citric acid, esters with dodecan-1-ol

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:

10 August 2016 - 01 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

28 July, 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Didodecyl hydrogen citrate

EC Number:

247-146-3

EC Name:

Didodecyl hydrogen citrate

Cas Number:

25637-88-1

Molecular formula:

C30H56O7

IUPAC Name:

didodecyl hydrogen citrate

Constituent 2

Reference substance name:

Tridodecyl 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

265-671-6

EC Name:

Tridodecyl 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

65277-53-4

Molecular formula:

C42H80O7

IUPAC Name:

tridodecyl citrate

Constituent 3

Reference substance name:

Dodecyl dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate

EC Number:

278-950-2

EC Name:

Dodecyl dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate

Cas Number:

78568-45-3

Molecular formula:

C18H32O7

IUPAC Name:

2-[2-(dodecyloxy)-2-oxoethyl]-2-hydroxysuccinic acid

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal: 1.26, 2.51, 5.07, 10.0 and 20.0 mg/L, mean measured test item concentrations:0.017, 0.038, 0.135, 0.188 and 0.621 mg test item/L (.
- Sampling method: For the determination of algae growth six replicates of the control (test medium only) and four replicates of each treatment were exposed to the test solutions with the concentrations given above.
- Sample storage conditions before analysis: To achieve the five test loadings, 40.06, 20.07, 10.14, 5.02 and 2.52 mg of the test item were transferred into 2 L sterile brown glass flasks (acetone washed and sterilised) with a bottom draining port. Sterile growth medium (2 L) and an acetone washed, sterilised star shaped
stirring bar were added to the bottles and stirred slowly avoiding bubble and foam formation for 68 h at room temperature (about 20°C) to achieve maximum solubility of the test item. To separate insoluble test item from the test medium the bottles were allowed to stand for 4.25 h. During this time most of the substance gathered on the surface of the medium. With
high loadings, a small part of the substance precipitated at the bottom of the flask. Thereafter, 300 mL were drained off and discarded. A visual observation of the discarded test media revealed the presence of small parts of the test item. The next slowly released 500 mL were used as test solution, here, the visual observation revealed no presence of the test item.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the very low water solubility of the test item individual Water Accommodated Fractions (WAFs) of the test item were prepared according to the OECD guidance No. 23.
To achieve the five test loadings, 40.06, 20.07, 10.14, 5.02 and 2.52 mg of the test item were transferred into 2 L sterile brown glass flasks (acetone washed and sterilised) with a bottom draining port. Sterile growth medium (2 L) and an acetone washed, sterilised star shaped stirring bar were added to the bottles and stirred slowly avoiding bubble and foam formation for 68 h 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.
To separate insoluble test item from the test medium the bottles were allowed to stand for 4.25 h. During this time most of the substance gathered on the surface of the medium. With high loadings, a small part of the substance precipitated at the bottom of the flask. Thereafter, 300 mL were drained off and discarded. A visual observation of the discarded test media revealed the presence of small parts of the test item. The next slowly released 500 mL were used as test solution, here, the visual observation revealed no presence of the test item.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- 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):
- Method of cultivation: Prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test. The culture duration of the pre-cultures was 3 days.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: The sterilised synthetic OECD medium according to OECD 201. The culture vessels were incubated at 21 to 24°C, controlled at ± 2°C with a light intensity (day light: OSRAM “day light”) adjusted between 60 - 120 μE m-² s-1 close to the surface of the liquid (equivalent 4440 and 8880 lux). Measurements were made of the direct light above the tray of the incubator. The cultures were oscillated by continuously stirring on a laboratory shaker with 150 rpm (Incubation Shaker Multitron®, INFORS, Switzerland). 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.
- Any deformed or abnormal cells observed: no information

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

no information

Test temperature:

21-22 °C

pH:

7.94-8.12 (initial); 8.28-8.35 (at test termination)

Dissolved oxygen:

no information

Salinity:

no information

Conductivity:

no information

Nominal and measured concentrations:

nominal: 1.26, 2.51, 5.07, 10.0 and 20.0 mg test item/L; measured: 0.017, 0.038, 0.135, 0.188 and 0.621 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical glass flasks covered with air-permeable siliconesponge caps.
- Initial cells density: 10,000 cells/mL
- Control end cells density: 1690000 cells/ml
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:purified water processed using an ELGA „PURELAB S7 + Classic UVF“
- Culture medium different from test medium: no
- Intervals of water quality measurement: no information

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 72 h
- Light intensity and quality: 96.24 μE m-2 s-1 to 99.67 μE m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (CASY® TT, Innovatis, Germany)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1.55, 3.13 and 10.2 mg/L
- Results used to determine the conditions for the definitive study: yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes

Any other information on results incl. tables

Cell number (x 10⁴) per mL dependent on test item and time.

