Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

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:

28 November 2016 - 26 January 2017

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:

Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, guideline No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission regulation (EC) No. 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C3: “Algal Inhibition Test”; Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item: 207258/AIdentification: MLA-3202Appearance: Clear amber-red liquidBatch: RC-1045Purity/Composition: UVCBTest item storage: At room temperature Stable under storage conditions until: 17 February 2019 (expiry date)Purity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3

Analytical monitoring:

yes

Details on sampling:

On the day of analysis, the test samples were thawed at room temperature. The samples were diluted in a 1:3 (v:v) ratio with methanol and analyzed. If necessary, the samples were further diluted with 75/25 (v/v) methanol/M2-medium to obtain concentrations within the calibration range.Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. Frequency: at t=0 h, t=24 h and t=72 hVolume: 1.0 mLStorage : Samples were stored in a freezer (≤-15°C) until analysis.At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.Additionally, reserve samples of 1.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis. Storage stability of samples under these conditions was demonstrated in project 511870.

Vehicle:

yes

Details on test solutions:

Preparation of test solutionsThe batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg/L in the combined limit/range-finding test and 15 mg/L in the full test. A one hour period of magnetic stirring was applied to accelerate the dissolving of the test item in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colorless up to and including 15 mg/L. The concentration of 100 mg/L in the combined limit/range-finding test was slightly hazy, but considered completely dissolved as no Tyndall effect was observed when using laser pointer. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

Species : Pseudokirchneriella subcapitata, strain: NIVA CHL 1Source : In-house laboratory culture.Reason for selection : This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Ca+Mg: 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C

pH:

6.0-9.0

Dissolved oxygen:

Not specified

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Combined limit/range-finding test Nominal concentrations: 0.10, 1.0 and 10 mg/L.Final test Nominal concentrations: 0.10, 0.35, 1.2, 4.3 and 15 mg/L

Details on test conditions:

Test duration: 72 hoursTest type: StaticTest vessels: 100 mL, all-glass, containing 50 mL of test solutionMedium: M2Cell density: An initial cell density of 1 x 104 cells/mL.Illumination: Continuously using TLD-lamps with a light intensity within the range of 95 to 96 µE.m-2.s-1.Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.16 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between: 0.13 - 0.20

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.66 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between: 0.56 - 0.76

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval between: 8.6 - 11

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between: 0.12 - 0.21

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between: 0.18 - 0.27

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.43 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% confidence interval between: 0.37 - 0.48

Details on results:

Combined limit/range-finding testMean cell densities, inhibition of growth rate and inhibition of yieldBased on these results samples taken from nominal concentrations of 0.10 and 100 mg/L were analyzed. The actual concentrations were at the level of nominal (94-102%) at the start of the exposure and at 5.7-98% of initial at the end of the test, respectively.Therefore, the expected EC50 for growth rate inhibition approximated the nominal concentration of 10 mg/L. The expected EC50 for yield inhibition was between 0.10 and 1.0 mg/L.All test conditions were maintained within the limits prescribed by the study plan.Final testMeasured test item concentrationsSamples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose. The concentrations measured in the solution incubated with algae (nominally 1.2 mg/L) was more stable than its counterpart incubated with algae (78 vs. 45% of initial at the end of the test, respectively). This indicates that the observed decrease could be (at least partly) caused by adsorption to the growing algal biomass. Based on these results, the Time Weight Average (TWA) concentrations were calculated.Inhibition of growth rate and inhibition of yield.Growth rates were in the range of the controls at the two lowest concentrations tested during the 72-hour test period, whereas the growth rate of algae exposed to a TWA concentration of 0.68 mg/L and higher were increasingly reduced. Inhibition of yield increased with increasing concentration of MLA-3202 from 12% at TWA concentration of 0.19 mg/L upwards resulting in 93% inhibition at the highest concentration tested. Statistically significant inhibition of both growth rate and yield was found at the TWA concentrations of 0.68, 3.3 and 14 mg/L.Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.Experimental conditionsBelow shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 unit). During the exposure period the temperature measured in the incubator was maintained at 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

Results with reference substance (positive control):

Potassium Dichromate significantly inhibited the growth rate of this fresh water algal species at nominal concentrations of 0.32 mg/L and higher.The EC50 for growth rate inhibition (72h-ERC50) was 1.3 mg/L with a 95% confidence interval ranging from 1.2 to 1.4 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range.The EC50 for yield inhibition (72h-EYC50) was 0.45 mg/L with a 95% confidence interval ranging from 0.39 to 0.53 mg/L. The historical ranges for yield inhibition lie between 0.20 and 1.1 mg/L. The observed 72h-EYC50 for the algal culture tested corresponds with this range.

