1-(2-bromo-4-chloro-phenyl)-4-(trifluoromethyl)triazole

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan - 28 Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted March 2006, corrected July 28, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(2-bromo-4-chlorophenyl)-4-(trifluoromethyl)-1H-1,2,3-triazole

EC Number:

863-757-3

Cas Number:

1445866-55-6

Molecular formula:

C9H4BrClF3N3

IUPAC Name:

1-(2-bromo-4-chlorophenyl)-4-(trifluoromethyl)-1H-1,2,3-triazole

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of two with acetonitrile.
- Sample storage conditions before analysis: All samples remained undiluted until sample preparation. All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION: The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 94.0 mg test item in 940 mL test water for preparing the test concentrations. The stock suspension was stirred for approx. 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:3.125, 1:10, 1:31.25 and 1:100 dilutions.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium: There were no remarkable observations.
Test concentration:
A filtrate with a loading rate of 100 mg test item/L and dilutions of 1:3.125, 1:10, 1:31.25 and 1:100, and a control, corresponding to following arithmetic mean measured concentrations of the test item:
27.0, 8.31, 2.56, 0.806 and 0.275 mg test item/L, and a control.
In the control, test water was used without addition of the test item.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)

The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

TEST ORGANISM
- Name/Strain: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
- Source (laboratory, culture collection): The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: no

Study design

Test type:

static

Water media type:

other: reconstituted water, OECD Medium

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L)
as CaCO3

Test temperature:

Water temperature was: 21.7 to 22.6 °C
(target: 21 – 24 °C, controlled at ± 2 °C).

pH:

The pH was measured in all test item concentrations and the control at the start (from excess solutions of each test medium) and the end of the test (via pooling of replicate test media; target pH: 8.1 ± 0.1 at test start in untreated test water).
pH value in the control at test start: 8.0,
pH value in the control at test end: 8.4
pH values in the test item treatments at test start: 8.0 to 8.1,
pH values in the test item treatments at test end: 8.2 to 8.5

Nominal and measured concentrations:

Nominal concentration:
A filtrate with a loading rate of 100 mg test item/L and dilutions of 1:3.125, 1:10, 1:31.25 and 1:100, and a control, corresponding to following arithmetic mean measured concentrations of the test item:
27.0, 8.31, 2.56, 0.806 and 0.275 mg test item/L, and a control.

Details on test conditions:

Test Vessels: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium loosely covered with watch glasses.
Introduction of Algae: The test was started (0 hours) by inoculation of a biomass of nominal 5000 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, which was set up 4 days prior to the test start under the same conditions as in the test.
Replicates: The test was performed with three replicates per test concentration and six replicates in the control.
Test Procedure: The test units were continuously stirred by magnetic stirrers. The flasks were incubated in a water bath, were placed in a random order and were repositioned each day to minimize differences in test conditions.

Blanks: Additionally, one replicate of each test concentration and of the control was prepared without algae to provide a blank for the spectrophotometric measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by the test item.

OTHER TEST CONDITIONS
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media (target: 4400 – 8800 lux ± 15 % of mean value).
Mean light intensity: 4997 lux (range: 4530 to 5440 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of the Cell Density: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae) at a wavelength of 685 nm.
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

No pre-experiments were performed.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.968 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.558 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.418 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.275 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.275 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.771 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.275 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.806 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth Inhibition: After 72 hours the inhibition of yield ranged from 11.4 to 99.8 % and the inhibition of growth rate ranged from 2.7 to 97.7 %.

Signs of Phytotoxicity evaluated by Microscopic Observations: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a loading rate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in July 2021

72-hour EC50 (Yield): 0.792 mg/L
72-hour NOEC (Yield): 0.2 mg/L
72-hour LOEC (Yield): 0.63 mg/L
72-hour EC50 (Growth rate): 1.62 mg/L
72-hour NOEC (Growth rate): 0.2 mg/L
72-hour LOEC (Growth rate): 0.63 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions), the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by a three parametric normal cumulative distribution function (CDF).
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Any other information on results incl. tables

Validity Criteria

Criterion	value required	value obtained	fullfilled
Cell Density Increase in control Cultures	at least by factor of 16 within 72 h	61.0-fold within 72 h	yes
Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures	< 35%	9.7 %	yes
Coefficient of Variation of Average Growth between Control Replicates	< 7%	3.7 %	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see any other information on results incl. tables

Conclusions:

The influence of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 0.968 mg test item/L and the 72-hour ErC50 value was calculated to be 1.80 mg test item/L. The 72-hour NOEyC was determined to be < 0.275 mg test item/L and the associated 72-hour LOEyC was 0.275 mg test item/L. The 72-hour NOErC was determined to be 0.275 mg test item/L and the associated 72-hour LOErC was 0.806 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean concentrations, since the test item concentrations were not within ± 20 % of the nominal concentrations during the test.

Executive summary:

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: A filtrate with a loading rate of 100 mg test item/L and dilutions of 1:3.125, 1:10, 1:31.25 and 1:100, and a control, corresponding to following arithmetic mean measured concentrations of the test item:

27.0, 8.31, 2.56, 0.806 and 0.275 mg test item/L, and a control.

Test Conditions: Water temperature: observed: 21.7 to 22.6 °C (target: 21 – 24 °C controlled at ± 2 °C); pH value in the control at test start: 8.0, pH value in the control at test end: 8.4; pH values in the test item treatments at test start: 8.0 to 8.1, pH values in the test item treatments at test end: 8.2 to 8.5; (target pH: 8.1 ± 0.1 at test start in untreated test water) continuous illumination: observed mean light intensity: 4997 lux (4530 to 5440 lux) (target: 4400 – 8800 lux ± 15 % of mean value)

Biological results

Parameter	Yield	Growth Rate
	mg test item/L	mg test item/L
72-hour EC50	0.968	1.80
72-hour EC20	0.558	1.03
72-hour EC10	0.418	0.771
72-hour NOEC	< 0.275	0.275
72-hour LOEC	0.275	0.806

Analytical results:The quantification of the test item in the test samples was performed using liquid chromatography with UV detection.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The mean measured values are

27.0 mg test item/L in the filtrate

8.31 mg test item/L in the 1:3.125 dilution of filtrate

2.56 mg test item/L in the 1:10 dilution of filtrate

0.806 mg test item/L in the 1:31.25 dilution of filtrate

0.275 mg test item/L in the 1:100 dilution of filtrate