Enzymatic hydrolysis products of Laminaria japonica, S. japonica, Laminariaceae

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:

August - September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: in accordance with guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Principles of method if other than guideline:

EU Commission Directive 440/2008, Part C.3.

GLP compliance:

yes (incl. QA statement)

Remarks:

by Italian Ministero della Salute

Test material

Test material information

Constituent 1

Reference substance name:

Ascophyllum nodosum, ext.

EC Number:

283-907-6

EC Name:

Ascophyllum nodosum, ext.

Cas Number:

84775-78-0

Molecular formula:

Not applicable; a generic molecular formula cannot be provided for this UVCB substance.

IUPAC Name:

Ascophyllum nodosum, extract

Details on test material:

- Name of test material (as cited in study report): Algea Fert Solid K+
- Substance type: fertilizer ( algal extract)
- Physical state: solid, black micro-flakes
- Lot/batch No.: 00680
- Expiration date of the lot/batch: March 2012
- Stability under test conditions: Stable under suitable storage conditions.
- Storage condition of test material: original tightly-closed containers in well- ventilated place.

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

The test concentrations were chosen based on the results of a pre-test (range-finding test).The main test included five concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L. A negative control without the test item was also run to check the acceptability of the algal population used in the assay.

The test medium was prepared as follows: prior to the start of the test, a stock solution of 100.0 mg/L was prepared by direct weighing into algal culture medium (0.1005 g of test item into 1000 mL of algal culture medium. The obtained solution appeared light-brown and it was manually mixed to achieve the totally solubilisation of test item; then the test concentrations were prepared by taking adequate aliquots of stock solution and diluting them into the algal medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchnerella subcapitata
- Source (laboratory, culture collection):cultured in the ChemService laboratory and originally obtained from the IRSA-CNR (Water Research Institute-Italian Research Council), Brugherio, Milano.
- Method of cultivation: the stock culture were weekly transferred to fresh medium and maintained in continuous shaking to ensure the necessary amount of CO2 and to keep algae in suspension.
- Culturing media and conditions (same as test or not): The culture medium was the same of the test medium.
- Any deformed or abnormal cells observed: Cultures containing deformed or abnormal cells were discarded. Only exponentially growing algal cultures have been used to start the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

from Sept 06, 2010 to Sept 09, 2010

Test conditions

Test temperature:

The test was incubated in a temperature-controlled room, under continuous illumination.
The ambient temperature under the lights was measured twice a day by the mean of a digital thermometer. It was in the range 24.5 – 25.5 °C during the test period.

pH:

At the beginning of the test, the pH of test item solutions was in the range 7.95 – 8.14 and the pH of algal culture medium was 7.93. At the end of the test the pH values of control ranged between 9.39 – 9.42, with a difference lower than 1.5 units as provided by OECD guideline, the pH mean values of test item solutions after 72 hours were in the range 9.03 – 9.42.

Details on test conditions:

All the described procedures were conducted under a laminar flow bench to ensure the sterility of the algal cultures.
Since the test item solutions were coloured, the test was performed as follows:
3 replicates for each tested concentration and 6 for the control were prepared and each flask was placed in a black cylinder covered with glass dishes containing purified water, so that the light was filtered by the solution contained into the glass dishes.
At the same time, an equal number of replicates for each concentration were prepared but in this case the glass dishes on the top of the flasks were filled with the coloured test item and the flasks below were filled with algal medium without test substance (as in the control). The depth of the test solution in the glass dishes was half of the depth of the test media in the Erlenmeyer flasks.
The first set of flasks allowed to asses the algal growth inhibition caused by the toxic effect of the test item and additionally to the reduced light intensity determined by the colour of the test media, while the second set was used to determine the growth inhibition caused only by the reduced light intensity determined by the colour.
For the test concentrations and for the negative control, 100 mL capacity conical glass flasks capped with air permeable stoppers were used.
Three replicates for each test concentration and six for control were prepared. At the start of the test, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain a starting cell density of around 104 cells/mL.
The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test in order to ensure that the assay was performed with exponentially growing algal population. The cell density of the inoculum was around 106 cells/mL.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol pestanal analytical standard

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield rate

Remarks on result:

other: range 9.52– 30.65

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

39.52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: range 19.29 – 55.60

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

25.99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield rate

Remarks on result:

other: range 8.98 – 43.03

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

63.51 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: range 55.58 – 69.63

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

60.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield rate

Remarks on result:

other: range 47.56 – 71.05

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Cell density was measured every 24 hours by fluorescent reading with a spectrofluorophotometer in few millilitre samples taken from each test concentration replicate and from controls. The observed values were used to calculate the percentage inhibition of cell growth as yield and as growth rate in comparison to the control.

At the end of the test the cell density in the negative control was increased on average by a factor of 58. 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 12.7% and a coefficient of variation of average growth in replicates control cultures during the test period of 6.0%.