Mean cell densities (x10⁴cells/mL) during the combined limit/range-finding test

Time (h)	Nominal concentration of MLA-3202 (mg/L)
	Control	0.10	1.0	10	100
0	1.0	1.0	1.0	1.0	1.0
24	3.9	n.d.	n.d.	n.d.	1.5
48	21.6	n.d.	n.d.	n.d.	3.5
72	101.6	129.6	22.7	8.8	5.5

n.a. – not applicable

Percentage inhibition of growth rate during the combined limit/range-finding test

MLA-3202 Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.537	0.0560	6
0.10	1.617	0.0687	3	-5.2
1.0	1.040	0.0155	3	32.3
10	0.725	0.0369	3	52.8
100	0.558	0.0792	6	63.7

           
Percentage inhibition of yield during the combined limit/range-finding test

MLA-3202 Nominal conc. (mg/L)	Mean	Std. Dev.	n	%Decrease
Control	100.6	15.90	6
0.10	128.6	27.73	3	-27.8
1.0	21.7	1.04	3	78.5
10	7.8	0.95	3	92.2
100	4.5	1.18	6	95.6

 

Measured concentrations versus nominal concentrations

MLA-3202 Nominal concentration (mg/L)	Measured concentration (mg/L)			TWA concentration (mg/L)
	t=0h	t=24h	t=72 h
0.10	0.0877	0.0279	0.0269	0.035
0.35	0.339	0.207	0.103	0.19
1.2	1.12	0.680	0.507	0.68
1.2 WA	1.16	0.946	0.903	0.97
4.3	4.07	3.35	2.76	3.3
15	14.6	14.1	13.2	14

 

Percentage inhibition of growth rate (total test period) during the final test

MLA-3202 TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.693	0.0563	6
0.035	1.721	0.0561	3	-1.6
0.19	1.652	0.0188	3	2.5
0.68	1.242*	0.0389	3	26.7
3.3	0.977*	0.0370	3	42.3
14	0.859*	0.0160	3	49.3

 

Percentage inhibition of growth rate at different time intervals during the final test

MLA-3202 TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.514	0.0	1.843	0.0	1.723	0.0
0.035	3	1.274	15.9	2.151	-16.8	1.737	-0.9
0.19	3	0.880	41.9	2.326	-26.2	1.749	-1.6
0.68	3	0.839	44.6	1.931	-4.8	0.956	44.5
3.3	3	0.772	49.0	1.778	3.5	0.382	77.8
14	3	1.134	25.1	0.849	53.9	0.595	65.5

           
Percentage inhibition of yield during the final test

MLA-3202 TWA conc. (mg/L)	Mean	Std. Dev.	n	%Decrease
Control	161.6	28.44	6
0.035	175.3	28.50	3	-8.4
0.19	141.0	7.95	3	12.8
0.68	40.7*	4.69	3	74.8
3.3	17.8*	2.02	3	89.0
14	12.2*	0.63	3	92.5

* - effect was statistically significant

pH levels recorded during the final test

MLA-3202 TWA conc. (mg/L)	pH
	t=0h	t=72h
Control	8.1	7.9
0.035	8.0	7.8
0.19	8.0	7.7
0.68	8.0	7.7
3.3	8.0	7.8
14	8.0	7.8

 

Individual cell densities (x10⁴cells/mL)

