calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

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:

January from 19th to 22nd, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Remarks:

temporary certificate

Analytical monitoring:

no

Details on test solutions:

Prior to the start of the test, a culture medium solution of 100 mg/l was prepared by direct weighting into algal culture medium. The obtained suspension was mixed by the means of magnetic stirrer for about 3 days to allow the maximum solubilisation of test item,then, it was filtered by a filtering cartridge (membrane pore: 0.45 um) to obtain a clear solution. The test concentrations were prepared by taking adequate aliquots of the mother solution and diluting them in the algal medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular, fresh-water green algae.
- Source: IRSA-CNR (Water Research Institute-Italian Research Council), Brugherio, Milano.

ACCLIMATION
- Culturing: test organisms were cultured in the testing laboratory.
- Culturing conditions: algae have been cultured in a temperature controlled room at 24.0 ± 2.0 °C under continuous uniform illumination of approximately 6000 Lux in the spectral range 400-700 nm. The culture medium was the same of the test medium; 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.
- 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Ranged between 23.6 and 25.3 °C

pH:

Control: 8.51 - 9.72
Test vessels: 7.97 - 9.82

Nominal and measured concentrations:

Nominal: 10.0, 17.8, 31.6, 56.2 and 100.0 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml capacity conical glass flasks.
- Type: flasks were capped with air permeable stoppers.
- Initial cells density: the cell density of the inoculum was around 10^6 cells/ml.
- No. of vessels per concentration: three replicates.
- No. of vessels per control: six replicates.

INOCULUM
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.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (OECD TG 201 medium) according to OECD Guideline 201, 2006.

OTHER TEST CONDITIONS
- Sterile test conditions: all the describe procedures were conducted under a laminar flow bench to ensure the axenicity of the algal cultures.
- Photoperiod: continuous illumination.
- Light intensity: 6000-10000 lux.

EFFECT PARAMETERS MEASURED
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 with control.
The CETIS v.1.026D software was used to carry out the statistical analyses. The statistical comparison with the control at each observation times was assessed by Dunnett's multiple comparison analysis and by Bonferroni Adjusted Test.
The EyCx was calculated by a non linear regression analysis, while the ErCx values were determined by a linear interpolation method.
The negative control was considered only to check that the growth of the algal population used in the test met the validity criteria. As input data nominal concentrations were used.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: five different concentrations in a geometric series, namely 10.0, 17.8, 31.6, 56.2 and 100.0 mg/l.
- Test concentrations: the test concentrations were chosen based on the results of a pre-test (range-finding test), which was not performed in compliance with the GLP Regulations.

Reference substance (positive control):

yes

Remarks:

3.5 Dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other: 63.77-67.56

Details on results:

ln the test solutions the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 3.5 % for the concentration of 31.6 mg/l to a maximum value of 31.1 % for the highest concentration tested, compared to the negative control. The two lowest concentrations did not show any significant effect after 72 hours of exposure (Dunnet’s multiple comparison analysis).

After 72 hours of exposure, yield (y) decreased by a minimum value of 2.4 % for the nominal concentration of 17.8 mg/l, to a maximum value of 83.4 % for the highest tested concentration, 100 mg/l, compared to the negative control.

ErC10 (72h): 53.86 mg/l (nominal) (CL 95 %: 52.05-55.82)
ErC20 (72h): 76.2 mg/l (nominal) (CL 95 %: 73.37 - 79.41)

EyC10 (72h): 28.31 mg/l (nominal) (CL 95 %: 23.33-32.43)
EyC20 (72h): 41.4 mg/l (nominal) (CL 95 %: 38.49-44.07)

NEGATIVE CONTROL
At the end of the test the cell density in the negative control was increased on average by a factor of 306. 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 34.4 % and a coefficient of variation of average growth in replicates control cultures during the test period of 3.8 %.

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 run on August 2008 with 3.5-Dichlorophenol as reference substance.
Testing laboratory data: ErC50 of 4.08 (3.04 -5.18) mg/l
Literature data: ErC50 of 3.38 (2.08-4.68) mg/l

The ErC10, the ErC20 and the ErC50 were assessed by a linear interpolation; the values for each observation time together with their confidence limits (LCL, Lower Confidence Level and UCL, Upper Confidence Level) based on nominal concentration are here reported

Time	ErC10 (mg/l)	ErC20 (mg/l)	ErC50 (mg/l)
24 h	> 100 ( n.d. - n.d.)	> 100 ( n.d. - n.d.)	> 100 ( n.d. - n.d.)
48 h	82.76 (73.58 - 93.67)	> 100 ( n.d. - n.d.)	> 100 ( n.d. - n.d.)
72 h	53.86 (52.05 - 55.82)	76.20 (73.37 - 79.41)	> 100 ( n.d. - n.d.)
Time	EyC10 (mg/l)	EyC20 (mg/l)	EyC50 (mg/l)
72 h	28.31 (23.33 - 32.43)	41.40 (38.49 - 44.07)	65.66 (63.77 - 67.56)

n.d. not determined

The EyCx calculation was performed using a non linear regression analysis after analyzing the normality of the distribution by Shapiro-Wilk W test and the homogeneity of the variances by modified Levene test.

