Fatty acids, C16-18 and C18-unsatd., reaction products with triethanolamine, di-Me sulfate-quaternized

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14.09.2004 - 08.06.2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The substance is reported to be dispersable which does not mean it is soluble. No information about the physcal state in the test solutions (dispersed or diluted) are available in the test report.
- Method: no data given
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data given

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21-25°C

pH:

initial: 7.74-7.94
end: 7.4-10.32

Nominal and measured concentrations:

0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L (spaced by a geometric factor of 2)
The average mean concentration during the test was about 37 % of the nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks with aluminium caps and two baffles
- Aeration: CO2 supply by shaking on a rotatory shaker
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 223.7 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- The cell density was determined by counting the cells with a Neubauer chamber and a microscope

GROWTH MEDIUM
- Standard medium used: Test medium composition according to OECD TG 201; pH after equilibration with air is approximately 8


OTHER TEST CONDITIONS
- Photoperiod: constant illumination from the top
- Light intensity and quality: approximatly 7000 lux (OSRAM Powerstar, HQI-T, 400 W)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth inhibition


TEST CONCENTRATIONS
Range finding study:
Test concentrations: 0, 0.01, 0.1, 1, 10, and 100 mg/L. The test flasks were inoculated with cells from semi-static liquid cultures to an initial cell density of 10^4 cells/mL.Results were used to determine the conditions for the definitive study. Inhibitory effects of the test item after 72 h of exposure were observed from 10 to 100 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.3 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: mean concentration reduction factor 0.37; 95% CL: 20.2-24.9 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

18.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: mean concentration reduction factor 0.37

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: mean concentration reduction factor 0.37

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.09 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: mean concentration reduction factor 0.1975; EC10 recalculated from raw data

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: EC10 recalculated from raw data 95% CL: 21.97-29.33

Details on results:

Results based on nominal concentrations:
72h-ErC50 = 60.4 mg/L (95% CL: 54.7 - 67.2 mg/L)
72h-LOEC = 50 mg/L
72h-NOEC = 25 mg/L

Recalculated after study report finalisation:
72h-ErC10 = 25.765 mg/l (95% CL: 21.966-29.333 mg/l)

Reported statistics and error estimates:

The EC50 value was determined by probability plot following normal distribution

Table 1: Average cell number for each sampling time and concentration of partially unsaturated TEA-Esterquat and inhibition of growth rate

Partially unsaturated TEA-Esterquat [mg/L], nominal	Average cell numbers/mL x 10^-4				Inhibition [%] of growth rate
	0h	24h	48h	72h
0	1.0	16.15	44.27	223.70	0
1.56	1.0	14.06	49.48	223.96	-0.1
3.13	1.0	8.33	64.58	259.38	-2.8
6.25	1.0	7.81	52.60	239.06	-1.3
12.5	1.0	4.69	42.71	213.54	0.8
25	1.0	3.65	22.40	156.77	6.5
50	1.0	2.08	3.65	30.73	36.8
100	1.0	1.56	1.56	3.13	80.7

Table 2: Growth rates, P-values of Dunnett test/ one-sided lower/p=0.05

Partially unsaturated TEA-Esterquat [mg/L], nominal	0-24h	0-48h	0-72h
1.56	0.80062	0.99558	0.93347
3.13	0.03151	1.00000	0.99767
6.25	0.04116*	0.99910	0.98264
12.5	0.00017*	0.79528	0.84997
25	0.00008*	0.00013*	0.05937
50	0.00000*	0.00000*	0.00000*
100	0.00000*	0.00000*	0.00000*

*significant difference to control (p ≤ 0.05

ANALYTICAL RESULTS

Range-finding test:

The range finding test was performed under static conditions. The concentration course of partially unsaturated TEA-Esterquat was verified in test medium by analysing over the whole test period. Samples were taken after initiation of the test, after 24 h, 48 h and at the end of the test after 72 h at the concentration levels of 1 mg/L and 100 mg/L and control. The mean recovery at 100 mg/l was 53.5%

Main test:

The main test was performed under static conditions. The concentration course of partially unsaturated TEA-Esterquat was verified in test medium during the main test by analysing over the whole test period. Samples were taken after initiation of the test and thereafter in 24 h intervals until the end of the test after 72 h at the concentration levels of 1.56, 12.5, and 100 mg/L and control. The measured initial concentration of partially unsaturated TEA-Esterquat was between 95.2 % and 99.7 % of nominal. The mean recovery over the 72h-period at 1.56, 12.5 and 100 mg/L was 30.2, 39.3 and 41%, respectively. Therefore, the mean measured concentration during the entire test period was calculated to be approx. 37 % of the nominal values.

