Fatty acids, C16-18 (even numbered), reaction products with diethylene triamine, di-Me sulfate quaternized

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:

2017-11-06 to 2018-04-xx

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:

adopted 23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

LC-MS

Details on sampling:

- Concentrations:
Control, 0.256, 0.640, 1.60, 4.00 and 10.0 mg test item/L,
equivalent to: control, 0.195, 0.486, 1.22, 3.04 and 7.60 mg a.i./L
- Sampling method: The concentrations of the test item in the water phase were measured by chemical analysis. Samples (2 x about 5 mL) were taken from all five test solutions and controls at the beginning of the test prior to addition of the algae (2 x 6 samples, analysis and retain samples).
At the end of the growth test, samples were taken directly from representative or pooled replicates per test concentration and controls (2 x 6 samples).
- Sample storage conditions before analysis: Samples which could not be measured on the day of sampling were immediately stored at ≤ - 18 °C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
To prepare a stock solution 100.004 mg of the test item were transferred into a 1 L sterile glass flask (acetone washed and sterilised). Sterile growth medium (1 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottle and stirred vigorously for 72 hours at room temperature (about 20°C) to achieve maximum solubility of the test item. Another flask was filled with growth medium only for the control replicates and stirred in the same way.
After 72 hours the test material appeared to be homogeneously dispersed within the test media. To separate insoluble test item from the test medium, the WAFs were filtered stepwise initially through a pleated filter, than through a 0.45 µm filter and in a last step through 0.22 µm PES filter. This test solution was diluted with sterile growth medium to obtain the other test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: Raphidocelis subcapitata, Selenastraceae, Chlorophycea, Chlorophyta. (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum)
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Catalog No 61.81 SAG
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Prior to testing a pre-culture will be established in standard OECD growth medium to obtain exponentially growing algae for the test. The culture duration of the pre-cultures was 3 days.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 -24 °C

pH:

at start 7.52 to 7.78 and at test end 7.63 to 7.80

Nominal and measured concentrations:

nominal: control, 0.195, 0.486, 1.22, 3.04 and 7.60 mg a.i./L
Measured: all concentrations < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical glass flasks
- Type (delete if not applicable): closed with air-permeable silicone-sponge caps
- Material, size, headspace, fill volume: 250 mL conical glass flasks,
- Initial cells density: 1E+4 cells/mL
- Control end cells density: 4.4E+4 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: light intensity (OSRAM Standard “cool white”) adjusted to 60 - 120 µE m-2 s-1 (4440 - 8880 lux) close to the surface of the liquid. The light intensity was measured daily using a cosine (2 π) receptor (LI-250A, LI-COR) in µE m-2 s-1 at level of the test media surface.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Other: The cell concentrations were determined in the inoculum culture prior to the addition to the test vessels at test start and after 24, 48 and 72 h in the test cultures.
The cell density was measured using an electronic particle counter (CASY TT, OMNI Life Science GmbH & Co. KG, Bremen, Germany)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Justification for using less concentrations than requested by guideline: The five loadings to be tested in the definitive test were selected on the basis of the results from the range-finding test and agreed upon with the sponsor (spacing factor 2.5)
- Range finding study
- Test concentrations: 1, 10, 100 mg/mL nominal loading
- Results used to determine the conditions for the definitive study: Inhibition of growth rate at 1 mg/mL = -2.2%; at 10 mg/mL = 98.3% and at 100 mg/mL = 100.0%

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

8.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.25 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6.24 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.08 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): at nominal concentrations of 4.00 and 10.0 mg test item/L Reduced appearance of algal cells and mainly cell debris was observed. At concentrations of 0.640 and 1.60 mg test item/L the appearance of the cells was normal but also some cells debris was observed.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The sensitivity of the test organism is routinely checked using 3,5-dichlorophenol as primary standard following internal SOPs in a non-GLP test twice a year. The latest (December 2017) ErC50 value of 2.90 mg/L (nominal; 95% confidence limits: 2.66 – 3.16 mg/L) for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L

Reported statistics and error estimates:

Since the test results showed inhibition above 50 % they were statistically analysed to determine an EC50 and EC10 values together with 95 % confidence intervals. The NOEC values were determined using appropriate statistical methods (Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm, significance level 0.05, one-sided smaller (growth rate) and Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm (yield))
The computer program ToxRat® was used for statistical evaluations

Inhibition of growth rate and yield compared to controls after 72 hours.

