6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

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:

1991-06-21 to 1991-06-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 mg/L
- Sampling: Water samples (200 mL in duplicate) were taken from test and solvent control cultures at 0 and 72 hours and sent to the HRC Department of Analytical Chemistry for analysis.

Vehicle:

yes

Remarks:

1% Tween 80-acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg test substance was dissolved in auxiliary solvent and the volume made up to 10 mL, 10 µL aliquots of the stock solution dispensed to 100 mL of algal suspension on six occasions to give the required number of replicates at the test level.
- Controls: untreated control
- Chemical name of vehicle: organic solvent

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain No: CCAP 276/20
- Source: Cultre Centre of Algae and Protozoa c/o Freshwater Biological Association, Cumbria, U.K.
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and incubated under continuous illumination ( = 7000 lux) and stirring (orbital shaker) at 24 °C to give an algal suspension in log phase growth characterised by an absorbance of 0.062 (at 665 nm). The suspension was diluted to an absorbance of 0.022 prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

ca. 8

Nominal and measured concentrations:

Nominal: 1.0 mg/L
Measured: 0.76 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type: loosely stoppered
- Material, size, headspace, fill volume: glass , 250 mL, 150 mL, 100 mL
- Aeration: No
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 0.08 mg/L
Na2EDTA.2H2O 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 3x10^-3 mg/L
CoCI2.6H2O 1.5x10^-3 mg/L
CuCI2.2H2O 10^-5 mg/L
Na2MoO4.2H2O 7x10^-3 mg/L
NaHCO3 50 mg/L



OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: absorbance measured at 665 nm, at 0, 24 48 and 72 h.

TEST CONCENTRATIONS
- Test concentrations: nominal 1.0 mg/L, measured 0.76 mg/L

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.76 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.76 mg/L

Nominal / measured:

meas. (not specified)

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 (for algal test): 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
- Effect concentrations exceeding solubility of substance in test medium: Yes

Table 1: Absorbance Values

Treatment	Conc., mg/L	Absorbance Values								pH
		0 h	Mean	24 h	Mean	48 h	Mean	72 h	Mean	0 h	72 h
Control	0	0.022	0.022	0.078	0.077	0.203	0.204	0.354	0.351	7.9	8.0
		0.022		0.078		0.213		0.359		7.9	8.1
		0.022		0.074		0.195		0.341		7.9	8.1
Test tem	0.76	0.026	0.026	0.087	0.085	0.185	0.208	0.362	0.345	7.9	8.1
		0.026		0.090		0.221		0.349		7.9	8.0
		0.026		0.087		0.215		0.348		7.9	8.0
		0.026		0.085		0.203		0.342		7.9	8.1
		0.026		0.079		0.221		0.337		7.9	8.1
		0.026		0.084		0.201		0.329		7.9	8.1
Solvent Control	0	0.022	0.022	0.068	0.072	0.179	0.196	0.395	0.371	7.9	8.1
		0.022		0.070		0199		0.347		7.9	8.1
		0.022		0.077		0.211		0.342		7.9	8.1

Table 2: Inhibition of Growth

Measured Conc., mg/L	Area under the Curve (72 h)	% Inhibition	Growth Rate (24 – 48 h)	% inhibition
Solvent Control	9.564	-	0.042	-
0.76	9.612	<0.5>	0.037	12

<Increase>

Validity criteria fulfilled:

not specified

Conclusions:

In this algae growth inhibition study a 72 h-EC50 of above 0.76 mg/L was determined with regard to growth rate. The respective NOEC was set to above or equal 0.76 mg/L.

Executive summary:

In a 72 hour acute study, cultures of Scenedesmus subspicatus were exposed to the test substance at a nominal concentration of 1 mg/L in accordance with OECD guideline 201 (Huntingdon, 1991). 1.0 mg/L was the highest test concentration that could be prepared due to the limited solubility of the test substance in water and having regard to the amount of auxiliary solvent permitted in the test. However, because of the unstable nature of the test substance in water under light conditions, the calculated mean measured value of 0.76 mg/L over the study period has been quoted. Analysis of test concentrations at 0 and 72 hours expressed as % of nominal were 93 % and 59 % respectively. No abnormalities were observed in any test or control culture inspected at 72 hours.