Time	Replicate	TWA concentration of MLA-3202 (mg/L)
		Control	0.035	0.19	0.68	3.3	14
0 h	1 2 3 4 5 6	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
n:		6	3	3	3	3	3
Mean:		1.0	1.0	1.0	1.0	1.0	1.0
Std.Dev.:		0.0	0.0	0.0	0.0	0.0	0.0
CV:		0.0	0.0	0.0	0.0	0.0	0.0
24 h	1 2 3 4 5 6	2.750	3.250	2.000	2.000	2.250	5.000
	2	6.000	3.750	2.000	2.250	1.500	3.000
	3	5.000	3.750	3.500	2.750	3.000	2.000
	4	4.750
	5	6.000
	6	3.750
n:		6	3	3	3	3	3
Mean:		4.7	3.6	2.5	2.3	2.3	3.3
Std.Dev.:		1.3	0.3	0.9	0.4	0.8	1.5
CV:		27.2	8.1	34.6	16.4	33.3	45.8
48 h	1 2 3 4 5 6	17.330	28.000	22.250	13.250	13.000	7.500
	2	25.000	30.500	27.250	12.500	15.000	6.000
	3	26.500	34.000	24.750	24.500	10.750	8.500
	4	35.750
	5	31.250
	6	43.500
n:		6	3	3	3	3	3
Mean:		29.9	30.8	24.8	16.8	12.9	7.3
Std.Dev.:		9.1	3.0	2.5	6.7	2.1	1.3
CV:		30.5	9.8	10.1	40.1	16.5	17.2
72 h	1 2 3 4 5 6	136.500	183.750	143.250	44.500	20.000	13.250
	2	153.000	144.750	149.250	36.250	20.000	13.750
	3	151.500	200.250	133.500	44.250	16.500	12.500
	4	192.000
	5	138.750
	6	204.000
n:		6	3	3	3	3	3
Mean:		162.6	176.3	142.0	41.7	18.8	13.2
Std.Dev.:		28.4	28.5	7.9	4.7	2.0	0.6
CV:		17.5	16.2	5.6	11.3	10.7	4.8

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 163. The test fulfils this validity criterion.

Individual growth rates (day^-1)

Time	Replicate	TWA concentration of MLA-3202 (mg/L)
		Control	0.035	0.19	0.68	3.3	14
0-24 h	1 2 3 4 5 6	1.012	1.179	0.693	0.693	0.811	1.609
	2	1.792	1.322	0.693	0.811	0.405	1.099
	3	1.609	1.322	1.253	1.012	1.099	0.693
	4	1.558
	5	1.792
	6	1.322
Mean:		1.514	1.274	0.88	0.839	0.772	1.134
Std.Dev.:		0.3016	0.0826	0.3231	0.161	0.3482	0.4592
n:		6	3	3	3	3	3
CV:		19.9	6.5	36.7	19.2	45.1	40.5
0-48 h	1 2 3 4 5 6	1.426	1.666	1.551	1.292	1.282	1.007
	2	1.609	1.709	1.653	1.263	1.354	0.896
	3	1.639	1.763	1.604	1.599	1.187	1.07
	4	1.788
	5	1.721
	6	1.886
Mean:		1.678	1.713	1.603	1.385	1.275	0.991
Std.Dev.:		0.1594	0.0487	0.0507	0.1864	0.0836	0.0882
n:		6	3	3	3	3	3
CV:		9.5	2.8	3.2	13.5	6.6	8.9
0-72 h	1 2 3 4 5 6	1.639	1.738	1.655	1.265	0.999	0.861
	2	1.677	1.658	1.669	1.197	0.999	0.874
	3	1.674	1.767	1.631	1.263	0.934	0.842
	4	1.752
	5	1.644
	6	1.773
Mean:		1.693	1.721	1.652	1.242	0.977	0.859
Std.Dev.:		0.0563	0.0561	0.0188	0.0389	0.037	0.016
n:		6	3	3	3	3	3
CV:		3.3	3.3	1.1	3.1	3.8	1.9

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 3.3%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%.The test fulfils this validity criterion.

Section-by-section growth rate(day^-1)

