Ethanol, 2-[(3-aminopyrazolo[1,5-a]pyridin-2-yl)oxy]-, hydrochloride (1:1)

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:

From 07-Jan-2011 to 20-Jan-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with international guidelines. Since test item was unstable, one would have expected a daily sampling and analysis of tested concentrations. The shading effect of dark colouration of the media has been determined but not interpreted as a possible impact on growth.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-({3-aminopyrazolo[1,5-a]pyridin-2-yl}oxy)ethan-1-ol hydrochloride

EC Number:

695-745-7

Cas Number:

1079221-49-0

Molecular formula:

C9 H11 N3 O2, ClH

IUPAC Name:

2-({3-aminopyrazolo[1,5-a]pyridin-2-yl}oxy)ethan-1-ol hydrochloride

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.22, 0.70, 2.2, 7.0, 22 and 70 mg/L. The concentrations of the test item were determined in all duplicate test medium samples from the test concentrations of 0.70 to 70 mg/L and in the control. The samples from the lowest nominal test concentration of 0.22 mg/L were not analyzed, since this concentration was well below the NOEC determined in this test and, thus, was not relevant for the interpretation of the biological results.
- Sampling method:duplicate samples were taken from the test media of all test concentrations and the control at the start of the test (without algae) and at the end of the test (containing algae). For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis:Immediately after sampling, sodium dithionite solution with a nominal concentration of 10 mg/L was added to each sample (100 μL per 10 mL of sample volume) in order to stabilize the latter for measurement. The samples were analyzed immediately after sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:The test medium of the highest nominal concentration of 70 mg/L was prepared by dissolving 70.1 mg of the test item completely in 1000 mL of test water by intense stirring for 3 minutes at room temperature in the dark. The test medium of the highest test concentration was serially diluted with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
In consultation with the Sponsor, the pH of the test water was lowered to 6.2 (using 1 M hydrochloric acid) to increase the stability of the test item in the test water. To keep the pH of the test water and test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to the test water. For equilibration of the pH the test water was stirred for 24 hours prior to use.
- Eluate:/
- Differential loading: No
- Controls: no other special treatment.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No vehicle
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):/
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period. At the two highest test concentrations (nominal 22 and 70 mg/L), the test media were dark colored by the test item over the entire test period. At the next lower concentration (nominal 7.0 mg/L) the coloration was observed after 24 hours of test duration until the end of the test.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Method of cultivation: The algae were cultivated under the test conditions. Reconstituted test water prepared according to the test guidelines was used. Analytical grade salts were dissolved in sterile purified water.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

The water hardness (calculated) of the test water was 0.24 mmol/L (= 24 mg/L as CaCO3).

Test temperature:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22 °C. The water temperature during the test was maintained at 22 °C.

pH:

In consultation with the Sponsor, the pH of the test water was lowered to 6.2 (using 1 M hydrochloric acid) to increase the stability of the test item in the test water. To keep the pH of the test water and test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to the test water. For equilibration of the pH the test water was stirred for 24 hours prior to use.
At the start of the test, the pH measured in the test media after addition of the algae was 6.5 in all treatments. At the end of the test, pH values of 6.9 to 7.0 were measured.

Dissolved oxygen:

Not recorded

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0.22, 0.70, 2.2, 7.0, 22 and 70 mg/L.
The measured concentrations of test item in the test media of the nominal test concentrations of 0.70 to 70 mg/L were between 60 and 93% of the nominal values at the start of the test. During the test period of 72 hours, the test item concentration in the test media decreased. At the end of the test, 8 to 9% of the nominal values were found in the three highest test concentrations of 7.0 to 70 mg/L. At the two lowest nominal concentrations of 0.70 and 2.2 mg/L, the measured concentrations were below LOQ (limit of quantification, LOQbio: 0.452 mg/L).