Nominal loading [mg test item/L]	Control	1.26	2.51	5.07	10.0	20.0
0 h	1	1	1	1	1	1
24 h	4.3	4.2	4.4	4.5	5.6	4.3
	4.8	4.7	4.8	4.7	4.8	5.0
	4.4	4.3	4.5	4.6	4.2	3.9
	4.4	4.7	4.4	4.5	4.6	5.0
	4.2
	4.5
Mean:	4.4	4.5	4.5	4.6	4.8	4.5
Std.Dev.:	0.20	0.26	0.18	0.13	0.61	0.56
Replicates:	6	4	4	4	4	4
CV:	4.6	5.9	4.0	2.8	12.8	12.3
48 h	23.6	22.3	25.8	24.6	36.0	30.8
	25.4	27.9	35.0	32.3	29.0	27.6
	23.8	22.1	25.4	25.4	31.2	20.2
	26.9	26.2	26.5	21.9	30.1	24.0
	26.1
	23.8
Mean	24.9	24.6	28.2	26.1	31.6	25.7
Std.Dev.:	1.39	2.88	4.62	4.44	3.06	4.59
Replicates	6	4	4	4	4	4
CV:	5.60	11.7	16.4	17.0	9.70	17.9
72 h	151.4	130.8	126.0	149.8	203.2	159.5
	157.7	175.4	172.5	160.6	173.5	159.0
	118.5	151.9	150.2	173.8	134.5	152.4
	141.7	156.9	135.6	191.7	132.8	171.4
	227.7
	152.2
Mean:	169.0	137.2	152.5	152.0	182.3	150.1
Std.Dev.:	29.7	16.6	20.4	15.3	18.9	19.8
Replicates:	6	4	4	4	4	4
CV:	17.6	12.1	13.4	10.0	10.4	13.2

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

Growth rates (1/d) dependent on the test item and time

Nominal loading [mg/L]	Control	1.26	2.51	5.07	10.0	20.0
24 h	1.466	1.429	1.484	1.494	1.724	1.462
	1.569	1.544	1.561	1.553	1.571	1.612
	1.474	1.468	1.510	1.535	1.425	1.353
	1.471	1.551	1.473	1.5	1.517	1.608
	1.444
	1.514
Mean:	1.49	1.498	1.507	1.52	1.559	1.509
Std.Dev.:	0.05	0.06	0.04	0.03	0.13	0.13
n:	6	4	4	4	4	4
CV:	3.00	4.00	2.60	1.90	8.00	8.30
48 h	1.580	1.553	1.624	1.602	1.792	1.714
	1.616	1.664	1.778	1.738	1.685	1.660
	1.585	1.548	1.617	1.618	1.720	1.503
	1.645	1.633	1.638	1.543	1.703	1.589
	1.631
	1.585
Mean:	1.607	1.599	1.664	1.625	1.725	1.616
Std.Dev.:	0.03	0.06	0.08	0.08	0.05	0.09
n:	6	4	4	4	4	4
CV:	1.70	3.60	4.60	5.00	2.70	5.70
72 h	1.673	1.625	1.612	1.670	1.771	1.691
	1.690	1.687	1.722	1.717	1.693	1.719
	1.676	1.592	1.674	1.671	1.719	1.634
	1.715	1.651	1.685	1.637	1.752	1.630
	1.809
	1.675
Mean:	1.706	1.639	1.674	1.673	1.734	1.668
Std.Dev.:	0.05	0.04	0.05	0.03	0.03	0.04
n:	6	4	4	4	4	4
CV:	3.10	2.50	2.70	2.00	2.00	2.60

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

Growth rate (G) and its inhibition relative to control (% I) as computed from the raw data for growth test intervals selected.