Nominal loading [mg/L]	Growth rate		Yield
	Growth rate [1/d]	% Inhibition	Yield [cells x 10^4/mL]	% Inhibition
Control	1.69	-	160.35	-
0.256	1.57	7.10 (-)	110.02	31.38 (+)
0.640	1.66	1.94 (-)	143.53	10.49 (+)
1.60	1.64	3.02 (-)	135.80	15.31 (+)
4.00	1.12	33.52(-)	37.86	76.39 (+)
10.0	0.77	54.65(-)	12.93	91.94(+)

(+) significantly/ (-) not significantly different from controls, Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm, significance level 0.05, one-sided smaller (growth rate) and Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm (yield).

Cell number (x 10⁴) per mL dependent on nominal loadings and time

Nominal loading [mg/L]	Control	0.256	0.640	1.60	4.00	10.0
0 h	1	1	1	1	1	1
24 h	3.6	3.7	4.1	4.3	3.7	1.0
	4.3	3.8	4.3	4.3	1.7	1.0
	3.9	3.5	3.9	4.1	1.8	1.0
	4.0	3.6	4.3	4.1	1.9	2.1
	4.8
	4.7
	5.0
	5.1
Mean:	4.4	3.7	4.1	4.2	2.3	1.3
Std.Dev.:	0.6	0.1	0.2	0.1	1.0	0.5
n:	8	4	4	4	4	4
CV:	12.6	3.9	4.9	2.5	42.0	42.2
48 h	23.6	20.6	26.4	26.9	16.5	1.0
	24.8	21.0	28.4	25.8	2.7	1.0
	22.5	20.5	22.6	23.4	4.1	1.0
	24.9	21.9	24.7	23.3	3.8	7.7
	31.6
	30.4
	31.9
	30.7
Mean:	27.5	21.0	25.5	24.8	6.8	2.7
Std.Dev.:	4.0	0.6	2.5	1.8	6.5	3.4
n:	8	4	4	4	4	4
CV:	14.4	3.0	9.6	7.1	95.6	125.5
72 h	129.4	109.5	141.7	146.9	94.9	6.5
	147.2	109.7	151.6	143.1	16.4	6.4
	125.5	109.3	130.3	124.8	27.3	6.5
	142.1	115.6	154.5	132.4	16.8	36.3
	193.4
	176.6
	194.2
	182.4
Mean:	161.3	111.0	144.5	136.8	38.9	13.9
Std.Dev.:	28.4	3.1	11.0	10.1	37.7	14.9
n:	8	4	4	4	4	4
CV:	17.6	2.8	7.6	7.4	97.1	107.0

Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Cell number at test start: 10 000 cells/mL

Note: Values in the highest test concentration which are at 1.0 were measured to be below 1.0 but set to be at 1.0, since 1.0 presents the start value and therefore a 100% inhibition of the algae growth in the replicate.

