Propylidynetrimethanol, ethoxylated, 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:

2010-05-24 to 2010-06-09

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 10 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: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 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
9.52 7.64 7.48
2.87 7.68 7.49
0.917 7.68 7.89
0.289 7.83 7.90
0.0718 7.90 7.92
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 97 100
1 32 77
0.1 -9 -47
0.01 -8 -41

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (1.87 - 2.61)

Remarks:

Confidence interval

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.323 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Confidence interval: 0.225 – 0.467 mg/L

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 the test item (0-72 h)

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

                               

	Rate-related Inhibition
NOEC	0.289
LOEC	0.917
ErC10	0.323 (0.225 – 0.467)
ErC20	0.656 (0.498 – 0.841)
ErC50	2.20 (1.87 – 2.61)
	Inhibition of Yield
NOEC	0.0718
LOEC	0.289
EyC10	0.137 (0.0918 – 0.209)
EyC20	0.210 (0.151 – 0.281)
EyC50	0.437 (0.352 – 0.542)

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
9.52		1		4254		9736	8715	7268
		2		4254		7620	9432	8588
		3		4254		7177	11085	6911
		Mean		4254		8178	9744	7589
2.87		1		4254		16677	29235	48686
		2		4254		18346	29580	55094
		3		4254		16177	25771	47357
		Mean		4254		17067	28195	50379
0.917		1		4254		14578	68206	140247
		2		4254		13721	52491	107599
		3		4254		12778	55976	181587
		Mean		4254		13692	58891	143144
0.289		1		4254		23840	135483	540489
		2		4254		21536	107139	384234
		3		4254		26017	119193	520231
		Mean		4254		23798	120605	481651
0.0718		1		4254		23639	166093	659975
		2		4254		21044	133322	671025
		3		4254		27977	153064	856345
		Mean		4254		24220	150826	729115
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]		[%]
9.52	1		0.179	89.5		3014	99.6
	2		0.234	86.3		4334	99.4
	3		0.162	90.5		2657	99.6
	Mean	(+)	0.191	88.8	(+)	3335	99.5
2.87	1		0.813	52.4		44432	93.7
	2		0.854	49.9		50840	92.8
	3		0.803	52.9		43103	93.9
	Mean	(+)	0.823	51.7	(+)	46125	93.5
0.917	1		1.17	31.7		135993	80.8
	2		1.08	36.9		103345	85.4
	3		1.25	26.6		177333	75.0
	Mean	(+)	1.16	31.7	(+)	138890	80.4
0.289	1		1.62	5.32		536235	24.3
	2		1.50	12.0		379980	46.4
	3		1.60	6.06		515977	27.2
	Mean	(+)*	1.57	7.79	(+)	477397	32.6
0.0718	1		1.68	1.41		655721	7.47
	2		1.69	1.09		666771	5.91
	3		1.77	-3.68		852091	-20.2
	Mean	(-)	1.71	-0.39	(-)	724861	-2.29
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 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 0.289 and 0.0718 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.917 and 0.289 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.20 (1.87 – 2.61) and 0.437 (0.352 – 0.542) mg/L, respectively. The EC10-values for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.137 (0.0918 – 0.209) and 0.323 (0.225 – 0.467) 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 study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 10³cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/Lcorresponding to geometric mean measured test item concentrations of 0.0718 - 0.289 - 0.917 - 2.87 - 9.52 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 102 to 111 % of the nominal values and at test end in the range of 47 to 86 % 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	0.289
LOEC	0.917
ErC10	0.323 (0.225 – 0.467)
ErC20	0.656 (0.498 – 0.841)
ErC50	2.20 (1.87 – 2.61)
	Inhibition of Yield
NOEC	0.0718
LOEC	0.289
EyC10	0.137 (0.0918 – 0.209)
EyC20	0.210 (0.151 – 0.281)
EyC50	0.437 (0.352 – 0.542)

Description of key information

Toxicity to aquatic algae - OECD 201

The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.289 and 0.0718 mg/L, respectively.

The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 0.917 and 0.289 mg/L, respectively.

The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 2.20 (1.87 – 2.61) and 0.437 (0.352 – 0.542) mg/L, respectively.

The EC10-values for inhibition of specific growth rate (ErC10) and yield (EyC10) after 72 h were 0.137 (0.0918 – 0.209) and 0.323 (0.225 – 0.467) mg/L, respectively.

All effect levels are given based on geometric mean measured concentrations of the test item.

PARAD consortium, NOACK, 2010, D. Scheerbaum)

Key value for chemical safety assessment

EC50 for freshwater algae:

2.2 mg/L

EC10 or NOEC for freshwater algae:

0.323 mg/L

Additional information

Toxicity to aquatic algae - OECD 201 – EC50: 2.2 mg/l, 72h - PARAD consortium, NOACK, 2010, D. Scheerbaum)

The toxicity of the test item (batch number: S700040031) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from May 24 to June 09, 2010 with the definitive exposure phase of the study from May 25 to 28, 2010. 

The study was conducted under static conditions with an initial cell density of approximately 2 – 5 x 10³cells/mL.Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 0.100 - 0.320 - 1.00 - 3.20 - 10.0 mg/L corresponding to geometric mean measured test item concentrations of 0.0718 - 0.289 - 0.917 - 2.87 - 9.52 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 102 to 111 % of the nominal values and at test end in the range of 47 to 86 % of the nominal values. All effect values are given based on geometric mean measured concentrations of the test item.The E_RC₅₀ after 72 h was determined to be: E_RC₅₀: 2.2 mg/l and the E_RC₁₀ after 72 h was determined to be: E_RC₁₀: 0.323 mg/l

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

 

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

Assessment: The test item is toxic to algae.