Time	Replicate	TWA concentration of MLA-3202 (mg/L)
		Control	0.035	0.19	0.68	3.3	14
0 - 24 h  h	1 2 3 4 5 6	1.012	1.179	0.693	0.693	0.811	1.609
	2	1.792	1.322	0.693	0.811	0.405	1.099
	3	1.609	1.322	1.253	1.012	1.099	0.693
	4	1.558
	5	1.792
	6	1.322
Mean:		1.514	1.274	0.88	0.839	0.772	1.134
Std.Dev.:		0.3016	0.0826	0.3231	0.161	0.3482	0.4592
n:		6	3	3	3	3	3
CV:		19.9	6.5	36.7	19.2	45.1	40.5
24 - 48 h	1 2 3 4 5 6	1.841	2.154	2.409	1.891	1.754	0.405
	2	1.427	2.096	2.612	1.715	2.303	0.693
	3	1.668	2.205	1.956	2.187	1.276	1.447
	4	2.018
	5	1.65
	6	2.451
Mean:		1.843	2.151	2.326	1.931	1.778	0.849
Std.Dev.:		0.3581	0.0543	0.3358	0.2387	0.5136	0.5378
n:		6	3	3	3	3	3
CV:		19.4	2.5	14.4	12.4	28.9	63.4
48 - 72 h	1 2 3 4 5 6	2.064	1.881	1.862	1.211	0.431	0.569
	2	1.812	1.557	1.701	1.065	0.288	0.829
	3	1.743	1.773	1.685	0.591	0.428	0.386
	4	1.681
	5	1.491
	6	1.545
Mean:		1.723	1.737	1.749	0.956	0.382	0.595
Std.Dev.:		0.2058	0.165	0.0981	0.3242	0.082	0.2229
n:		6	3	3	3	3	3
CV:		11.9	9.5	5.6	33.9	21.4	37.5

The mean of the replicate coefficients of variation for section-by-section growth rate in the control was: 18%. According to OECD 201, the mean coefficient of variation, measured in the control from 0 to 72 h, must not be higher than 35%.The test fulfils this validity criterion.

Individual yields(x10⁴cells/mL)

Time	Replicate	TWA concentration of MLA-3202 (mg/L)
		Control	0.035	0.19	0.68	3.3	14
24 h	1 2 3 4 5 6	1.8	2.3	1	1	1.3	4
	2	5	2.8	1	1.3	0.5	2
	3	4	2.8	2.5	1.8	2	1
	4	3.8
	5	5
	6	2.8
Mean:		3.7	2.6	1.5	1.3	1.3	2.3
Std.Dev.:		1.28	0.29	0.87	0.38	0.75	1.53
n:		6	3	3	3	3	3
CV:		34.5	11.2	57.7	28.6	60	65.5
48 h	1 2 3 4 5 6	16.3	27	21.3	12.3	12	6.5
	2	24	29.5	26.3	11.5	14	5
	3	25.5	33	23.8	23.5	9.8	7.5
	4	34.8
	5	30.3
	6	42.5
Mean:		28.9	29.8	23.8	15.8	11.9	6.3
Std.Dev.:		9.11	3.01	2.5	6.72	2.13	1.26
n:		6	3	3	3	3	3
CV:		31.5	10.1	10.5	42.7	17.8	19.9
72 h	1 2 3 4 5 6	135.5	182.8	142.3	43.5	19	12.3
	2	152	143.8	148.3	35.3	19	12.8
	3	150.5	199.3	132.5	43.3	15.5	11.5
	4	191
	5	137.8
	6	203
Mean:		161.6	175.3	141	40.7	17.8	12.2
Std.Dev.:		28.44	28.5	7.95	4.69	2.02	0.63
n:		6	3	3	3	3	3
CV:		17.6	16.3	5.6	11.5	11.3	5.2

 

STATISTICS: GROWTH RATE

Test for normality of distribution

Shapiro-Wilk´s Test on Normal Distribution

Treatm. [mg/L]	Mean	s	n
Control	1.693	0.0563	6
0.035	1.721	0.0561	3
0.19	1.652	0.0188	3
0.68	1.242	0.0389	3
3.3	0.977	0.0370	3
14	0.859	0.0160	3

                      

Results:                    

Number of residuals = 19  

Shapiro-Wilk´s W = 0.964  

p(W) = 0.645            

p(W) is greater than the selected significance level of 0.010; thus treatment data do not

significantly deviate from normal distribution.

 

Test for homogeneity of variance

Levene´s Test on Variance Homogeneity (with Residuals)

Source	SS	df	MSS	F	p(F)
Treatment	0.00385	5	0.00077	2.330	0.094
Residuals	0.00496	15	0.00033
Total	0.00880	20

The Levene test indicates variance homogeneity (p > 0.010).

Variance homogeneity check passed (p > 0.01).          

Normal-distribution and variance-homogeneity requirements are fulfilled.

A parametric multiple test is advisable.   

           

Determination of NOEC

The analysis of contrasts revealed a linear trend, thus the selected Williams test was performed.

