Bumetrizole

Toxicity to terrestrial arthropods

Administrative data

Endpoint:

toxicity to terrestrial arthropods: long-term

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

The test substance is a solid material. The water solubility is not enough to prepare a
sufficient stock solution with deionized water. Therefore, the test substance was added
to the quartz sand (25 g), thoroughly mixed and later added to 2500 g (dry weight) of
prepared test substrate.

The required amounts of artificial soil were weighed into a mixing tray. The test substance
/ quartz sand mixtures were added and thoroughly mixed with the soil. Thereafter, the
water content of the soil was adjusted to about 50% WHCmax using deionized water and
was mixed once again. The test mixtures were distributed to the test units.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Species: Eisenia fetida
Reason for selection of species: Recommended by the test guideline
Breeding facility: BASF SE, Experimental Toxicology and Ecology,
67056 Ludwigshafen, Germany
Only adult worms (with clitellum) with a fresh weight between 300 and 600 mg were used.
The worms that were introduced into the test were bred in horse manure and had an age
from about 11 months.
Acclimatization:
Before starting the test, the worms selected were acclimatized in the test substrate in a
plastic box under test conditions through overnight under permanent light and without
feeding. For this, a plastic box was filled up with approx. 750 g test substrate (Dw) and
added with a sufficient number of adult worms that were sorted out from the breeding
boxes.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

19.6° C – 22.0° C

pH:

pH value of the dry test substrate: 6.4 (mean value of 2 single values; 6.4/6.4)

Moisture:

Water content in the test: 42.5% WHCmax (mean value)

Details on test conditions:

Test temperature: 19.6° C – 22.0° C
Measurement of the temperature: In a separate vessel, filled with about 750 g
test substrate (dry weight), around two times
per week during the exposure time with an
electronic thermometer. The water content of
this vessel was adjusted initially (on day 0) to
the water content of test vessels.
Test container: Plastic dishes with a volume of about 1.0 L,
dimensions 11×15.5×6.5 [cm] with transparent
and punctured lid
Test substrate per test container: Approx. 667 g artificial soil (wet weight)
Number of animals / test container: 10
Worm weight at start of exposure: Body weight of all added worms in the
required range of 300-600 mg
Number of replicates: 8 replicates for control and 4 replicates for
each test substance concentration
Number of test substance
concentrations: 5
Determination of water content of
the dry test substrate: 0.5% (mean of 2 single values: 0.5%/0.5%)
Illumination: Light/dark cycle of 16:8 hours, over the
exposure period
Measurement of light intensity,
once at start of exposure:
532 Lux, (mean of 5 single measurements:
733, 503, 460, 513 and 453 [Lux])
Measurement of the temperature at
adaptation: 21.6° C
Measurement of light intensity,
once at start of adaption: 705 Lux
pH value of the dry test substrate: 6.4 (mean value of 2 single values; 6.4/6.4)
Water content in the test: 42.5% WHCmax (mean value)


