Amorphophallus campanulatus dry purified extract

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:

2009

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Test article name : Amorphophallus campanulatus dry purified extract
Batch code : 29895/M1
Chemical class : Botanical extract
Preparation date : April, 2009
Expiry date : April, 2011
Physical form at 20°C : Powder
Colour : Pale brown
Storage conditions : The product was stored at room temperature, in well ventilated room, in its own original container
Safety precautions : Routinely hygienic procedures

Sampling and analysis

Analytical monitoring:

yes

Test organisms

Test organisms (species):

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

Study design

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

the ambient temperature under the lights was continuously measured by the mean of a data logger. It was in the range 22.9 – 25.0 °C.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.3 mg/L

Basis for effect:

other: yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

67.64 mg/L

Basis for effect:

growth rate

Any other information on results incl. tables

A full test was performed in accordance with the OECD Guideline No. 201, 2006 to evaluate the effect of the test itemAmorphophallus campanulatusdry purified extract onPseudokirchneriella subcapitata.

Cell density was measured every 24 hours by fluorescent reading with a spectrofluorophotometer in few millilitre samples taken from each test concentration replicate, from controls and from the screened solution replicate. The obtained cell densities for each replicate are reported in Table 2 while, in Table 3, the effect due to the light absorption on the growth ofPseudokirkneriella subcapitatais shown.

Table 2           Effect ofAmorphophallus campanulatusdry purified extract on the growth ofPseudokirchneriella subcapitata

Nominal test concentration [mg/L]		replicate		Cell density (n. cells x 10^4/ml)
			0 h		24 h		48 h		72 h
Negative control		A B C D E F Average value		1 1 1 1 1 1 1		4.4366 4.1781 4.7081 4.0745 5.2250 5.8142 4.7394		24.6571 23.1341 25.8591 20.5951 30.5911 26.7931 25.2716		108.5761 97.7271 100.4801 103.5571 96.2481 95.2161 100.3008
10.0		A B C Average value		1 1 1 1		7.1843 6.8256 7.1586 7.0562		35.8485 32.6505 32.2175 33.5722		66.8259 74.6289 73.1539 71.5362
17.8		A B C Average value		1 1 1 1		7.2384 7.0005 6.8546 7.0312		25.7441 29.6791 24.7501 26.7244		59.3372 57.3452 61.0382 59.2402
31.6		A B C Average value		1 1 1 1		7.0105 7.7756 6.8038 7.1966		18.7332 15.3892 17.4572 17.1932		29.3407 33.4007 30.8317 31.1910
56.2		A B C Average value		1 1 1 1		5.9786 5.4058 5.7304 5.7049		9.2845 8.1475 10.6835 9.3718		10.4731 12.3351 13.0241 11.9441
100.0		A B C Average value		1 1 1 1		5.4599 3.7495 4.6668 4.6254		4.7105 7.6995 5.5585 5.9895		6.8650 3.6790 5.1190 5.2210

Table 3      Effect of light absorption of different concentration ofAmorphophallus campanulatusdry purified extract on the cells growth ofPseudokirchneriella subcapitata

Nominal test concentration [mg/L]		replicate		Cell density (n. cells x 10^4/ml)
			0 h		24 h		48 h		72 h
10.0		A B C Average value		1 1 1 1		4.1217 4.5441 5.5193 4.7284		25.5941 29.7601 30.3451 28.5664		115.8421 114.5141 112.3181 114.2248
17.8		A B C Average value		1 1 1 1		5.1389 5.7563 6.3397 5.7450		26.3251 31.8061 32.6961 30.2758		107.1571 110.1811 112.7431 110.0271
31.6		A B C Average value		1 1 1 1		3.5398 4.9475 4.9214 4.4696		30.0461 24.1771 25.0631 26.4288		101.4901 100.9891 98.7921 100.4238
56.2		A B C Average value		1 1 1 1		5.0620 8.0731 4.0552 5.7301		32.2501 27.1951 28.5851 29.3434		108.0861 116.2961 112.3621 112.2481
100.0		A B C Average value		1 1 1 1		4.5882 8.9107 2.8642 5.4544		24.2611 26.3421 28.7721 26.4584		106.1041 100.7901 106.8681 104.5874

The data obtained by the modified test, reported in Table 3, were analysed for the statistical comparison with control by Dunnett’s Multiple Comparison for the algal growth rate and by Bonferroni adjusted test for the yield, after analysing the normality of distribution and the homogeneity of variance by Shapiro-Wilk test and Barlett test respectively. No significant effect due to test item light absorption on the cells growth was observed, however the data will be recorded with the raw data under the code of this study.