Validity criteria fulfilled:

yes

Conclusions:

The 72-hour toxicity of the test item to the unicellular green alga Raphidocelis subcapitata was determined in a static system (OECD 201) exposed to nominal loadings of 0.256, 0.640, 1.60, 4.00 and 10.0 mg test item/L. The concentrations of the test item in the media were confirmed by measurements of the test item concentrations before and after the test by LC-MS (LOQ = 0.05 mg/L). At test initiation and test termination the measured concentrations were below the LOQ in all treatments. Therefore, the evaluation was based on the nominal loadings of the test item. A concentration-dependent inhibiting effect on the growth of the freshwater green algae was observed over the range of the tested concentrations. The respective 72-hour ErL10 and ErL50 for the inhibition of growth rate were estimated to be 1.65 mg test item/L and 8.21 mg test item/L equivalent to 1.25 mg a.i./L and 6.24 mg a.i./L. For yield, an EyL10 and EyL50 of 1.31 and 2.74 mg test item/L were determined, respectively, equivalent to 1.00 mg a.i./L and 2.08 mg a.i./L. A NOEL of 1.60 mg test item/L (1.22 mg a.i./L) was determined for the inhibition of growth rate, whereas a NOEL of below the lowest test concentration of 0.256 mg test item/L (0.19 mg a.i./L) was derived for the inhibition of yield.

Executive summary:

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of  Raphidocelis subcapitata were exposed to Imidazolium compounds, 4,5-dihydro-2-(hydrogenated nortallow alkyl)-1-[2-(hydrogenated tallow amido)ethyl]-1-methyl, Me sulfates at nominal loadings of 0.256, 0.640, 1.60, 4.00 and 10.0 mg test item/L in growth medium, corresponding to nominal concentrations of control, 0.195, 0.486, 1.22, 3.04 and 7.60 mg a.i./L under static conditions.

The test item has a khown poor water solubility. Due to these properties the test item is difficult to test in aqueous test medium and results from such tests depend from the test settings applied. These condictions normally lead to low analytical recoveries at the end of the exposure. Nevertheless the test item was dispersed in the test medium and therefore available for exposure.

All test concentrations and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours), 24, 48 hours and the end of the exposure (72 hours).

The measured concentrations of the test item at the start of the exposure were all below the LOQ. Therefore, all values given are nominal exposure concentrations.

The NOEL, EL10 and EL50 based on growth rate inhibition were 0.0202 mg a.i./L, 0.205 mg a.i./L (95% c.l. 0.131 – 0.881 mg a.i./L) and 0.407 mg a.i./L (95% c.l. 0.213 – 1.12 mg a.i./L), respectively.

The % growth inhibition in the treated algal culture as compared to the control ranged from 2% to 100%.

The NOEC, EC10 and EC50 based on yield were 1.22 mg a.i./L, 1.25 mg a.i./L and 6.24 mg a.i./L , respectively.

Microscopic evaluation of the cells at start and the end of the incubation period revealed the following morphological abnormalities: At nominal concentrations of 0.486 and 1.22 mg a.i./L appearance of algal cells was normal but cell debris occurs and at 3.04 and 7.60 mg a.i./L appearance of algal cells was reduced and mainly cells debris was observed.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Raphidocelis subcapitata.

 

Results Synopsis

Test Organism: Raphidocelis subcapitata

Test Type: Static

 

72 hr ErL10: 1.25mg a.i./L,(95% c.l. 0.42 -2.05 mg a.i./L)

72 hr ErL50: 6.24 mg a.i./L (95% c.l. 4.72 -7.79 mg a.i./L)

72 hr NOEL: 1.22 mg a.i./L

Endpoint(s) Effected:  growth rate

72 hr EyL10: 1.00 mg a.i./L (95% c.l. 0.27 -1.45 mg a.i./L)

72 hr EyL50: 2.08 mg a.i./L (95% c.l. 1.43 -2.94 mg a.i./L)

72 hr NOEL: < 0.19 mg a.i./L

Endpoint(s) Effected:  yield

Description of key information

- study according to OECD guideline 201 (adopted 23 March, 2006), GLP, RL1, 10000 cells/mL ( Raphidocelis subcapitat) were exposed for 72h to nominal concentrations of control (0), 0.195, 0.486, 1.22, 3.04 and 7.60 mg a.i./L of Imidazolium compounds, 4,5-dihydro-2-(hydrogenated nortallow alkyl)-1-[2-(hydrogenated tallow amido)ethyl]-1-methyl, Me sulfates f under static conditions,