In the test item solution the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 2.4% for the nominal concentration of 17.8 mg/L to a maximum value of 45.3% for the highest concentration tested, compared to the negative control.
The lowest concentration of 10.0 mg/L did not shown any inhibitory effect, being the algal growth rate higher than the control (mean % inhibition : negative value).

The yield (y) decreased by a minimum value of 9.2% for the nominal concentration of 17.8 mg/L, to a maximum value of 85.6% for the highest concentration, compared to the negative control.
The lowest concentration of 10.0 mg/L did not shown any inhibitory effect, being the algal growth rate higher than the control (mean % inhibition : negative value).

Results with reference substance (positive control):

The results of the most recently performed test with a reference substance are reported and compared with literature data to demonstrate that the algal strain used has got an adequate level of sensitivity to that substance. In the current case the results refer to a test performed on April 2010 (CHE-I-003/2010) with 3,5-Dichlorophenol.

Effect of 3,5-dichlorophenol on the growth of Pseudokirchneriella subcapitata
ErC50 (mg/L) in Chemservice study CHE-I-003/2010: 4.12 (3.32 -4.98)
ErC50 (mg/L) in Literature data (ref.ISO 8962, 2004): 3.38 (2.08-4.68)

Reported statistics and error estimates:

The CETIS v.1.026D software was used to carry out the statistical analysis.
The statistical comparison with the control at each observation time was determined by Bonferroni adjusted test for both the end-points, growth rate and yield, after analyzing the normality of distribution and the homogeneity of variance by Shapiro-Wilk and Barlett’s test respectively.

The EyCx and the ErCx values were determined, respectively, by a linear interpolation analysis, chosen by the PC software as more appropriate.
The negative control was considered to check that the growth of the algal population used in the test meets the validity criteria.
As input data nominal concentrations were used.

Any other information on results incl. tables

Table 3      Effects of Algea Fert Solid K+ on the growth ofPseudokirchneriella subcapitata

Nominal test item 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	4.1525 5.5916 5.0122 4.2801 4.4333 4.6894 4.6932	17.1655 17.5605 18.6935 16.4315 19.6465 15.6895 17.5312	56.4972 60.2282 60.3152 58.5442 61.6072 53.3802 58.4287
10.0	A B C Average value	1 1 1 1	11.7871 6.4638 9.2301 9.1603	37.1473 33.3263 37.6223 36.0320	63.2406 60.4476 55.0386 59.5756
17.8	A B C Average value	1 1 1 1	4.9173 3.6157 6.1038 4.8789	28.5387 24.5847 21.2507 24.7914	46.1587 53.0567 60.2447 53.1534
31.6	A B C Average value	1 1 1 1	3.9049 4.8206 4.1652 4.2969	22.8028 16.9368 13.1108 17.6168	37.9150 50.3660 42.0900 43.4570
56.2	A B C Average value	1 1 1 1	3.3782 3.8174 4.8609 4.0188	10.1922 16.8776 11.4339 12.8346	27.9553 32.7373 35.8073 32.1666
100.0	A B C Average value	1 1 1 1	5.1789 3.2978 4.3991 4.2919	5.7739 7.6071 5.9551 6.4454	9.5444 8.2524 10.0660 9.2876

Table 4      Effect of light absorption of different concentration of Algea Fert Solid K+ on the cells growth of Pseudokirchneriella subcapitata

Nominal test item 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	4.6986 8.3976 6.8812 6.6591	24.5315 23.2155 17.9445 21.8972	58.5772 61.6432 56.7162 58.9789
17.8	A B C Average value	1 1 1 1	8.2356 6.4985 7.0686 7.2676	17.0415 21.3525 21.1655 19.8532	58.8482 62.4772 54.9112 58.7455
31.6	A B C Average value	1 1 1 1	4.7050 6.1830 6.3378 5.7419	22.1925 27.3625 23.5625 24.3725	58.8792 64.8672 67.7562 63.8342
56.2	A B C Average value	1 1 1 1	8.3633 7.4305 7.6430 7.8123	18.1535 18.9295 17.8585 18.3138	59.2012 59.8792 55.7392 58.2732
100.0	A B C Average value	1 1 1 1	7.4103 7.7378 8.5144 7.8875	19.6145 17.3635 18.3235 18.4338	60.0722 62.1312 55.2462 59.1499

Table 5      Algal growth rate and inhibition caused by Algea Fert Solid K+ 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)	64.2	-----	59.6	-----	56.5	-----
10.0	91.1	-41.8	74.6	-25.2	56.7	-0.5*
17.8	65.1	-1.4	66.7	-12.0	55.1	2.4
31.6	60.6	5.6	59.2	0.6	52.3	7.4
56.2	57.5	10.5	52.7	11.6	48.1	14.8
100.0	60.0	6.6	38.7	35.1	30.9	45.3

