Glycerol, propoxylated, esters with acrylic acid

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:

2010-05-03 to 2010-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration of the test item was analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Separate samples at start (provided without alga) were prepared. Samples at the end of the test were taken directly from the test replicates.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 32 mg/L was freshly prepared with dilution water. The stock solution was stirred with approx. 1100 rpm for 2 h at room temperature.
- Eluate: Dilution water
- Differential loading: 0.0320 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 - 32.0 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not measured

Test temperature:

Mean: 22.8 °C, Min: 22.1 °C, Max: 23.5 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
33.9 7.84 7.71
9.57 7.90 7.92
3.11 7.89 7.89
0.921 7.92 7.91
0.306 7.88 7.92
0.0832 7.83 7.91
0.0288 7.86 7.93
Control 7.90 7.94

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 4254 cells/mL
- Control end cells density: Mean 712901 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Tests (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition[%] Yield Inhibition [%]
10 81 99
1 30 76
0.1 -1 -6
0.01 -2 -9

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72

Dose descriptor:

EC10

Effect conc.:

2.06 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (1.5-2.8)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (10.5-14.2)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.614 (CI 0.551 - 0.679) mg/L
Yield Inhibition: 0.333 (CI 0.318 - 0.350) mg/L

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO.WILK-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05. Equal variance test failed for growth rate calculation. The data were transformed using Graph Pad Prism function Y = Y sqrt.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

NOEC, LOEC and EC - values of test item (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	0.921
LOEC	3.11
ErC10	2.06 (1.50 – 2.83)
ErC20	3.96 (3.13 – 4.92)
ErC50	12.2 (10.5 – 14.2)
	Inhibition of Yield
NOEC	0.306
LOEC	0.921
EyC10	0.565 (0.389 – 0.838)
EyC20	0.925 (0.684 – 1.21)
EyC50	2.15 (1.77 – 2.59)

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
33.9		1		4254		13409	9949	10461
		2		4254		14061	10908	13996
		3		4254		13262	12126	7723
		Mean		4254		13577	10994	10727
9.57		1		4254		18907	38161	108275
		2		4254		21925	38715	123736
		3		4254		19625	38813	111785
		Mean		4254		20152	38563	114599
3.11		1		4254		23504	106278	244785
		2		4254		26677	98944	214666
		3		4254		26271	102170	211472
		Mean		4254		25484	102464	223641
0.921		1		4254		20064	114819	616007
		2		4254		18219	109419	553617
		3		4254		24328	125036	581546
		Mean		4254		20870	116425	583723
0.306		1		4254		21384	106155	618131
		2		4254		23372	95225	729421
		3		4254		23012	121018	806874
		Mean		4254		22589	107466	718142
0.0832		1		4254		22651	118730	685453
		2		4254		24032	107426	706363
		3		4254		21372	117939	636126
		Mean		4254		22685	114698	675981
0.0288		1		4254		24201	160435	735018
		2		4254		22991	138808	622140
		3		4254		27148	143080	675814
		Mean		4254		24780	147441	677657
Control		1		4254		24180	190308	751450
		2		4254		30137	140030	598541
		3		4254		19178	194465	723189
		4		4254		31113	176798	787924
		5		4254		23987	160903	646593
		6		4254		25927	145450	769708
		Mean		4254		25754	167992	712901

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
33.9	1		0.300	82.4		6207	99.1
	2		0.397	76.7		9742	98.6
	3		0.199	88.4		3469	99.5
	Mean	(+)	0.299	82.5	(+)	6473	99.1
9.57	1		1.08	36.7		104021	85.3
	2		1.12	34.1		119482	83.1
	3		1.09	36.1		107531	84.8
	Mean	(+)	1.10	35.7	(+)	110345	84.4
3.11	1		1.35	20.8		240531	66.1
	2		1.31	23.4		210412	70.3
	3		1.30	23.7		207218	70.8
	Mean	(+)	1.32	22.6	(+)	219387	69.0
0.921	1		1.66	2.76		611753	13.7
	2		1.62	4.85		549363	22.5
	3		1.64	3.89		577292	18.5
	Mean	(+)*	1.64	3.83	(+)	579469	18.2
0.306	1		1.66	2.69		613877	13.4
	2		1.72	-0.54		725167	-2.33
	3		1.75	-2.51		802620	-13.3
	Mean	(-)	1.71	-0.12	(-)	713888	-0.74
0.0832	1		1.69	0.67		681199	3.87
	2		1.70	0.09		702109	0.92
	3		1.67	2.13		631872	10.8
	Mean	(-)	1.69	0.96	(-)	671727	5.21
0.0288	1		1.72	-0.69		730764	-3.12
	2		1.66	2.57		617886	12.8
	3		1.69	0.95		671560	5.23
	Mean	(-)	1.69	0.94	(-)	673403	4.97
Control	1		1.73			747196
	2		1.65			594287
	3		1.71			718935
	4		1.74			783670
	5		1.68			642339
	6		1.73			765454
	Mean		1.71			708647

