Phenolphthalein

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:

2010-05-12 to 2010-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted March 23, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken at the beginning of the study and after 72 hours. The samples were delivered to the analytic laboratory for a quantitative analysis.

- Sample storage conditions before analysis: The test medium (reconstituted water and test material) was freshly prepared.

Vehicle:

no

Details on test solutions:

The test medium (reconstituted water and test material) was freshly prepared. Therefore, the calibrated flask with test material and vehicle (reconstituted water) was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was given through a nutsch filter (pore size 10 -16 µm) and the filtrate was used for the study.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: SAG 86.81
- Source: Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

250 mg CaCO3

Test temperature:

22 - 23 °C

pH:

7.94 - 9.48

Nominal and measured concentrations:

The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: open
- Material, size, headspace, fill volume: 300 mL, 100 mL
- Aeration: no
- Initial cells density: 10,000 algae cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: between 4440 and 8880 Lux

EFFECT PARAMETERS MEASURED: The algae cell densities were determined with an electronic particle counter

TEST CONCENTRATIONS
- Test concentrations: 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 3.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.54 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence interval 2 20 - 3.04

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 0.49 - 0.94

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence interval 1.63 - 2.10

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.13 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval 0.87 - 1.34

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval 2.93 - 3.61

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.57 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.57 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control: 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 observations that might cause a difference between measured and nominal values: no

Validity criteria
In the control group the biomass increased by a factor of 107 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 group (section-by-section growth rates) during 72 hours was 8.9 %. 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 1.6 %. According to the OECD test guideline, the coefficient of variation must not be higher than 7 %. Thus, the validity criterion was fulfilled.

Table 1: Analytical concentrations measured in the test media

Nominal concentration [mg/L]	Time after preparation [hours]	Analytical concentrations measured		Mean analytical concentration^1) [mg/L]	Recovery from nominal [%]
		Sample 1	Sample 2
0.0	0 (start)	n.d. n.d. n.d.	-	0.00	-
	72 (end)	n.d. n.d. n.d.	-	0.00	-
10.0	0 (start)	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	<LOQ	-
	72 (end)	<LOQ <LOQ <LOQ	<LOQ <LOQ <LOQ	< LOQ	-
17.8	0 (start)	<LOQ <LOQ <LOQ	<LOQ < LOQ <LOQ	<LOQ	-
	72 (end)	<LOQ <LOQ <LOQ	<LOQ < LOQ <LOQ	<LOQ	-
31.6	0 (start)	<LOQ <LOQ <LOQ	<LOQ 1.032 1.001	<LOQ	-
	72 (end)	<LOQ <LOQ < LOQ	<LOQ <LOQ <LOQ	<LOQ	-
56.2	0 (start)	1.859 1.786 1.738	1.893 1.804 1.787	1.81	3.2 %
	72 (end)	1.844 1.832 1.803	1.811 1.821 1.808	1.82	3.2 %
100.0	0 (start)	3.332 3.366 3.340	3.271 3.395 3.335	3.34	3.3 %
	72 (end)	3.311 3.262 3.437	3.342 3.311 3.288	3.33	3.3%

¹⁾ rounded value

²⁾ n.d. = not detected

³⁾ LOQ = 1 mg/L

 

Table 2: EC values determined for the test item

Parameter (0-72h)	Growth rate	Yield
ECio (mg/L)	1.89	0.74
EC20 (mg/L)	3.22	1.13
EC50 (mg/L)	>3.33	2.54
NOEC (mg/L)	0.57	0.57
LOEC (mg/L)	1.01	1.01

 

 

Validity criteria fulfilled:

yes

Conclusions:

Under the given conditions of this study the 72h EC50 value was 2.5 mg/L for Yield. The EC50 value for the growth rate could not be determined as it exceeds the highest concentration used in this study (> 3.33 mg/L).

Executive summary:

The effect of the test item on the growth and growth rate of the green algae specie Desmodesmus subspicatus was tested in a 72 hour static test according to OECD 201 and EU method C.3. For this purpose, the algae were exposed to different concentrations of the test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water. The study design included one control with six replicates and five test material groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analyzed at 0 (start) and 72 hours after start of the experimental part. At the start and 72 hours after start of the experimental part the recovery rates of the test item in the test media with nominally 56.2 and 100.0 mg/L were 3.2 or 3.3 %, respectively. The test item concentrations were stable in these test media during the test period of 72 hours. The nominal concentrations 10.0, 17.8 and 31.6 mg/L were below the limit of quantification of 1 mg/L. Based on a recovery rate of 3.2 % the analytical concentrations in the three lower concentrated test media (10.0, 17.8 and 31.6 mg/L) were calculated. Under the given conditions of this study the 72h EC50 value was 2.5 mg/L for Yield. The EC50 value for the growth rate could not be determined as it exceeds the highest concentration used in this study (> 3.33 mg/L).

Description of key information

The test item was prepared in reconstituted water and tested in an open static test system. Under the given conditions of this study the 72h EC₅₀ value was 2.5 mg/L for Yield. The EC₅₀ value for the growth rate could not be determined as it exceeds the highest concentration used in this study (> 3.33 mg/L) (reference 6.1.5-1).

Additional information

The effect of the test item on the growth and growth rate of the green algae specie Desmodesmus subspicatus was tested in a 72 hour static test according to OECD 201 and EU method C.3. For this purpose, the algae were exposed to different concentrations of the test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. The study was performed with nominal concentrations of 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L in reconstituted water. The study design included one control with six replicates and five test material groups with three replicates, each containing 100 mL reconstituted water or test medium and about 10,000 cells/mL at the start of the experimental phase. The study was performed in an open static test system. The test material concentrations were analyzed at 0 (start) and 72 hours after start of the experimental part. At the start and 72 hours after start of the experimental part the recovery rates of the test item in the test media with nominally 56.2 and 100.0 mg/L were 3.2 or 3.3 %, respectively. The test item concentrations were stable in these test media during the test period of 72 hours. The nominal concentrations 10.0, 17.8 and 31.6 mg/L were below the limit of quantification of 1 mg/L. Based on a recovery rate of 3.2 % the analytical concentrations in the three lower concentrated test media (10.0, 17.8 and 31.6 mg/L) were calculated. Under the given conditions of this study the 72h EC50 value was 2.5 mg/L for Yield. The EC50 value for the growth rate could not be determined as it exceeds the highest concentration used in this study (> 3.33 mg/L).