Nominal loading [mg/L]	0-24 h		0-48 h		0-72 h
	G	% I	G	% I	G	% I
Control	1.490	0.0	1.607	0.0	1.706	0.0
1.26	1.498	-0.6	1.599	0.5	1.639	4.0
2.51	1.507	-1.2	1.664	-3.5	1.674	1.9
5.07	1.520	-2.1	1.625	-1.1	1.673	1.9
10.0	1.559	-4.7	1.725	-7.3	1.734	-1.6
20.0	1.509	-1.3	1.616	-0.6	1.668	2.2

negative inhibition indicates increase in the observed parameter

Yield (cell number increase) dependent on the test item and time

Nominal loading [mg/L]	Control	1.26	2.51	5.07	10.0	20.0
24 h	3.30	3.20	3.40	3.50	4.60	3.30
	3.80	3.70	3.80	3.70	3.80	4.00
	3.40	3.30	3.50	3.60	3.20	2.90
	3.40	3.70	3.40	3.50	3.60	4.00
	3.20
	3.50
Mean:	3.40	3.50	3.50	3.60	3.80	3.50
Std.Dev.:	0.20	0.26	0.18	0.13	0.61	0.56
n:	6	4	4	4	4	4
CV:	5.90	7.60	5.10	3.60	16.1	15.7
48 h	22.6	21.3	24.8	23.6	35.0	29.8
	24.4	26.9	34.0	31.3	28.0	26.6
	22.8	21.1	24.4	24.4	30.2	19.2
	25.9	25.2	25.5	20.9	29.1	23.0
	25.1
	22.8
Mean:	23.9	23.6	27.2	25.1	30.6	24.7
Std.Dev.:	1.39	2.88	4.62	4.44	3.06	4.59
n:	6	4	4	4	4	4
CV:	5.8	12.2	17.0	17.7	10.0	18.6
72 h	150.4	129.8	125.0	148.8	202.2	158.5
	158.0	156.7	174.4	171.5	159.6	172.5
	151.4	117.5	150.9	149.2	172.8	133.5
	170.4	140.7	155.9	134.6	190.7	131.8
	226.7
	151.2
Mean:	168.0	136.2	151.5	151.0	181.3	149.1
Std.Dev.:	29.7	16.6	20.4	15.3	18.9	19.8
n:	6	4	4	4	4	4
CV:	17.7	12.2	13.4	10.1	10.4	13.3

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

Yield (Y) and its inhibition relative to control (% I) as computed from the raw data for growth test intervals selected.

Nominal loading [mg/L]	0-24 h		0-48 h		0-72 h
	Y	% I	Y	% I	Y	% I
Control	3.4	0.0	23.9	0.0	168.0	0.0
1.26	3.5	-1.2	23.6	1.2	136.2	19.0
2.51	3.5	-2.3	27.2	-13.5	151.5	9.8
5.07	3.6	-4.0	25.1	-4.8	151.0	10.1
10.0	3.8	-10.0	30.6	-27.9	181.3	-7.9
20.0	3.5	-3.2	24.7	-3.1	149.1	11.3

negative values indicate increase in the observed parameter

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour ErL50 and EyL50 were set to be above a nominal loading of 20.0 mg/L. The NOEL for growth rate and yield was determined to be ≥ 20.0 mg/L nominal loading.

Executive summary:

The toxicity of the substance was investigated in a study according to OECD guideline 201. Uni-cellular freshwater green algal species Raphidocelis subcapitata was exposed to the substance for 72 hours.

The nominal test concentrations were prepared in sterile growth medium under sterile conditions. Since the test item is a UVCB substance and of low water solubility, toxicity was determined using eluates made from different test item loadings, prepared individually in accordance to the OECD guidance for difficult substances No. 23. Individual Water Accommodated Fractions (WAFs) of the test item were prepared. nominal loadings of 1.26, 2.51, 5.07, 10.0 and 20.0 mg test item/L were used, corresponding to measured concentrations of 0.017, 0.038, 0.135, 0.188 and 0.621 mg test item/L.

Since the test item is a UVCB substance the evaluation of the test was based on the nominal loadings.

There were no concentration-dependent inhibiting effects on the growth of the green algae over the range of the tested concentrations. Since the test results of the growth inhibition test (exposure period) did not show any inhibitions above 50% compared to controls, they were not statistically analysed to determine EL50 and EL10 values for yield and growth rate. Therefore, the 72 hour ErL50 and EyL50 were set to be above a nominal loading of 20.0 mg/L. The NOEL for growth rate and yield was determined to be ≥ 20.0 mg/L nominal loading.