In the control group, eight test units and in each further treatment group, four test units
were used. These were closed with perforated lids and placed in a temperature-controlled
incubation room and a photoperiod of 16 h light and 8 h dark.
Day 0 (test preparation)
For each test concentration, required aliquots of the test substance were mixed with
25 g quartz sand. The quartz/substance mixtures were blended with approx. 2500 g
(Dw) of artificial soil. After adjusting the water content to 50 ±10% WHCmax (nominal
value), the test mixtures were distributed to the test units. The test mixture of the
control contained only quartz sand without test substance. Eight replicates were
prepared for the control assay and four replicates were prepared for each test
substance concentration. The pH-values and the moisture of the test assays were
determined once for each concentration (samples were taken from the mixing
container).
Day 0 (Introduction of the worms)
The acclimatized worms were weighed and distributed into 28 test units, each unit
containing 10 individuals. The animals were put onto the soil surface of each test
unit. No behavioural changes were observed during the introduction of worms.
Day 1 of the Test:
No abnormalities or behavioural changes were observed.
Feeding: About 5 g dried mixture of cow manure were sprayed on to the soil
surface of each test container. The food was moistened with about 5-6 mL
deionized water per test container.
Determination of the initial weight of the test vessels were carried out for later
comparison with its actual initial weights.
Day 7 of the Test
No abnormalities or behavioural changes were seen.
The weight of the test containers was verified.
Feeding and moistening: The consumption of food was recorded for each test
container. No decreased feeding activity was recorded. About 5g dried cow manure
was spread on the soil surface of each test container. The food was moistened with
5-6 mL deionized water per test container. The total weights of the test containers
were determined which is used on day 14 for comparison.
Day 14 of the Test:
No abnormalities or behavioural changes were reported.
The weight of each test container was compared with the weight of the previous
feeding days.
Feeding and moistening: The consumption of food was recorded for each test
container and no decreased feeding activity was recorded. About 5 g dried cow
manure was spread on the soil surface of each test container. The food was
moistened with 5-6 mL deionized water per test container to compensate the loss
of water in comparison with the initial weight of the test container. The test
containers were weighed again.
Day 21 of the Test:
No abnormalities or behavioural changes were seen.
The weight of each test container was compared with the weight of the previous
feeding day i.e., day 14.
Feeding and moistening: The consumption of food was recorded for each test
container and no decreased feeding activity was recorded. About 5 g dried cow
manure was spread on to the soil surface of each test container. The food was
moistened with 5-6 mL deionized water per test container to compensate the water
loss compared to the previous feeding day. Later, the new weight was recorded.
Day 28 of the Test:
The feeding activity was recorded and no changes in food consumption
compared to previous days were documented.
Moistening: The weight of each test container was compared with the previous
feeding day. The loss of water was compensated by addition of deionized water.
The test container was emptied to another tray and the adult worms were
recovered from each of the test containers. During this process, no morphological
or behavioral changes were found. The number of surviving adult worms in each
test container was documented. Worms were classified as dead when they did
not respond to a gentle mechanical stimulus to the front end. The individual weight
of the surviving adult worms from each replicate was recorded.
Feeding: Dried food material (5 g per test container separately) was gently
blended into the test substrate. Then the test substrate (including cocoons and
juvenile worms, if any) were placed back into the test containers. The new initial
weight of the test vessels for the comparison of the actual weight was noted.
The test containers were closed again. For another 28 days, they were
investigated under the same conditions as described above.
Day 36 of the Test:
No abnormal changes were observed.
Moistening: The weight of each test container was determined, and the loss of
water was compensated by the addition of deionized water.
Day 42 and 49 of the Test:
No abnormal morphological or behavioral changes were observed.
Moistening: The weight of each test container was determined, and the loss of
water was compensated by the addition of deionized water.
Day 56 of the Test:
The juvenile worms were brought to the surface of the test substrate by using the
water bath method (at a setting of 59.6 °C). During this process morphological and
behavioral changes and the number of juveniles in each test container were
recorded. No morphological and behavioral changes were observed. The
juveniles from the control units were larger and healthy compared to those from
the test concentration groups.
The pH-value and the moisture content of the test substrate were also determined
as single determinations from a representative test substrate sample, taken from
one of the replicates of each test concentration.

Nominal and measured concentrations:

nominal: 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg Dw

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass change

Details on results:

In this 56-day study assessing the toxicity of 2-(5-chloro(2H)-benzotriazole-2-yl)-4-
(methyl)-6-(tert-butyl)phenol, adult earth worms of the species Eisenia fetida were
exposed to nominal concentrations of 0 mg (control), 62.5 mg, 125 mg, 250 mg, 500 mg
and 1000 mg/kg dry weight in accordance with the OECD 222 guideline.
The mortality and the body weight change of the adult worms were observed and
recorded on day 28.
The reproduction, number of juveniles per test vessel 56 days after application was
observed.
Adult worms:
No mortality of adult worms was observed in the controls and in all replicates with
test substance
NOECMortality: ≥ 1000 mg test substance/kg soil (dw)
LOECMortality: > 1000 mg test substance /kg soil (dw).
NOECBiomass change: ≥ 1000 mg test substance/kg soil (dw)
LOECBiomass change: > 1000 mg test substance /kg soil (dw)
No significant differences were obtained in the statistical analysis (Williams Test one
sided and Dunnett test one sided p ≤0.05) concerning the mean body weight change of
individual adults per replicate over 28 days between the control and all replicates of the
tested test substance concentrations.
Juvenile worms:
Reproduction by count of juvenile worms:
NOECReproduction: ≥ 1000 mg test substance/kg soil (dw)
LOECReproduction: > 1000 mg test substance/kg soil (dw)
No ECx calculations were performed, because there was no dose response relationship.
The results in this study are consistent with all validity criteria and the test is valid
according to the test guideline of this study. No deviations from the test guidelines or other
incidents occurred during the reported test, which may have influenced the results.

Results with reference substance (positive control):

The reference substance BAS 346 F (Carbendazim) was tested by a non-GLP project
68E0902/00G027 (start of exposure 15 April 2019). The result of the reference substance is presented below.
ECx values [mg/kg soil (dw)], reproduction by counting juvenile worms, conf. limits 95%
EC10: 0.56 LCL: 0.45 UCL: 0.71
EC50: 1.03 LCL: 0.91 UCL: 1.16

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria:
Mean mortality in the control should be ≤ 10 %. The reproduction in the controls should
be at least 30 juveniles per container and the coefficient of variance of reproduction in
the control should be ≤ 30 %.
All validity criteria were met. After 28 days, no adult worm in the control assay was dead.
Around 280 juveniles (mean value) were counted in the test units of the control assay.
The mortality of the adult worms in the control was <10 % and the coefficient of variation
concerning to the offspring juvenile worms in the pooled control assay was 7.6%.