Williams Multiple Sequential t-test Procedure

Treatm. [mg/L]	Mean	s	df	LhM	%MDD	t	t*	Sign.
Control	1.693	0.0441
0.035	1.721	0.0441	15	1.721	-3.2	0.89	-1.75	-
0.19	1.652	0.0441	15	1.652	-3.4	-1.33	-1.83	-
0.68	1.242	0.0441	15	1.242	-3.4	-14.48	-1.85	+
3.3	0.977	0.0441	15	0.977	-3.4	-22.97	-1.86	+
14	0.859	0.0441	15	0.859	-3.4	-26.77	-1.87	+

+: significant; -: non-significant

A NOEC of 0.19 mg/L is suggested by the program.

 

STATISTICS: YIELD

Shapiro-Wilk´s Test on Normal Distribution

Treatm. [mg/L]	Mean	s	n
Control	161.6	28.44	6
0.035	175.3	28.50	3
0.19	141.0	7.95	3
0.68	40.7	4.69	3
3.3	17.8	2.02	3
14	12.2	0.63	3

Results:

Number of residuals = 20

Shapiro-Wilk´s W = 0.936

p(W) = 0.204

p(W) is greater than the selected significance level of 0.010; thus treatment data do not

significantly deviate from normal distribution.

 

Test for homogeneity of variance

Levene´s Test on Variance Homogeneity (with Residuals)

Source	SS	df	MSS	F	p(F)
Treatment	2098.453	5	419.691	6.029	0.003
Residuals	1044.247	15	69.616
Total	3142.700	20

The Levene test indicates variance heterogeneity (p <= 0.010)

Variance homogeneity check failed                                            

However, normal distribution requirements are fulfilled.                                 

The Welch-t-test for non-homogeneous variances with Bonferroni-Holm-adjustment is advisable.

 

Determination of NOEC

Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm

Treatm. [mg/L]	Mean	s	df	%MDD	t	p(t)	Alpha(i)	Sign.
Control	161.6	28.44
0.035	175.3	28.50	4	-26.6	0.68	0.732	0.050	-
0.19	141.0	7.95	6	-18.9	-1.65	0.075	0.025	-
0.68	40.7	4.69	5	-21.5	-10.15	< 0.001	0.017	+
3.3	17.8	2.02	5	-22.8	-12.32	< 0.001	0.013	+
14	12.2	0.63	5	-24.2	-12.87	< 0.001	0.010	+

+: significant; -: non-significant

A NOEC of 0.19 mg/L is suggested by the program.

 

Calibration Curves

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration²weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

QC Samples

During the range-finding test, the chromatograms of the blank QC samples showed no peak at the retention time of the test item. During the final test, a small response at the retention time of the test item was detected in the chromatograms of the blank QC samples. Concentration was below the limit of detection which was 0.00008 mg/L during the range-finding test and 0.00007 mg/L during the final test.

During the range-finding test, the mean accuracy of the 100 mg/L QC samples was slightly above the criterion range of 70 - 110% (i.e. 113% of target). The mean accuracy of the 0.01 mg/L QC samples was within the criterion range. During the final test, the mean accuracies of QC samples containing test item fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test item concentration in the test samples.

Test Samples

During the final test, a small response at the retention time was observed in the chromatograms of nominal 0 mg/L samples. Concentration was just above the limit of detection.

QC Samples

Date of preparation	Date of analysis	Concentration [mg/L]			Accuracy [%]
		Target	Nominal	Analyzed	Individual	Mean (coefficient of variation)
07 Dec 2016	07 Dec 2016	0	0.00	< LOD	n.a.	n.a.
			0.00	< LOD	n.a.
07 Dec 2016	07 Dec 2016	0.01	0.0100	0.0108	108	107
			0.0100	0.0107	107	(n.a.)
07 Dec 2016	07 Dec 2016	100	100	112	112	113
			100	107	107	(4)
			100	119	119
			100	114	114
			100	116	116
26 Jan 2017	26 Jan 2017	0	0.00	< LOD	n.a.	n.a.
			0.00	< LOD	n.a.
26 Jan 2017	26 Jan 2017	0.01	0.0100	0.0103	103	103
			0.0100	0.0103	103
26 Jan 2017	26 Jan 2017	100	100	97.5	98	100
			100	102	102

LOD   The limit of detection of the method, taking a dilution factor of four into account, was determined to be 0.00008 mg/L on 07 Dec 2016 and 0.00007 mg/L on 26 Jan 2017.

n.d.              Not detected.

n.a.               Not applicable.