At the end of the test the cell density in the negative control was increased on average by a factor of 100. This value complies with the validity criteria of the test, according to the mentioned guidelines, which indicate a minimum increase factor of 16.

The negative control met also the other validity criteria, with a coefficient of variation of daily growth rates of 9.5% and a coefficient of variation of average growth in replicates control cultures during the test period of 6.8%.

The growth curves for each replicate of the negative control are represented in Figure 1, the growth curves for each concentration are represented in Figures 2 to 6, while Figure 7 represents the comparison between the growth curves of the different concentrations and of the control calculated on the mean density values.

In the test solutions the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 7.3% for the concentration of 10.0 mg/L to a maximum value of 64.1% for the highest tested concentration, compared to the negative control.

The percentages of growth inhibition for the tested concentrations are reported in Table 4.

Table 4      Algal growth rate and inhibition caused byAmorphophallus campanulatusdry purified extract over 72 hours of exposure in comparison with control

Nominal test item concentration [mg/L]		24 h			48 h			72h
		Mean growth rate (x10^-3)		Mean inhibition %		Mean growth rate (x10^-3)		Mean inhibition %		Mean growth rate (x10^-3)		Mean inhibition %
0 (negative control)		64.8		-----		67.3		-----		64.0		-----
10.0		81.4		-25.6		73.2		-8.8		59.3		7.3
17.8		81.3		-25.4		68.5		-1.7		56.7		11.4
31.6		82.2		-26.8		59.3		11.9		47.8		25.3
56.2		72.6		-11.9		46.6		30.7		34.5		46.2
100.0		63.8		1.6		37.3		44.6		23.0		64.1

The statistical analysis was carried out by the mean of CETIS elaboration software v1.026D, which automatically selects the more appropriated statistic analysis. In this case the E_rC calculation was performed using a linear interpolation at 24 and 48 hours observation and a non linear regression analysis at 72 hours.

The E_rC₁₀, the E_rC₂₀and the E_rC₅₀for each observation time together with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) based on nominal product concentrations were reported in the following table:

Time		ErC10(mg/L)		ErC20(mg/L)		ErC50(mg/L)
24 h		70.06 (47.94 – n.d.)		> 100.0 (n.d. – n.d.)		> 100.0 (n.d. – n.d.)
48 h		25.73 (20.23 – 31.49)		37.61 (29.15 – 45.12)		> 100.0 (n.d. – n.d.)
72 h		12.52 (5.41 – 19.85)		24.61 (16.97 – 32.58)		67.64 (57.35 – 78.96)

n.d. not determined

After 72 hours of exposure, yield (y) decreased by a minimum value of 29.0% for the nominal concentration of 10.0 mg/L, to a maximum value of 95.7% for the highest concentration, compared to the negative control.

The percentages of growth inhibition in terms of biomass for the tested concentrations are reported in Table 5.

Table 5      Algal yield and growth inhibition caused byAmorphophallus campanulatusdry purified extract over 72 hours of exposure in comparison with negative control

Nominal test item concentration (mg/L)	Mean biomass at 72h (cell/ml)	Mean growth inhibition %
0 (Negative control)	1003008	-----
10.0	715362	29.0
17.8	592402	41.3
31.6	311910	69.6
56.2	119441	89.0
100.0	52210	95.7

The E_yC_xcalculation was performed by a linear interpolation analysis. The E_yC₁₀, the E_yC₂₀and the E_yC₅₀at 72 hours together with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) were as reported below :

Time	EyC10(mg/L)	EyC20(mg/L)	EyC50(mg/L)
72 h		3.45 (2.49 – 4.70)		6.90 (4.98 – 9.39)		22.03 (19.29 – 24.16)

The concentration-response plots at 72 hours for growth rate and yield end-points are shown respectively in Figures 8 and 9 for the results expressed as nominal product concentrations,

At the beginning of the test period, the pH values of test medium ranged between 7.87 and 8.02, and at the end the range was between 8.13 and 9.02. The mean values are reported in Table 6.

The pH in the control varied by 1.11 units, so the pH validity criterium was met.

Table 6      pH values in the test media at the start and at the end of the test.

Nominal test item concentration	Start ( 0 hours)	End ( 72 hours)
(mg/L)	pH	pH (mean values)
Negative control	7.89	9.02
10.0	8.02	8.45
17.8	7.90	8.41
31.6	7.93	8.18
56.2	7.88	8.17
100.0	7.87	8.13

The temperature under the test light was in the range 22.9 – 25.0 °C along the exposure time, with a mean value of 24.0 °C and a standard deviation of 0.5 °C during the test period, according to the OECD recommended range (24.0±2.0 °C).

The registration of the temperature is reported in Figure 10.