Butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo-, tetradecyl ester

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 January 2004 to 19 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Concentration determinations were made by at time 0 (test initiation), 24, 48 and 72 hours intervals during the test to evaluate the test substance stability under test conditions.

Vehicle:

yes

Details on test solutions:

A stock solution was prepared by weighing 0.1006 g of the test substance into an acid-washed 10-mL glass volumetric flask and diluted to 10 mL with dimethylfomamide (DMF).
(concentration = 10,060 mg/L) .
A limit test exposure solution was prepared by first weighing 999.7 g of sterile algal medium (AM) into a clean, sterile 1000 mL polystyrene flask (Nalgene). Then, 100 µL of the 10,060 mg/L stock solution were then pipetted directly into the AM with constant stirring and without touching the flask itself.
(concentration = 1.01 mgL)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): American Type Culture Collection, Rockville, MD, USA
- Culture: A 3 day culture passage in liquid algal medium. Previous passages were performed in order to confirm exponential growth under the conditions of the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24.7 ºC

pH:

7.51 (test start)
8.38 (test end)

Nominal and measured concentrations:

1.01 mg/L (nominal)
0.88 mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile 250 mL Erlenmeyer flasks
- Material, size, headspace, fill volume: plastic

GROWTH MEDIUM
Sterile algal medium was prepared using high quality distilled water. The pH of the algal medium was adjusted to 7.5 ± 0.1 using NaOH prior to use.

PREPARATION OF TEST FLASKS
100 mL of test exposure solution was added to flasks #1-5. Flasks #1 and #2 served as light-exposed and light-shielded chemical controls respectively. 100 mL of algal growth medium were added to control flasks #6-11. Then solvent controls were prepared by adding 10 µL of DMF to flasks #6-8. Then 769 µL of stock algal suspension was added to flasks #3-11.

TEST PROCEDURES
Flasks were conditioned by adding a few millilitres of the appropriate solution, swirling to coat the flask and them removing the solution by inverting into a waste beaker. A total of eleven flasks were prepared. Five flasks were conditioned for the test substance. Three flasks were conditioned for the negative control using growth medium and three flasks were conditioned as solvent controls.

A hemacytometer count was made to determine the number of algal cells in the culture flask serving as the inoculum source for the test. Sterile 100 mL volumetric flasks were used to dispense the test substance solutions into the appropriate sets of the conditioned flasks. Enough cells were inoculated into each flask containing the test substance solution and into the negative control flasks to give a cell concentration of 1 x 10E+4 cells/mL. The flasks were secured with foam stoppers and then transferred to the incubator were they were maintained at a temperature of approximately 24.7 ºC and shaken at approximately 100 rpm. Mean illumination of 741.5 ± 1.0 foot-candles was maintained during the test.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Details on results:

Loss of the test substance concentration from flasks containing algae nominally exposed to 1.0 mg/L was determined to be 33 %. Losses in test substance concentrations from the chemical control flasks (without algae) were determined to be 8.5 % in the light-exposed flasks and 4.7 % in the light-shielded flasks. The analytical results suggest that some of the test substance may have adsorbed to the algae as greater losses in concentrations were observed in the flasks containing algae than in either of the chemical controls.

The cell count results indicate that the algae in the solvent control flasks exhibited normal log growth increasing from 1 x 10E+4 cells/mL at time 0 to a mean of 1.66 x 10E+6 at 72 hours. A growth curve for the controls and the test substance concentration throughout the test is shown in Figure 1.

Two measures of growth (biomass and growth rate) are illustrated in Figures 2 and 3. The EbC50 (0-72 h) value was determined to be >0/88 mg/L. The ErC50 (0-72 h) value was determined to be >0.88 mg/L. The 72 h NOEC value was estimated to be 0.88 mg/L

Test validity
The cell concentration in the control cultures increased by a factor of 166-fold within three days. This growth factor meets the OECD criteria for a valid test which is a 16-fold increase within 3 days.

Reported statistics and error estimates:

Statistical analyses were not performed on the data as no inhibition of growth was observed in flasks exposed to the test substance.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the 72 h EC50 value (biomass and growth rate) of the test substance to algae is >0.88 mg/L. The NOEC of the test substance to algae is 0.88 mg/L.

Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 201, the acute toxicity of butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester to algae was investigated. The 72 hour EC50 of the test substance was determined to be >0.88 mg/L. The NOEC of the test substance was determined to be 0.88 mg/L.

Description of key information

Key study:- Hirsch (2004) 'A growth inhibition limit test with the alga, Selenastrum capricornutum' was conducted in line with OECD Guideline 201. The 72 hour EC50 of the test substance was determined to be >0.88 mg/L. The NOEC of the test substance was determined to be 0.88 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.88 mg/L

Additional information

The key study (Hirsch, 2004) was performed in compliance with GLP and to OECD Guideline 201 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard in line with an accepted, standardised guideline and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The 72 hour EC50 of the test substance was determined to be >0.88 mg/L. The NOEC of the test substance was determined to be 0.88 mg/L.