 

Range-finding Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis^1	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	28 Nov 2016	07 Dec 2016	0.1	0.0940	94
			100	102	102
24	29 Nov 2016	07 Dec 2016	0.1	0.0176	18	19
			100	108	108	106
72	01 Dec 2016	07 Dec 2016	0.1	0.0053^2	5.3	5.7
			100	100	100	98

¹ Samples were stored in the freezer (≤ -15°C) until the day of analysis.

² Estimated value, calculated by extrapolation of the calibration curve.

Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis^1	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	16 Jan 2017	26 Jan 2017	0	0.000089^3
			0.1	0.0877	88
			0.35	0.339	97
			1.2	1.12	94
			1.2^2	1.16	97
			4.3	4.07	95
			15	14.6	97
24	17 Jan 2017	26 Jan 2017	0	0.000084^3
			0.1	0.0279	28	32
			0.35	0.207	59	61
			1.2	0.680	57	60
			1.2^2	0.946	79	82
			4.3	3.35	78	82
			15	14.1	94	96
72	19 Jan 2017	26 Jan 2017	0	0.000095^3
			0.1	0.0269	27	31
			0.35	0.103	30	31
			1.2	0.507	42	45
			1.2^2	0.903	75	78
			4.3	2.76	64	68
			15	13.2	88	90

¹ Samples were stored in the freezer (≤ -15°C) until the day of analysis.

² Without algae.

³ Estimated value, calculated by extrapolation of the calibration curve.

n.a.    Not applicable.

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, MLA-3202 reduced growth rate and inhibited the yield of this fresh water algae species significantly at a TWA concentration of 0.68 mg/L and higher.The EC50 for growth rate inhibition (72h-ERC50) was 9.8 mg/L with a 95% confidence interval ranging from 8.6 to 11 mg/L.The EC50 for yield inhibition (72h-EYC50) was 0.43 mg/L with a 95% confidence interval ranging from 0.37 to 0.48 mg/L.The 72h-NOEC for both, growth rate and yield inhibition was 0.19 mg/L.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with MLA-3202.

The study procedures were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 2016/266, Part C.3, 2008; Amended by EC No. 2016/266.

The batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely soluble in test medium at the concentrations tested.No correction was made for the purity/composition of the test item.

A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.10, 0.35, 1.2, 4.3 and 15 mg MLA-3202 per liter. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose.

Based on these results, the Time Weight Average (TWA) concentrations were 0.035, 0.16, 0.68, 3.3 and 14 mg/L in nominally 0.10, 0.35, 1.2, 4.3 and 15 mg/L, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

 

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.19	0.16	0.66	9.8
	lower 95%-cl		0.13	0.56	8.6
	upper 95%-cl		0.20	0.76	11
Yield	Value	0.19	0.17	0.23	0.43
	lower95%-cl		0.12	0.18	0.37
	upper 95%-cl		0.21	0.27	0.48

 

Description of key information

Key value determined in GLP accredited laboratory study according to the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 2016/266, Part C.3, 2008; Amended by EC No. 2016/266.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.43 mg/L

EC10 or NOEC for freshwater algae:

0.19 mg/L

Additional information

The batch of MLA-3202 tested was a clear amber-red liquid and a UVCB substance. Test item was completely

soluble in test medium at the concentrations tested.No correction was made for the purity/composition of the test item.

A final test was performed based on the results of a combined limit/range-finding test. Six exponentially growing

algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to 0.10, 0.35, 1.2, 4.3 and 15 mg MLA-3202 per liter. The initial algal cell density was 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all concentrations were analyzed. The actual concentrations were at the level of nominal (88-97%). The measured concentrations were at the level of 31-90% of initially measured at the end of the exposure. The stability of the concentrations increased with the applied dose.

Based on these results, the Time Weight Average (TWA) concentrations were 0.035, 0.16, 0.68, 3.3 and 14 mg/L in nominally 0.10, 0.35, 1.2, 4.3 and 15 mg/L, respectively.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

 

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.19	0.16	0.66	9.8
	lower 95%-cl		0.13	0.56	8.6
	upper 95%-cl		0.20	0.76	11
Yield	Value	0.19	0.17	0.23	0.43
	lower95%-cl		0.12	0.18	0.37
	upper 95%-cl		0.21	0.27	0.48