3-(p-cumenyl)propionaldehyde

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:

23 Jan 2017 to 17 Feb 2017

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:

2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Stock solution preparation: A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100 mg/L stock solution.
- Preparation dilutions: A series of dilutions was made from this stock solution to give stock solutions of 1.0, 3.2, 10 and 32 mg/L. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with 12.2 mL of algal suspension to give the required test concentrations. Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

STOCK MAINTENANCE
- Master culture: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.

ACCLIMATION
- Pre-culture: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1E+03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 1E+04 - 1E+05 cells/mL.
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

- Test start 7.7 - 8.4
- Test end: 8.4 - 10.1

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L (based on a range-finding study)
- Measured concentrations (geometric means): 0 (control), 0.49, 2.3, 8.1, 30 and 96 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL ground glass conical flasks, completely filled and sealed with ground glass stoppers
- Type: closed
- Aeration: no
- Initial cells density: 5E+03 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3
- Test environment: an incubator (INFORS Multitron Version 2 incubator), where vessels were constantly shaken at approximately 150 rpm

GROWTH MEDIUM
- Standard medium used: AAP-medium with additional sodium bicarbonate (250 mg/L instead of 15 mg NaHCO3/L).

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: The culture medium used for both the range finding and definitive tests was the same as that used to maintain the stock culture. The media used in both the range finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system.
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5E+03 cells/mL) was taken as the starting cell density.
- Appearance of cells: To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- Other: pH (start and end of the test), temperature (daily) and appearance of medium (daily) were determined.

RANGE-FINDING STUDY
- Test concentrations: nominal test concentrations of 0 (control),0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L.: 9.1 - 13 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Growth data: The cell numbers recorded during the final test indicate a clear concentration-response relationship. Growth rate was inhibited from a test concentration of 10 mg/L (32% inhibition) and increased in a dose-dependent manner up to 93 and 106% inhibition at 32 and 100 mg/L, respectively. See 'Any other information on results incl. tables' for algal densities measured during the test and derived data on growth rate and yield.
- Observation of abnormalities: After 72 hours there were no abnormalities detected in the control or test cultures at 0.49, 2.3 and 8.1 mg/L, however, no intact cells were observed to be present in the test cultures at 30 and 96 mg/L.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period the control, 0.49 and 2.3 mg/L test cultures were observed to be green dispersions, the 8.1 mg/L test cultures were observed to be pale green dispersions and the 30 and 96 mg/L test cultures were observed to be clear colorless solution.

Results with reference substance (positive control):

The most recent test performed between 28 November 2016 and 01 December 2016, determined a 72h-ErC50 of 1.4 mg/L (95% C.L.: 1.2 - 1.5 mg/L).

Reported statistics and error estimates:

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line. Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table: Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 h	24 h	48 h	72 h	72 h
Control	R1	7.7	3.35E+04	1.52E+05	3.98E+05	10.1
	R2		3.33E+04	1.68E+05	4.21E+05
	R3		2.93E+04	1.36E+05	3.94E+05
	R4		2.95E+04	1.71E+05	4.36E+05
	R5		2.78E+04	1.53E+05	3.60E+05
	R6		3.05E+04	1.63E+05	4.30E+05
	Mean		3.06E+04	1.57E+05	4.07E+05
1	R1	7.8	3.12E+04	1.79E+05	3.72E+05	10.3
	R2		3.50E+04	1.79E+05	4.20E+05
	R3		2.96E+04	1.59E+05	3.73E+05
	Mean		3.19E+04	1.72E+05	3.89E+05
3.2	R1	7.8	2.70E+04	1.46E+05	4.12E+05	10.3
	R2		2.47E+04	1.55E+05	4.31E+05
	R3		2.39E+04	1.32E+05	4.23E+05
	Mean		2.52E+04	1.44E+05	4.22E+05
10	R1	7.8	1.31E+04	3.86E+04	1.12E+05	9.4
	R2		1.33E+04	3.14E+04	8.07E+04
	R3		1.36E+04	3.54E+04	1.09E+05
	Mean		1.33E+04	3.51E+04	1.01E+05
32	R1	7.9	5.87E+03	5.84E+03	1.00E+04	8
	R2		5.63E+03	4.34E+03	6.51E+03
	R3		5.43E+03	6.66E+03	4.50E+03
	Mean		5.64E+03	5.61E+03	7.01E+03
100	R1	8.4	4.11E+03	3.40E+03	4.45E+03	8.4
	R2		4.55E+03	4.58E+03	4.48E+03
	R3		6.07E+03	4.46E+03	2.93E+03
	Mean		4.91E+03	4.15E+03	3.95E+03

*Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6 = Replicates 1 to 6

Table: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 - 72 h	% Inhibition	0 - 72 h	% Inhibition*
Control	R1	0.061		3.93E+05
	R2	0.062		4.16E+05
	R3	0.061		3.89E+05
	R4	0.062	-	4.31E+05	-
	R5	0.059		3.55E+05
	R6	0.062		4.25E+05
	Mean	0.061		4.02E+05
	SD	0.001		2.83E+04
1	R1	0.06	2	3.67E+05
	R2	0.062	[2]	4.15E+05
	R3	0.06	2	3.68E+05
	Mean	0.061	1	3.84E+05	4
	SD	0.001		2.75E+04
3.2	R1	0.061	0	4.07E+05
	R2	0.062	[2]	4.26E+05
	R3	0.062	[2]	4.18E+05
	Mean	0.062	[1]	4.17E+05	[4]
	SD	0.001		9.17E+03
10	R1	0.043	30	1.07E+05
	R2	0.039	36	7.57E+04
	R3	0.043	30	1.04E+05
	Mean	0.042	32	9.55E+04	76
	SD	0.002		1.73E+04
32	R1	0.01	84	5.02E+03
	R2	0.004	93	1.51E+03
	R3	-0.001	102	-5.04E+02
	Mean	0.004	93	2.01E+03	99
	SD	0.006		2.79E+03
100	R1	-0.002	103	-5.52E+02
	R2	-0.002	103	-5.20E+02
	R3	-0.007	111	-2.07E+03
	Mean	-0.004	106	-1.05E+03	100
	SD	0.003		8.87E+02

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 – R6 =Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

ADDITIONAL EFFECT PARAMETERS

- NOEC: The No Observed Effect Concentrations based on growth rate and yield were 2.3 and 2.3 mg/L respectively. 

- LOEC: The Lowest Observed Effect Concentrations based on growth rate and yield were 8.1 and 8.1 mg/L respectively.

- ErC20: the EC20 -value based on growth rate and yield were 6.5 and 6.4 mg/L, respectively.

Validity criteria fulfilled:

yes

Remarks:

see 'Any other information on materials and methods incl. tables'

Conclusions:

The 72-h ErC50 and 72-h ErC10 are 11 mg/L and 4.8 mg/L, respectively, in green algae (Pseudokirchneriella subcapitata).

Executive summary:

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at the t=0h (start), t=24h, t=48h and t=72h (end). A >20% decline in measured test concentrations was observed in the lower concentrations throughout the duration of the test and therefore the results are expressed in terms of geometric means of the exposure concentration. The average exposure concentrations (geometric means) were calculated to correspond to: 0 (control), 0.49, 2.3, 8.1, 30 and 96 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the algae were inspected microscopically for abnormalities. At the end of the test the percentage inhibition of yield and average specific growth rate were calculated. The pH increased up to 10.1, but was considered not to have adversely affected the study outcome as no effects on controls were observed. In the test no abnormal cells were observed, although no intact cells were observed to be present in the test cultures at 30 and 96 mg/L (measured concentrations). The cell numbers recorded during the final test indicate a clear concentration-response relationship. Growth rate was inhibited from a test concentration of 10 mg/L (32% inhibition) and increased in a dose-dependent manner up to 93 and 106% inhibition at 32 and 100 mg/L, respectively. Yield was also inhibited from 10 mg/L. Based on these findings, the 72-h EC50 values for growth rate and yield were determined at 11 mg/L and 7.3 mg/L, respectively. The 72-h EC10 for growth rate and yield were determined at 4.8 and 5.9 mg/L, respectively.

Description of key information

The toxicity towards freshwater algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours under static conditions. Test concentrations were analytically verified at the t=0h (start), t=24h, t=48h and t=72h (end). A >20% decline in measured test concentrations was observed in the lower concentrations throughout the duration of the test and therefore the results are expressed in terms of geometric means of the exposure concentration. The average exposure concentrations (geometric means) were calculated to correspond to: 0 (control), 0.49, 2.3, 8.1, 30 and 96 mg/L. At the start of the test and after 24, 48 and 72 hours exposure duration, cell densities were determined and the algae were inspected microscopically for abnormalities. At the end of the test the percentage inhibition of yield and average specific growth rate were calculated. The pH increased up to 10.1, but was considered not to have adversely affected the study outcome as no effects on controls were observed. In the test no abnormal cells were observed, although no intact cells were observed to be present in the test cultures at 30 and 96 mg/L (measured concentrations).The cell numbers recorded during the final test indicate a clear concentration-response relationship. Growth rate was inhibited from a test concentration of 10 mg/L (32% inhibition) and increased in a dose-dependent manner up to 93 and 106% inhibition at 32 and 100 mg/L, respectively. Yield was also inhibited from 10 mg/L.Based on these findings, the 72-h EC50 values for growth rate and yield were determined at 11 mg/L and 7.3 mg/L, respectively. The 72-h EC10 for growth rate and yield were determined at 4.8 and 5.9 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L

EC10 or NOEC for freshwater algae:

4.8 mg/L