Sodium 4(or 5)-methyl-1H-benzotriazolide

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2020- 03 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2016/266, 2015, Part C.33: Earthworm Reproduction Test (Eisenia fetida/ Eisenia andrei)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 29, 2016:

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

unknown

Molecular formula:

unknown

IUPAC Name:

unknown

Constituent 1

Reference substance name:

Methyl-1H-benzotriazole

EC Number:

249-596-6

EC Name:

Methyl-1H-benzotriazole

Cas Number:

29385-43-1

Molecular formula:

C7H7N3

IUPAC Name:

4(or5)-methyl-1H-1,2,3-benzotriazole

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor of Test, Batch 190733
- Purity, including information on contaminants, isomers, etc.: 99.98 % Tolyltriazole

Sampling and analysis

Analytical monitoring:

no

Remarks:

Due to the high stability of Tolyltriazole as shown in previous studies, no analytical monitoring was performed.

Test substrate

Details on preparation and application of test substrate:

Preparation of artificial soil:
The artificial soil substrate was prepared according to the OECD test guideline 222. More details of soil substrate characteristics under the heading details on test condition.

Preparation of Tolyltriazole test solution and test substrate:
1 Day before test started, the test solutions at the required test concentrations are prepared.
Due to the low solubility in water, the Tolyltriazol was dispersed homogeneously in 25 mL of the organic solvent acetone, before dropped on a constant amount of quartz sand (50g) and left for complete evaporation of the solvent for 18 hours under a hood. After, the test item-sand mixture was mixed with premoistened artificial OECD soil to the final test substrate.
The test substrate was moistened a second time to achieve maximum water holding capacity of 52.64 % and mixed by means of a laboratory mixer for five minutes.
preparation controls
The solvent control treatment was prepared in the same way as the test substrate but without Tolyltriazole. The control treatment was prepared in the same way as the solvent control treatment but without solvent.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia fetida (Annelida: Lumbricidae) with an age of five months and a clitellum were used for the study. They came from a stock culture maintained at IES Ltd. The wet mass of individual worms, unsing in the test, was between 300 and 600 mg. Earthworms were acclimatized for two days in the artificial soil used for the test. No mortality during acclimation were observed.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Total exposure duration:

56 d

Remarks:

The adult individuals of the worm species Eisenia fetida were removed from the test units after 28 Days. After additional four weeks, the number of offspring present in the soil were counted to assess the effect on reproduction.

Post exposure observation period:

four weeks (further explanantion are given under remarks above)

Test conditions

Test temperature:

Mean temperature: 20.4 °C (range 18.8 – 23.6 °C); temperature was one
time for a short-time period above 22.0 °C due to insufficient
temperature control of the climate chamber. These deviation from the
required range for temperature (i.e. 18 – 22 °C) did not impact the study
as shown by the performance of the control.

pH:

pH range from 5.5 to 6.1

Moisture:

Water content at start: 27.93 to 30.43 % corresponding to 51.5 and 56.1 % of the maximum water holding capacity.
Water content at test termination: 29.36 to 40.17 % corresponding to 54.1 and 74.1 % of the maximum water holding capacity in the controls and in the test item treatments.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic containers (approximately 17 cm x 12 cm x 11.8 cm) were used
- Amount of soil or substrate: 656 g artificial soil with a water content of 28.55 % at test start
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Composition (if artificial substrate): approximately 70 % fine quartz-sand, 20 % kaolin clay (kaolinite content > 30 %), 10 % sphagnum peat (air dried and finely ground, with no visible plant remains), Calcium carbonate: < 1 % calcium carbonate (CaCO3, pulverised, analytical grade) to obtain a pH of 6.0 ± 0.5.
- Maximum water holding capacity (in % dry weigth): 54.23 % dry weigth

recorded soil quality parameter and measuring intervals:
-Temperature was continuously measured in climate chamber
-pH-Value was determined in two samples per treatment at test initiation and termination
-Water content of the soil was measured per treatment at test initiation and termination


OTHER TEST CONDITIONS
- Photoperiod: 16/8 hour light/dark cycle
- Light intensity: intensity 578-666 Lux.
- Feeding during test: 6 g of horse manure was added once a week, starting on day after test initiation until day 28. After adults are removed from the test units further 6 g of horse manure moistened with 12 g ultrapure water are added once for the remaining four weeks.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Mortality, body weight and symptoms of toxicity of adults (once at Day 28)
-Number of juveniles and number of cocoons (once at Day 56)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: not applicable: Due to the results of the range-finding test, the number of test concentrations of the main test was increased from eight to nine in order to increase the probabilityof achieving a reliable NOEC.

Range finding study:
56 Day Range-finding study was performed, with one control and four different concentration of Tolyltriazole (1,10,100,100 mg Tolyltriazole /kg dry soil), containing of two replicates each, while using 10 adult Eisenia fetida per replicate.
Due to the results of the range-finding test, the number of test concentrations of the main test was increased from eight to nine in order to increase the probability of achieving a reliable NOEC.

Nominal and measured concentrations:

Nominal test concentrations: 1.6, 2.8, 5.0, 9.1, 16.3, 29.1, 52.9, 95.3, 171 mg Tolyltriazole /kg dry soil (spacing factor: 1.8)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

95.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of juveniles

Effect concentrations 2

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

52.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

number of juveniles

Effect concentrations 3

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

6.75 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Endpoint specific resultes:

Mortality: No mortality in all treatments up to and including the highest test concentration (171 mg/kg dry soil).

Body Weigth: no effect on body weight up to and including the highest test concentration (171 mg/kg dry soil).

Number of Juveniles: At the two highest test concentrations of 95.3 and 171 mg/kg dry soil, the average number of juveniles decreased to 62 and 55 % of the solvent control and was statistically significantly smaller than the solvent control. From these results the NOEC and LOEC of the number of juveniles were 52.9 and 95.3 mg Tolyltriazole/kg dry soil, respectively, while the EC 10 was calculated at 6.75 mg Tolyltriazole/kg dry soil. At the other test concentrations, no effect was observed.

Reported statistics and error estimates:

Differences between the controls as well as between the test item treatments and the pooled
control (for body weight) and solvent control (for number of juveniles), respectively, were tested:
- Mortality: no statistics necessary, since no mortality in all test
item treatments up to and including 171 mg/kg dry soil.
- Student t-test and Dunnett t-test for adult body weight at Day 0
and Day 28 and for body weight change (α = 0.05, two-sided)
- Student t-test and Williams t-test for number of juveniles
(α = 0.05, two-sided and one-sided smaller, respectively).
NOEC and LOEC for reproduction were determined based on the outcome of the statistics. ECx calculations for number of juveniles were performed using Probit analysis with linear maximum
likelihood regression.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on experimental observations the effect concentration for reproduction of EC10(56d) = 6.75 mg/L were calculated. All validity criteria were fulfilled and hence the study is considered adequate for regulatory purposes.

Executive summary:

Conclusion:
In the present valid and reliable study following OECD 222 (2016), the earthworm species Eisenia fetida was exposed for 56 days to artificial soil treated with the test item Tolyltriazole. The NOEC and LOEC for the most sensitive endpoint number of juveniles were 52.9 and 95.3 mg Tolyltriazole/kg dry soil. The EC₁₀ for reproduction was 6.75 mg Tolyltriazole/kg dry soil, respectively. Tolyltriazol did not effect mortality and body weight of E.fetida adults.