72 hr ErL10: 1.25mg a.i./L,(95% c.l. 0.42 -2.05 mg a.i./L), 72 hr ErL50: 6.24mg a.i./L (95% c.l. 4.72 -7.79 mg a.i./L), 72 hr NOEL: 1.22mg a.i./L, 72 hr EyL10: 1.00mg a.i./L(95% c.l. 0.27 -1.45 mg a.i./L), 72 hr EyL50: 2.08mg a.i./L (95% c.l. 1.43 -2.94 mg a.i./L), 72 hr NOEL: < 0.19mg a.i./L

Key value for chemical safety assessment

EC50 for freshwater algae:

6.24 mg/L

EC10 or NOEC for freshwater algae:

1.25 mg/L

Additional information

In a 72 hour toxicity study according to OECD Guideline 201 (2011), the cultures of Raphidocelis subcapitata were exposed to Imidazolium compounds, 4,5-dihydro-2-(hydrogenated nortallow alkyl)-1-[2-(hydrogenated tallow amido)ethyl]-1-methyl, Me sulfates at nominal loadings of 0.256, 0.640, 1.60, 4.00 and 10.0 mg test item/Lin growth medium, corresponding to nominal concentrations of control, 0.195, 0.486, 1.22, 3.04 and 7.60 mg a.i./L under static conditions.

The test item has a known poor water solubility. Due to these properties the test item is difficult to test in aqueous test medium and results from such tests depend from the test settings applied. These conditions normally lead to low analytical recoveries at the end of the exposure. Nevertheless, the test item was dispersed in the test medium and therefore available for exposure.

All test concentrations and the control were analytically verified by LC-MS/MS analysis at the start of the exposure (0 hours), 24, 48 hours and the end of the exposure (72 hours).

The measured concentrations of the test item at the start of the exposure were all below the LOQ. Therefore, all values given are nominal exposure concentrations.

Based on the statistical analysis the reduction of cell growth at nominal concentration of 3.04 and 7.60 mg a.i./L of 33.52% and 54.65% were not significant. However, due to the strong inhibition the NOEL was set to 1.60 mg test item/L (1.22 mg a.i./L) and the LOEL was set to 4.00 mg test item/L (3.04 mg a.i./L) by expert judgement. Effects below 10 %, as found in the three lower loadings, compared to the control are generally not considered to be ecotoxicologically relevant, but strong effects even if they were not statistically significant different from the control should be considered as relevant. Generally, it is recommended by OECD and EFSA to use the EL₁₀approach in preference to the NOEL approach for the environmental risk assessment.

The NOEL, EL10 and EL50 based on growth rate inhibition were 1.22 mg a.i./L, 1.25 mg a.i./L (95% c.l. 0.42 -2.05 mg a.i./L) and 6.24 mg a.i./L (95% c.l. 4.72 -7.79 mg a.i./L), respectively.

The NOEL, EL10 and EL50 based on yield were 72 hr EyL10: 1.00mg a.i./L(95% c.l. 0.27 -1.45 mg a.i./L), 72 hr EyL50: 2.08mg a.i./L (95% c.l. 1.43 -2.94 mg a.i./L) and 72 hr NOEL: < 0.19mg a.i./L, respectively.

The % growth inhibition in the treated algal culture as compared to the control ranged from -2.2% to 100%.

Microscopic evaluation of the cells at start and the end of the incubation period revealed the following morphological abnormalities: At nominal concentrations of 0.486 and 1.22 mg a.i./L appearance of algal cells was normal but cell debris occurs and at 3.04 and 7.60 mg a.i./L appearance of algal cells was reduced and mainly cells debris was observed.

