the hives was determined at the day of the start of feeding (shortly before feeding) and after (once a day during flight activity). The number of worker bees entering and leaving the hive during a period of 60 second was counted.

The condition of the colonies and the development of the bee brood was checked approx. 24 hours before the start of feeding and on day +3, +7, +14 and, +21 (± 1 day, respectively) after the start of feeding. In order to record effects of the test substance, the following parameters were assessed in all treatments:

• Strength of the colony: Number of combs covered by adult bees
assessed as number of gaps between the combs filled with bees
directly after opening of the hive.
• Presence of a healthy queen.
• Estimate of comb area containing eggs, larvae and capped cells
(in %)
• Drawing up of folio plans (using overhead folio) from pin marked
areas on different comb sides (with at least 100 brood cells); the
different brood stages were assessed by using different coloured
pens or using different symbols.

For the individual brood assessment a brood comb was taken out of
each colony at the first brood assessment before application to mark
areas with at least 100 cells containing eggs or young larvae. The first
folio was attached to the wooden frame with drawing pins and the outline of the chosen area was copied to the folio by using a permanent pen. After the assessment the folio was removed from the frame and the brood comb was returned to the hive. At the following assessment the folio of the previous assessment was used as a template for the current assessment and both folios together (the new one above) were attached in the same position to the same frame again and the new observations (content of each cell) were marked on the new folio. This enabled to follow the pre-imaginal development which lasted 21 days in average. The different brood stages were coded by using different
coloured pens and different signs. The development stages were valued from 0 to 5 as follows:

0: Empty at the beginning / termination of the development
1: Egg stage
2: Larval stage
3: Coiled larval
4: Pupal stage
5: Empty after the hatch

Cells filled with nectar and pollen were titled as "N" and "P" and the respective cells were not included in further evaluations. The actual stage of development for at least 100 cells per colony of the same brood area was recorded during each brood assessment date. To see the development in each treatment group and to compare the different treatments a brood index was calculated. The values of all cells in each treatment group, assessed at the same date, were summed up and divided by the number of observed cells in order to obtain the brood index. Assumed that at the first assessment preferably eggs or young
larvae were marked the brood index can be expected to be low (between 1 and 2) at the first assessment. An increase of the brood index during the following two or three assessments can be observed if a normal development of the brood is presumed. This increase is caused by the development from egg to larval stages to a pupae and finally to the adult, emerged bee and furthermore due to the rising numbers which are assigned to the brood stages (as described above).

Behaviour of the Bees at the Entrance of the Hives: In addition to the assessments of mortality and brood development, the behaviour of the bees around the hive was observed on the days before as well as after start of feeding once a day for the entire test period.

Results and discussion

Toxic reference:

Fenoxycarb.

Effect concentrations

Key result

Remarks on result:

not measured/tested

Remarks:

Bees are not considered for the risk assessment under REACH (NB are not considered to be terrestrial according to ECHA)

Details on results:

Increased mortality of pupae was seen at 41.7 mg/colony; slightly increased mortality was seen at 7.5 mg/colony.
No effects were seen on flight intensity in any groups
Bee brood development was markedly affected at 41.7 mg/colony
No effects on behaviour were seen in bees treated with 2.5 or 41.7 mg/colony. At 7.5 mg/colony, an increased number of bees with coordination problems, deformed legs and wings were seen

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

This study was performed to investigate the effects of the product Triflumuron SC 480 on the honey bee (Apis mellifera) under field conditions. The test material was mixed with sugar solution at concentrations of 25, 7.5 and 41.7 mg a.s./colony and was directly fed to the test colonies. Effects of treatment were assessed over a period of 21 days.
Increased mortality of pupae was seen at 41.7 mg/colony; slightly increased mortality was seen at 7.5 mg/colony.
No effects were seen on flight intensity in any groups
Bee brood development was markedly affected at 41.7 mg/colony
No effects on behaviour were seen in bees treated with 2.5 or 41.7 mg/colony. At 7.5 mg/colony, an increased number of bees with coordination problems, deformed legs and wings were seen.