Details on test conditions:

TEST SYSTEM
- Test vessel, Type (open / closed), Material, size, headspace, fill volume: Since the test item caused a dark coloration of the test media, 17 mL of test solution were placed in 100-mL Erlenmeyer flasks achieving a reduced layer thickness of 0.9 cm and enhancing the light supply for the algae. The Erlenmeyer flasks were covered with a glass dish.
- Aeration: During exposure, the test solutions were continuously stirred by magnetic stirrers.
- Type of flow-through (e.g. peristaltic or proportional diluter):/
- Renewal rate of test solution (frequency/flow rate):/
- Initial cells density: 10000 cells/mL
- Control end cells density: In the control, the biomass increased by a factor of 127 over 72 hours.
- No. of organisms per vessel:/
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per vehicle control (replicates):/

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:/

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water prepared according to the test guidelines was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.
- Total organic carbon:/
- Particulate matter:/
- Metals:/
- Pesticides:/
- Chlorine:/
- Alkalinity:/
- Ca/mg ratio:/
- Conductivity:/
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH was measured and recorded in each treatment at the start and end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: In consultation with the Sponsor, the pH of the test water was lowered to 6.2 (using 1 M hydrochloric acid) to increase the stability of the test item in the test water. To keep the pH of the test water and test media as constant as possible 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) was added to the test water. For equilibration of the pH the test water was stirred for 24 hours prior to use.
- Photoperiod:24H light
- Light intensity and quality: fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 5900 Lux (range: 5320 to 6470 Lux, measured at nine places in the experimental area). The light intensity over the incubation area deviated by less than ±15% from the average light intensity as recommended by the guideline.
- Salinity (for marine algae):/

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algal biomass in the samples was determined by fluorescence measurement (BIO-TEK® Multi-Detection Microplate Reader, Model FLx800). The measurements were performed at least in duplicate.
- Chlorophyll measurement:/
- Other:/

TEST CONCENTRATIONS
- Spacing factor for test concentrations:3.2
- Justification for using less concentrations than requested by guideline:/
- Range finding study: yes, not GLP, results not provided in the study report, but used for the selection of test concentrations of the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 22 mg/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Unusual cell shape:/
- Colour differences:/
- Flocculation:/
- Adherence to test vessels:/
- Aggregation of algal cells:/
- Other: Since the test item caused a dark coloration of the test media (intensifying with the test duration and increasing test item concentration), the shading effect for each treatment was determined by measurement of the light intensity under each test solution at the start and at the end of the tests(see below).
- Any stimulation of growth found in any treatment:/
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:/
- Effect concentrations exceeding solubility of substance in test medium:/

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in October 2010 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.99 mg/L (study C97564), range of the 72-hour EC50 for the growth rate from 2000 to 2010: 0.71-1.7 mg/L).

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t-test and Welch t-test.

Any other information on results incl. tables

The test item had a statistically significant inhibitory effect on the growth of the algae (average growth rate and yield) after the test period of 72 hours at the concentration of 1.7 mg/L and at all higher test concentrations (results of Welch t-tests, one-sided smaller, α = 0.05, Table 2 and Table 3). However, the beginning of the concentration-effect-relationship is assumed to be at the next lower concentration of mean measured 0.55 mg/L. Furthermore, the inhibition values at the concentration of 0.55 mg/L, 4.3% for growth rate and 20% for yield, were seen as biologically relevant. Therefore, the mean measured concentration of 0.55 mg/L was determined to be the 72-hour LOEC.

The 72-hour NOEC was determined to be 0.38 mg/L, since up to and including this test concentration, the growth of the algae (average growth rate and yield) were not inhibited by the test item after 72 hours test duration.

In the control, the biomass increased by a factor of 127 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 9.6%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 4.4%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see above

Executive summary:

The influence of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006), and the Commission Regulation (EC) No. 761/2009, C.3.

The nominal concentrations of the test item of 0.22, 0.70, 2.2, 7.0, 22 and 70 mg/L were tested in parallel with a control.

The measured concentrations of the test item in the test media of the nominal test concentrations of 0.70 to 70 mg/L were between 60 and 93% of the nominal values at the start of the test. During the test period of 72 hours, the test item concentration in the test media decreased. At the end of the test, 8 to 9% of the nominal values were found in the three highest test concentrations of 7.0 to 70 mg/L. At the two lowest nominal concentrations of 0.70 and 2.2 mg/L, the measured concentrations were below LOQ (limit of quantification, LOQbio: 0.452 mg/L).

Due to the instability of the test item in the test water, the biological results were related to the mean measured test item concentrations (calculated as the geometric means of the concentrations measured at the start and end of the test):

EC50 = 12 mg/L (growth rate)

NOEC = 0.38 mg/L (growth rate)

LOEC = 0.55 mg/L (growth rate)