Supporting information:

In a 72 hour acute toxicity study, the cultures ofPseudokirchneriella subcapitatawere exposed to Partially unsaturated IQAC, DMS quaternised at nominal concentrations of (0.010, 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L) under static conditions in accordance with the according to OECD Guideline 201 (2006). The 72-hour LOEC for the growth rate was determined to be 1.0 mg/L (corresponding to a mean measured concentration of 21 μg/L) and the 72-hour NOEC for the growth rate was determined to be 0.32 mg/L (corresponding to a mean measured concentration of 7.6 μg/L) based on water accommodated fractions (WAFs). The 72-hour LOEC for yield was determined to be 0.10 mg/L (corresponding to a mean measured concentration of 2.8 μg/L) and the 72- hour NOEC for yield was determined to be 0.032 mg/L (corresponding to a mean measured concentration of 0.52 μg/L) based on water accommodated fractions (WAFs).

The measured concentrations in the test media of the two lowest test item treatments with loading rates of 0.010 and 0.032 mg/L were <LOQbio (limit of quantification, 0.614 μg/L). The measured concentrations at the loading rates of 0.10, 0.32, 1.0, 3.2, 10 mg/L were 2.2, 6.2, 11, 14 and 22 μg/L, respectively. The biological results were related to the loading rates of the test item and to mean measured test item concentrations.

There were no compound related phytotoxic effects.

Results Synopsis

Test Organism: Pseudokirchneriella subcapitata

Test Type: Static

Growth

72hr EC10: 2.1 mg/L (31 μg/L measured) 95% C.I.: 1.7 to 2.5 mg/L (27 to 34 μg/L measured)

72hr EC20: 2.9 mg/L (39 μg/L measured) 95% C.I.: 2.6 to 3.3 mg/L (36 to 42 μg/L measured)

72hr EC50: 4.8 mg/L (56 μg/L measured) 95% C.I.: 4.3 to 5.5 mg/L (52 to 61 μg/L measured)

72hr NOEC: 0.32 mg /L (7.6 μg/L measured)

72hr LOEC: 1.0 mg /L (21 μg/L measured)

In a 72 hour acute toxicity study, the cultures ofPseudokirchneriella subcapitatawere exposed to oleic acid based IQAC, DMS quaternised at nominal concentrations of 79, 119, 178, 267, 400 µg a.i./L under static conditions in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test).  The EC₅₀values based on growth rate and yield arecalculated to be > 400 µg a.i./L (highest test concentration) and 356 µg a.i./L. The EC₁₀values were calculated to be 277 and 181 µg a.i./L for growth rate and yield, respectively.The NOEC values for growth rate and yield were estimated to be 178 and 79 µg a.i./L.However, at the LOEC concentration of 267 µg a.i./L for effects on growth rate the inhibition was only 8.1% compared to the controls.This value was statistically significant due to the low variability of the control replicates (1.5 % CV).Since effects below 10 % are generally not considered to be ecotoxicologically relevant, it is recommended to use the growth rate EC₁₀of 277 µg a.i./L as relevant endpoint for the long-term aquatic risk assessment.

No cell abnormalities were noted: 

This toxicity study is classified as acceptable and satisfies the guideline requirements for OECD Guideline 201 (Alga, Growth Inhibition Test) toxicity study.

 

Results Synopsis

 

Test Organism:

Test Type: Static:

72 hr ErC₅₀:  >400 μg a.i./L                 95% CL  not applicable

72 hr ErC₁₀: 277 μg a.i./L                    95% CL:  239 - 303 µg a.i./L

72.hr NOECr:  178 μg a.i./L               Probit Slope:  -2.01

72 hr EyC₅₀:  356μg a.i./L                   95% CL: 95% CL: 320 - 413 µg a.i./L

72 hr EyC₁₀: 181 μg a.i./L                   95% CL:  95% CL: 126 - 217 µg a.i./L

72.hr NOECy:  79 μg a.i./L                 Probit Slope:  -3.343

Endpoint(s) Effected:  growth rate, yield