Executive summary:

The study was designed to determine the effects of Triflumuron SC 480 on the honey bee (Apis mellifera L.) under field conditions. The study was carried out in Germany near Pforzheim at the test location Niefern.The test substance Triflumuron SC 480 was mixed with 50 % aqueous sugar solution at three concentrations (41.7 mg a.s.; 7.5 mg a.s. and 2.5 mg a.s. per colony) and was directly fed to the test colonies. The control group was fed with pure sugar solution and sugar solution containing Insegar 25 WG was offered to the colonies of the toxic standard treatment at a concentration of 25 mg a.s./hive. A quantity of 1 L test solution was offered to each colony. Two runs were performed with tree colonies (replicates) per treatment group. In the 1st run (30MAY2001 - 20JUN2001) the test substance treatment T1 (41.7 mg a.s./colony), the control and the toxic standard treatment was tested. In the 2ncr run (28JUN2001 - 19JUL2001) two additional test substance concentrations (T2: 7.5 mg a.i./colony and T3: 2.5 mg a.s./colony) were tested together with another control group and another toxic standard treatment. The mortality of bees and pupae, the condition of the colonies, the development of the bee brood and the behaviour of the bees were assessed over a period of 21 days. The flight activity in front of the hive was observed on three assessment days. The influence of the test substance Triflumuron SC 480 to the bees and their brood was evaluated by comparing the bees of the test substance treatment to the control bees and those treated with the toxic standard.

Effect on honey bee mortality:

The feeding of the test solution containing Triflumuron SC 480 (T1: 41.7 mg a.s./colony) did not cause an acute increased mortality in the first run but the number of dead bees counted in the test substance treatment group T1 was higher compared to the control group on DAF 13 to 15 and on DAF 21. In the test substance treatment group T1 (41.7 mg a.s./colony) a maximum mean mortality of 123.3 dead bees/colony /day was found on DAF 14. From DAF 16 until DAF 20 the mean number of dead adult bees was on a low level. The total number of dead pupae which were found in front of all hives of the Triflumuron SC 480 treatment group T1 over the entire postfeeding period was increased (232 dead pupae) compared to the control (10 dead pupae). In the toxic standard treatment a total number of 1977 dead pupae were found in all three colonies over the postfeeding period.

In test substance treatment group T2 (7.5 mg a.s./colony) a slightly increased mortality could be observed on DAF 14 to 18 compared to the observations made the days before and after this period of time and compared to the control. The mortality ranged between 27.0 and 56.3 dead bees/colony/day from DAF 14 to 18. No remarkable observations regarding the mortality were made from DAF 19 until the end of the test.

No difference could be observed regarding the mortality which occurred after start of feeding in the Triflumuron SC 480 treatment T3 (2.5 mg a.s./colony) compared to the control group.

Especially the total number of dead pupae found during the entire postfeeding period in all three colonies of the toxic standard group was considerably high (853 dead pupae) as in the first run of this study which validates the design of this study.

Effects on honey bee flight intensity:

No remarkable differences were observed regarding the flight intensity observed in front of the hives between the test substance treatment groups T1 (41.7 mg a.s./colony), T2 (7.5 mg a.s./colony) and T3 (2.5 mg a.s./colony), the toxic standard and the control group.

Effects on honev bee brood development:

A clear harmful effect on the bee brood was noticed in the colonies of the Triflumuron SC480 treatment group T1 (41.7 mg a.s./colony) from DAF 13 to 21. Between DAF 6 and 13 a drastically decrease of the percentage of larval stages was observed until the final brood assessment and only 0.0 to 1.3 % of all brood stages observed were larval stages. Additionally the number of combs containing brood was reduced in replicate No. 1 and 3 at the end of the test period (only 5 and 4 brood combs were counted on DAF 21). From DAF 6 until the end of the observation period the brood indices of all colonies in the test substance treatment group T1 (41.7 mg a.i./colony) decreased.

The observations made in the toxic standard treatment showed that the feeding of Insegar 25 WG as toxic standard has the expected harmful effect to honey bee brood.

No remarkable observations were made regarding the percentage of pre-imaginal stages during the assessment period in the colonies of all treatment groups in the second run. No abnormal difference which could be attributed to the influence of the test substance were observed in the treatment groups T2 (7.5 mg a.s./colony) and T3 (2.5 mg a.i./colony) compared to the control group. In the toxic standard no considerable differences regarding the brood development were made based on the results of the brood assessments and the appraisals of individual brood cells. Nevertheless a few pupae were noticed which showed an abnormal development and remained too long within their capped cells and possibly died juvenile.

Behaviour of the Bees:

Regarding the behaviour of the bees in front of the hives no differences could be observed between the test substance treatment T1 (41.7 mg a.s./colony), T3 (2.5 mg a.s./colony) and the toxic standard compared to the control group. In the Triflumuron SC 480 treatment T2 (7.5 mg a.i./colony), 45 young adult living bees with co-ordination problems, deformations of legs and wings which were unable to fly and to survive were found on DAF 14.