Algal growth rate and inhibition caused by test item over 72 hours of exposure in comparison with control.

Nominal test item concentration [mg/l]	24 h		48 h		72 h
	Mean growth rate (x 10^-3)	Mean inhibition %	Mean growth rate (x 10^-3)	Mean inhibition %	Mean growth rate (x 10^-3)	Mean inhibition %
0	96.7	-	93.3	-	79.5	-
10.0	101.1	-4.5	92.0	1.3	78.9	0.7
17.8	97.0	-0.3	92.2	1.1	79.1	0.4
31.6	98.4	-1.7	90.8	2.7	76.7	3.5
56.2	101.3	-4.7	88.7	4.9	71.0	10.7
100.0	103.9	-7.4	80.8	13.3	54.8	31.1

Algal yield and growth inhibition caused by test item over 72 hours of exposure in comparison with negative control.

Nominal test item concentration [mg/l]	Mean biomass at 72 h (cell/ml)	Mean growth inhibition %
0	3056003	-
10.0	2938986	3.8
17.8	2983689	2.4
31.6	2494312	18.4
56.2	1657764	45.9
100.0	516896	83.7

Effect of Set Retard Plus on the 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
Negative control	A	1	9.8875	83.5931	309.3186
	B	1	10.7180	87.0031	304.4886
	C	1	8.8414	85.7431	301.0086
	D	1	10.4811	93.0731	312.5086
	E	1	10.2900	88.3431	305.2286
	F	1	10.9300	89.8131	301.0486
	Average	1	10.1913	87.9281	305.6003
10.0	A	1	11.4055	82.5520	297.3286
	B	1	11.1355	83.8920	292.0286
	C	1	11.3877	82.1020	292.3386
	Average	1	11.3096	82.8487	293.8986
17.8	A	1	10.1941	81.7965	298.2789
	B	1	10.2861	88.2465	296.1789
	C	1	10.3001	80.5265	300.6489
	Average	1	10.2601	83.5232	298.3689
31.6	A	1	9.8271	75.4641	246.6579
	B	1	10.6721	81.3741	252.6879
	C	1	11.3361	77.0141	248.9479
	Average	1	10.6118	77.9508	249.4312
56.2	A	1	10.6422	69.2180	168.2731
	B	1	12.3272	73.2860	163.4231
	C	1	11.1612	69.2490	165.6331
	Average	1	11.3769	70.5843	165.7764
100.0	A	1	11.4089	51.0052	55.9893
	B	1	11.9569	49.4632	53.4103
	C	1	12.9449	44.8282	45.6693
	Average	1	12.1036	48.4322	51.6896

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h) > 100 mg/l (nominal)
EyC50 (72h): 65.66 mgl (nominal)

Executive summary:

Method

The influence of the test item 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 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 test item was also prepared to check the acceptability of the algal inoculum.

Observations

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. The pH values of test medium at the beginning of the test ranged between 7.97 and 8.51. Therefore, the pH of the test medium was not adjusted before the start of the test.

The room temperature was in the range 23.6 – 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 306. 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 (34.4 %) and the coefficient of variation of average growth in replicates control cultures during the test period (3.8 %).

In the test item solution the algal growth rate (r) after 72 hours of exposure was inhibited by a minimum value of 3.5 % for the nominal concentration of 31.6 mg/l to a maximum value of 31.1 % for the highest concentration tested, compared to the negative control. The two lowest concentrations did not show any significant effect after 72 hours of exposure (Dunnet’s multiple comparison analysis)

The yield (y) decreased by a minimum value of 2.4 % for the nominal concentration of 17.8 mg/l, to a maximum value of 83.4 % for the highest concentration, compared to the negative control.

Results

The EC10, EC20 and EC50 for both the end-points after 72 hours, calculated based on the nominal concentrations tested were as follows:

Growth rate

EC10 (72h): 53.86 (52.05 -55.82)mg/l (nominal)

EC20 (72h): 76.20 (73.37 – 79.41)mg/l (nominal)

EC50 (72h) > 100 mg/l (nominal)

Yield (biomass)

EC10 (72h): 28.31 (23.33 – 32.43)mg/l (nominal)

EC20 (72h): 41.40 (38.49 – 44.07) mg/l (nominal)

EC50 (72h): 65.66 (63.77 – 67.56)mg/l (nominal)

Description of key information

ErC50 (72h) > 100 mg/l (nominal)

EyC50 (72h): 65.66 mgl (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

53.86 mg/L

Additional information

The influence of the test item 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 OECD Guideline No. 201, 2006.

The EC10, EC20 and EC50 for both the end-points after 72 hours were calculated based on the nominal concentrations tested.

Growth rate

EC10 (72h): 53.86 (52.05 -55.82)mg/l (nominal)

EC20 (72h): 76.20 (73.37 – 79.41)mg/l (nominal)

EC50 (72h) > 100 mg/l (nominal)

Yield(biomass)

EC10 (72h): 28.31 (23.33 – 32.43)mg/l (nominal)

EC20 (72h): 41.40 (38.49 – 44.07) mg/l (nominal)

EC50 (72h): 65.66 (63.77 – 67.56)mg/l (nominal)