Section-by-Section and Average Specific Growth Rates of the Control Group

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	1.74	2.06	1.37	1.73	0.345	20.0	16.9
	2	1.96	1.54	1.45	1.65	0.271	16.4
	3	1.51	2.32	1.31	1.71	0.532	31.1
	4	1.99	1.74	1.49	1.74	0.248	14.2
	5	1.73	1.90	1.39	1.68	0.261	15.6
	6	1.81	1.73	1.67	1.73	0.071	4.10
				Mean	1.71
				SD ±	0.04
				CV [%]	2.12

Validity criteria
Criteria according to current OECD 201	Validity criteria according to OECD Guideline 201	Current Study	Fulfilled
Factor of biomass control increase in 72 h	≥16	168	Yes
Mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures	≤35%	13.5 %	Yes
Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures	≤7%	2.1 %	Yes

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.921 and 0.306 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 3.11 and 0.921 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 12.2 (10.5 – 14.2) and 2.15 (1.77 – 2.59) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary test and a first definitive study 7 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.0320 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 - 32.0 mg/Lcorresponding to geometric mean measured test item concentrations of 0.0288 - 0.0832 - 0.306 - 0.921 - 3.11 -9.57 - 33.9 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of the test item were analysed at all concentration levels after 0 and   72 h via LC-MS/MS analysis. At test start the recoveries of the test item were in the range of 98 to 110 % of the nominal values and at test end in the range of 69 to 102 % of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.

 

 

NOEC, LOEC and EC - values of the test item (0-72 h)

                   based on geometric mean measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	0.921
LOEC	3.11
ErC10	2.06 (1.50 – 2.83)
ErC20	3.96 (3.13 – 4.92)
ErC50	12.2 (10.5 – 14.2)
	Inhibition of Yield
NOEC	0.306
LOEC	0.921
EyC10	0.565 (0.389 – 0.838)
EyC20	0.925 (0.684 – 1.21)
EyC50	2.15 (1.77 – 2.59)

Description of key information

Toxicity to aquatic algae - OECD 201 – EC50: 12.2 mg/l, EC10: 2.06 mg/l, 72h - (PARAD consortium, NOACK, 2010, D. Scheerbaum) key study

Key value for chemical safety assessment

EC50 for freshwater algae:

12.2 mg/L

EC10 or NOEC for freshwater algae:

2.06 mg/L

Additional information

A reliable toxicity study on aquatic algae is available:

Toxicity to aquatic algae - OECD 201 – EC50: 12.2 mg/l, 72h - (PARAD consortium, NOACK, 2010, D. Scheerbaum) key study

The toxicity of the registered substance (test item) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL.Based on a preliminary test and a first definitive study 7concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.0320 - 0.100 - 0.320 - 1.00 - 3.20 - 10.0 - 32.0 mg/Lcorresponding to geometric mean measured test item concentrations of 0.0288 - 0.0832 - 0.306 - 0.921 - 3.11 -9.57 - 33.9 mg/L.Three replicates were tested for each test item concentration and six replicates for the control.Environmental conditionswere determined to be within the acceptable limits.The concentrations of test item were analysed at all concentration levels after 0 and   72 h via LC-MS/MS analysis. At test start the recoveries of the test item test item were in the range of 98 to 110 % of the nominal values and at test end in the range of 69 to 102 % of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.After 72 h an E_rC₅₀: = 12.2 mg/l was determined.

Evaluation: valid without restrictions, Klimisch-I , Key study

The test item is harmful to algae.