2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide

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:

23 November 2017 - 08 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide

EC Number:

253-021-4

EC Name:

2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide

Cas Number:

36411-52-6

Molecular formula:

C9H8N4O2

IUPAC Name:

2-hydroxy-N-1H-1,2,4-triazol-3-ylbenzamide

Test material form:

solid: particulate/powder

Details on test material:

Batch:102Z5
Purity: 99.9%
white powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.
- Sampling method: Samples were taken at 22, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded
- Sample storage conditions before analysis: frozen

Test solutions

Vehicle:

yes

Remarks:

Culture medium

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 56, 32, 18 and 10% v/v saturated solution. An aliquot (1 liter) of each of the stock solutions was separately inoculated with 19 mL of algal suspension to give the required test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution.
The control group was maintained under identical experimental conditions but not exposed to the test item.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: The master cultures were maintained in the laboratory under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10e3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10e4 to 10e5 cells/mL.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not specified

Test temperature:

24 ±1 °C

pH:

8.5 - 9.4

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

- nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution
- geometric mean measured concentrations of 0.49, 0.89, 1.7, 3.4, 6.3 mg/L

Details on test conditions:

TEST SYSTEM
250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and 3 flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.63 x 10e5 cells per mL. Inoculation of 1 liter of test medium with 19 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10e3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

GROWTH MEDIUM
- Standard medium used: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 22, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter

TEST CONCENTRATIONS
- Range finding study: 0.10, 1.0, 10 and 100% v/v saturated solution
- Test concentrations: 10, 18, 32, 56 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 2.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

ca. 5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 6.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an EC50 value as at the maximum attainable dissolved test item concentration of 6.3 mg/L, no more than 27% inhibition of growth rate occurred.

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

ca. 3.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
The growth rate of Pseudokirchneriella subcapitata (CCAP 278/4) was affected by the presence of the test item over the 72-Hour exposure period. All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
EC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Reported statistics and error estimates:

n/a

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 hour period and based on the geometric mean measured test concentrations gave the following results:

EC10 (0 to 72 hour) : 2.4 mg/L
EC20 (0 to 72 hour) : 5.0 mg/L
EC50 (0 to 72 hour) : >6.3* mg/L
No Observed Effect Concentration: 1.7 mg/L
Lowest Observed Effect Concentration: 3.4 mg/L

*It was not possible to calculate an ErC50 value as at the maximum attainable dissolved test item concentration of 6.3 mg/L, no more than 27% inhibition of growth rate occurred.