Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-09-17 to 2019-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 23, 2006; Annex 5 corrected July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) 2016/266.

Version / remarks:

December 7, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analytical measurement a sample from the freshly prepared test medium from the concentration of the closed system was taken at the start of the study. For the determination of the maintenance of the test material concentration during the exposure period a sample was taken from the medium at the end of the exposure period.

Vehicle:

no

Details on test solutions:

The test media (reconstituted water) was freshly prepared. A stock preparation with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle, reconstituted water was added. The preparation was stirred with a magnetic stirrer for 1 hour. Then the formulation was passed through a filter membrane (pore size: 0.2 pm). The filtrate was used for the preparation of the different concentrations. The pH was not adjusted.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 6181
- Source: Pflanzenphysiologisches Institut der Universität Göttingen, Germany
ACCLIMATION
- Acclimation period: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21.0 - 24.0°C

pH:

5.32 to 8.68

Nominal and measured concentrations:

Nominal measured: 0.0, 1.0, 3.2, 10.0, 31.6, 100,0

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: open
- Material, size, fill volume: glass, 300 mL, 100 mL
- Aeration: No
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: according to OECD 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: Continuous illumination

EFFECT PARAMETERS MEASURED: Algae cell density of the pre-culture was determined with an electronic particle counter (Coulter-Counter Z2, Beckman Coulter GmbH, Krefeld, Germany).

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.52 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
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no

Validity criteria
In the control group, the biomass increased by a factor of 274.8 over 72 hours. Therefore, 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 group (section-by-section growth rates) during 72 hours was 25.34%. 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 group after 72 hours was 1.30%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled

Table 1: Measured concentrations

Nominal test item concentration [mg/L]	Analytical results (samples inoculated with algae) including extrapolated values									Geometric Mean [mg/L]
	0h		24 h			48 h		72 h
	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]		[%]	Meas. Conc. [mg/L]	[%]
0.0	<LOQ	-	<LOQ	-	<LOQ		-	<LOQ	-	-
1.0	0.752	75.2	0.165	-	0.036		-	0.016	-	0.09
3.2	2.305	72.0	0.483	15.1	0.115		-	0.051	-	0.26
10.0	7.322	73.2	1.662	16.6	0.305		3.1	0.160	-	0.81
31.6	24.860	78.7	5.188	16.4	1.175		3.7	0.423	1.3	2.69
100.0	78.300	78.3	17.998	18.0	4.055		4.1	1.790	1.8	9.55

Table 2: EC values determined for the test item

Parameter (0-72h)	Growth Rate	Yield
EC10 [mg/L] 95% confidence interval	0.52 (0.32 - 0.72)	0.21 (0.15-0.26)
EC20 [mg/L] 95% confidence interval	0.89 (0.63-1.15)	0.32 (0.25-0.38)
EC50 [mg/L] 95% confidence interval	2.54 (2.09 - 3.09)	0.72 (0.63 - 0.82)

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study according to OECD 201, the test item showed a 72h EC50 of 2.54 mg/L for the growth rate and 0.72 mg/L for the yield of the unicellular green alga with 72-h EC10 of 0.52 mg/L for growth and 0.21 mg/L for the yield.

Executive summary:

The objective of the present study was to determine the effect of the test item on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata according to OECD 201. For this purpose, exponentially growing cultures of green algae were exposed to five different concentrations of the test item for a period of 72 hours. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC values for growth rate and yield were calculated. Samples of the test and control media were taken directly after preparation and then daily and the test item concentration were analyzed via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. As the test item concentrations could not be maintained >80% of the measured initial concentrations, the EC values were calculated based on the calculated geometric mean concentrations. For measured concentrations <LOQ the concentrations used for calculation of the geometric mean were extrapolated with the mean recovery of the higher concentrations at the same time point where analytical values were >LOQ. Under the conditions of the present study, the test item showed a 72h EC₅₀ of 2.54 mg/L for the growth rate and 0.72 mg/L for the yield with a 72-h EC₁₀ of 0.52 mg/L for growth and 0.21 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Please refer to attached QMRF and QPRF Documents

Qualifier:

according to guideline

Guideline:

other: Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals

Deviations:

not applicable

Principles of method if other than guideline:

Calculation of toxicity in Algae. Software used: ECOSAR 1.11 (EPISUITE) for chemical class "Neutral Organics"

GLP compliance:

no

Specific details on test material used for the study:

Smiles: c12c(cccc1)N(OP(N1CCCC1)(N1CCCC1)N1CCCC1)N=N2

Analytical monitoring:

not required

Vehicle:

no

Test organisms (species):

other: Algae

Water media type:

freshwater

Remarks on exposure duration:

acute and chronic exposure

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1.531 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.691 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

ECOSAR v1.11 Class-specific Estimations

--------------------------------------

Benzotriazoles

Predicted

ECOSAR Class                Organism           Duration End Pt  mg/L (ppm)

=========================== ================== ======== 

Benzotriazoles            : Green Algae         96-hr     EC50      1.531

Benzotriazoles            : Green Algae          96 -hr ChV      0.691

Conclusions:

sing ECOSAR v1.11 the toxicity 96-h EC50 and NOEC values for Algae were determined to be 1.531 mg/L, and 0.691 mg/L, respectively. The substance is within the applicability domain of the model.

Executive summary:

Using ECOSAR v1.11 the toxicity 96-h EC50 and NOEC values for Algae were determined to be 1.531 mg/L, and 0.691 mg/L, respectively. The substance is within the applicability domain of the model.

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Description of key information

Under the conditions of the present study according to OECD 201, the test item showed a 72h EC₅₀ of 2.54 mg/L for the growth rate and 0.72 mg/L for the yield of the unicellular green alga with 72-h EC₁₀ of 0.52 mg/L for growth and 0.21 mg/L for the yield (reference 6.1.5 -2).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.54 mg/L

EC10 or NOEC for freshwater algae:

0.52 mg/L

Additional information

The objective of the present study was to determine the effect of the test item on growth rate and yield of the unicellular green alga Pseudokirchneriella subcapitata according to OECD 201. For this purpose, exponentially growing cultures of green algae were exposed to five different concentrations of the test item for a period of 72 hours. Growth of the algae was determined after 24, 48 and 72 hours exposure and compared with the growth of the control group. From the results, EC values for growth rate and yield were calculated. Samples of the test and control media were taken directly after preparation and then daily and the test item concentration were analyzed via HPLC with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. As the test item concentrations could not be maintained >80% of the measured initial concentrations, the EC values were calculated based on the calculated geometric mean concentrations. For measured concentrations <LOQ the concentrations used for calculation of the geometric mean were extrapolated with the mean recovery of the higher concentrations at the same time point where analytical values were >LOQ. Under the conditions of the present study, the test item showed a 72h EC₅₀ of 2.54 mg/L for the growth rate and 0.72 mg/L for the yield with a 72-h EC₁₀ of 0.52 mg/L for growth and 0.21 mg/L for the yield of the unicellular green alga Pseudokirchneriella subcapitata. The accuracy and reliability of the test method is demonstrated periodically as recommended by OECD Guideline No. 201 and the Commission Regulation (EU) 2016/266.

 

For additional supporting data, QSAR was used for the evlaution of the test item:

Using ECOSAR v1.11 the toxicity 96-h EC50 and NOEC values for Algae were determined 1.531 mg/L and 0.691 mg/L, respectively. The substance is within the applicability domain of the model.

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

 

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.