* the cells growth rate was greater than the negative control

  

Table 6: E_rC₁₀, E_rC₂₀and E_rC₅₀for each observation time together with their confidence limits

Time	ErC10(mg/L)	ErC20(mg/L)	ErC50(mg/L)
24 h	14.84 (10.74 – 38.25)	26.95 (3.49 – n.d.)	> 100.0
48 h	29.39 (18.09 – 54.48)	52.44 (8.26 – 76.02)	> 100.0
72 h	39.52 (19.29 – 55.60)	63.51 (55.58 – 69.63)	> 100.0

 n.d. not determined

Table 7: Algal yield and growth inhibition caused by Algea Fert Solid K+ 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)	584287	-----
10.0	595756	-2.0*
17.8	531534	9.2
31.6	434570	26.1
56.2	321666	45.7
100.0	92876	85.6

* the cells growth was greater than the negative control

 

Table 8: E_yC₁₀, E_yC₂₀and E_yC₅₀at 72 hours together with their confidence limits

 

Time	EyC10(mg/L)	EyC20(mg/L)	EyC50(mg/L)
72 h	17.74 (9.52– 30.65)	25.99 (8.98 – 43.03)	60.35 (47.56 – 71.05)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

•A test is not acceptable if the cell density in the control cultures has not increased by a factor of at least 16 within the test period. •The coefficient of variation of daily growth rates in the control cultures must not exceed 35%. •The coefficient of

Conclusions:

The toxicity of test item Algea Fert Solid K+ was tested on Pseudokirchneriella subcapitata. The EC10, EC20 and EC50 with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) for both the end-points after 72 hours, calculated based on the nominal concentrations were as follows:

Endpoint 72 h EC10 (mg/L) 72 h EC20 (mg/L) 72 h EC50 (mg/L)
Yield 17.74 (9.52– 30.65) 25.99 (8.98 – 43.03) 60.35 (47.56 – 71.05)
Growth rate 39.52 (19.29 – 55.60) 63.51 (55.58 – 69.63) > 100.0

Executive summary:

The influence of the test item Algea Fert Solid K+ on the growth of the green algal species Pseudokirchneriella subcapitata, formerly known as Selenastrum capricornutum, was investigated in a 72-hour full test according to the EU Commission Directive 440/2008,Part C.3., and to the OECD Guideline No. 201, 2006. 

For this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous test medium containing the test item at five nominal concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/L, under defined conditions. Besides the five concentrations, a negative control without the test item was also prepared to check the acceptability of the algal inoculum.

Algal cell density was measured every 24 hours by fluorescent reading with a spectrofluorophotometer in few millilitre samples taken from each test concentration replicate and from controls.

At the beginning of the test, the pH of test item solutions was in the range 7.95 – 8.14 and the pH of algal culture medium was 7.93. At the end of the test the pH values of control ranged between 9.39 – 9.42, with a difference lower than 1.5 units as provided by OECD guideline, the pH mean values of test item solutions after 72 hours were in the range 9.03 – 9.42.

 Light intensity during the test period was in the range 4873 - 5042 Lux (within the range 4000 - 6000 Lux).

The room temperature was in the range 24.5 –during the test period, according to the OECD’s recommended range (24.0±2.0 °C).

In the negative control the cell density increased on average by a factor of 58. This value complies with the validity criteria of the test according to the mentioned guidelines, such as the coefficient of variation of daily growth rates (12.7%) and the coefficient of variation of average growth in replicates control cultures during the test period (6.0%).

In the test item solution the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 2.4% for the nominal concentration of 17.8 mg/L to a maximum value of 45.3% for the highest concentration tested, compared to the negative control.

The yield (y) decreased by a minimum value of 9.2% for the nominal concentration of 17.8 mg/L, to a maximum value of 85.6% for the highest concentration tested, compared to the negative control.

After 72 hours of exposure, the lowest concentration (10.0 mg/L) did not show any inhibitory effects on the algal growth for both end-points. The 72-hour EC₁₀, EC₂₀and EC₅₀with their 95% confidence limits calculated on the basis of the nominal concentrations of product, for both the end-points, are reported in the following table:

Endpoint	72 h EC10(mg/L)	72 h EC20(mg/L)	72 h EC50(mg/L)
Yield	17.74 (9.52– 30.65)	25.99 (8.98 – 43.03)	60.35 (47.56 – 71.05)
Growth rate	39.52 (19.29 – 55.60)	63.51 (55.58 – 69.63)	> 100.0

t the end of test period the two lowest concentrations (10.0 and 17.8 mg/L) did not shown any significant biological effects due to test item (Bonferroni adjusted test).

Since the test item solutions were not clear, a parallel modified test was performed in order to define the inhibition caused by light absorption rather than by toxicity of test item.

Based on results of modified test, no adverse effects due to light absorption were showed (Bonferroni Adjusted test).