The NOEC and EC50 values based on growth rate were above or equal to 0.76 mg/L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: The stock solutions were prepared with Tween 80 which was diluted into test water. Appropriate amounts of this stock were added to test water to achieve the required concentration.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.65 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 3.65 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

The substance is poorly water soluble and was tested above the solubility limit. Concentrations higher than those tested could not be maintained in dispersion. No significant inhibition was observed at the highest concentration tested. The bioavailability of the substance cannot be guaranteed during the test and care should be taken in interpreting the results. The figures for EbC50 and NOEC are based on measured concentrations. ErC50 and NOErC are not given.

Validity criteria fulfilled:

not specified

Conclusions:

In this algae growth inhibition study a 72h-EC50 of above 3.65 mg/L was determined with regard to biomass. The respective NOEC was set to above or equal 3.65 mg/L.

Executive summary:

In a 72 hour acute toxicity study, culture of Scenedesmus subspicatus were exposed to the test substance under semi-static conditions in accordance with EU method C.3 as cited in Commission Directive 92/69/EEC (RCC, ECHA RSS, 2017). The stock solutions were prepared with Tween 80 which was diluted into test water. Appropriate amounts of this stock were added to test water to achieve the required concentration.

The substance is poorly water soluble and was tested above the solubility limit. Concentrations higher than those tested could not be maintained in dispersion. No significant inhibition was observed at the highest concentration tested. The bioavailability of the substance cannot be guaranteed during the test and care should be taken in interpreting the results. The figures for EbC50 and NOEbC are above or equal to 3.65 mg/L (biomass) and are based on measured concentrations. EC50 and NOEC are not given for growth inhibition.

Description of key information

In an algae test according to OECD guideline 201 (Huntingdon, 1991), an EC50 of >0.76 mg/L and a NOEC of 0.76 mg/L based on growth rate was observed.

Additional information

Key study

In a 72 hour acute study, cultures of Scenedesmus subspicatus were exposed to the test substance at a nominal concentration of 1 mg/L in accordance with OECD guideline 201 (Huntingdon, 1991). 1.0 mg/L was the highest test concentration that could be prepared due to the limited solubility of the test substance in water and having regard to the amount of auxiliary solvent permitted in the test. However, because of the unstable nature of the test substance in water under light conditions the calculated mean measured value of 0.76 mg/L over the study period has been quoted. Analysis of test concentrations at 0 and 72 hours expressed as % of nominal were 93 % and 59 % respectively. No abnormalities were observed in any test or control culture inspected at 72 hours.

The NOEC and EC50 values based on growth rate were above or equal to 0.76 mg/L and 0.76 mg/L, respectively.

In accordance with Article 25(3) of the REACH Regulation the robust study summaries submitted 12 years previously, are additionally used for the purpose of the registration.

Supporting study

In a 72 hour acute toxicity study, culture of Scenedesmus subspicatus were exposed to the test substance under semi-static conditions in accordance with EU method C.3 as cited in Commission Directive 92/69/EEC (RCC, ECHA RSS, 2017). The stock solutions were prepared with Tween 80 which was diluted into test water. Appropriate amounts of this stock were added to test water to achieve the required concentration.

The substance is poorly water soluble and was tested above its solubility limit. Concentrations higher than those tested could not be maintained in dispersion. No significant inhibition was observed at the highest concentration tested. The bioavailability of the substance cannot be guaranteed during the test and care should be taken in interpreting the results. The figures for EbC50 and NOEbC are above or equal to 3.65 mg/L (biomass) and are based on measured concentrations. EC50 and NOEC are not given for growth inhibition.