RECALCULATION OF 72h-ErC10 (after finalisation of study report)

The growth rate inhibition after 72 h was given in this study report:

 

Concentration, nominal	%inhibition
0	0
1.56	-0.1
3.13	-2.8
6.25	-1.3
12.5	0.8
25	6.5
50	36.8
100	80.7

 

ErC10 values were calculated from the %inhibition using Probit analysis:

72 h ErC10[mg/L],nominal	95% c.i. [mg/L], nominal
25.765	21.966 – 29.333

RECALCULATION OF CONCENTRATION DECREASE (after finalisation of study report)

time (h)		measured concentration of AE425/03 in % of nominal
0		99,7	98,8	95,2
24		12,8	38,5	44,7
48		8,33	14,9	14,2		geometric mean of %-decrease
72		1,92	5,04	10		(values used for mean calculation)
							19,75	%
LOD=	0,03	mg/l

For t=72h and c=1,56mg/l: measurement was <LOD.

For geometric mean calculation, LOD was used.

Validity criteria fulfilled:

yes

Remarks:

fulfils validity criteria OECD201: cells no, meas. controls 0-72h, increase fact. 223.7 coef. of variat. average growth in replicate control cultures < 15 % coef. of variat.daily growth rates in controls < 35 % pH control cultures increase < 1.5

Conclusions:

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test), the cultures of the green algae Desmodesmus subspicatus were exposed to partially unsaturated TEA-Esterquat (CAS No. 157905-74-3). Results: 72h-ErC50: 22.3 mg /L (mean measured concentration), 72h-NOEC: 9.25 mg/L (mean measured concentration) 72h-ErC10: 5.09 mg/l (geometric mean measured concentration)

Executive summary:

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test), the cultures of the green algae Desmodesmus subspicatus were exposed to partially unsaturated TEA-Esterquat (CAS No. 157905-74-3) at nominal concentrations of 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L under static conditions. The average mean (geometric mean) concentration during the test was about 37 % (19,75 %) of the nominal concentrations. The 72 h NOEC and 72 h EC50 values based on growth rate were 9.25 and 22.3 mg/L (mean measured concentrations), respectively.

After study finalisatinon, ErC10 was recalculated from raw data with probit analysis after study report finalisation.

The 72-h ErC10 was 25.8 mg/l (nominal) resp. 5.09 mg/l (geometric mean measured concentration).

This study is classified as reliable without restriction and satisfies the guideline requirements for an algae toxicity study.

Results Synopsis
Test Organism: Desmodesmus subspicatus
Test Type: Static

72h-ErC50: 22.3 mg /L       95% C.I.: 20.2 to 24.9 mg/L
72h-NOEC: 9.25 mg/L (mean measured concentrations each)

72h-ErC10: 55.77 mg/L       95% C.I.: 21.97 to 29.33 mg/L

72h-ErC10: 5.09 mg/L

72h-ErC10: 5.09 mg/L (geometric mean measured concentration)

Endpoint(s) effected: growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-01-23 to 2004-02-06

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:

June, 1994

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- At the beginning of the main test, after application of test substance, 4 x 500 ml (for all time points of 0, 24, 48, 72 h) of each of the 5 test concentrations were filled into 500 ml vessels. At day 0 five vessels with the corresponding test concentrations were firmly closed by screw caps. Additionally two vessels were prepared, one with algae medium and one with the 10 g/l stock solution (only day 0). These samples were immediately brought to thelaboratory for analysis.
The remaining vessels (24 h, 48 h, 72 h, with the five test concentrations for each time point) were placed into the incubator under the same conditions as the test vessels. They were stirred by magnetic stirrer and closed with a cotton-wool plug.
At 24, 48 and 72 hours the vessels with the five test substance concentrations were brought as well to the laboratory.

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

-

Test temperature:

25 +/- 1 °C

pH:

0 h: 8.00 - 8.21
72 h: 7.65 - 8.41

Dissolved oxygen:

-

Salinity:

-

Nominal and measured concentrations:

- Nominal concentrations: 0.25, 0.55, 1.21, 2.662, 5.856 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: conical flasks, closed with cotton-wool plugs
- Material, size, headspace, fill volume: 300 ml (filled with 100 ml of fresh sterile culture medium)
- Aeration: continuously stirred
- Initial cells density: cell concentration had to be at least 16 x 10E4 cells per ml.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 7, one vessel was used as blind value
- Light intensity and quality: 8000 +/- 1000 Lux


GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
For the sterile culture
No.1 Sodium nitrate, NaN03: 49.6 g/l
No.2 Dipotassium hydrogen phosphate, K2HP04: 3.9 g/l
No.3 Magnesium sulphate heptahydrate, MgS04 x 7 H2O: 7.5 g/l
No.4 Calcium chloride dihydrate, CaCl2 2 H2O: 3.6 g/l
NO.5 Ferric-II I-citrate, C6H5FeO7 x H2O 0.3 g/l
NO.6 Trace elements
Boric acid, H3BO3: 2.860 g/l
Manganese chloride tetrahydrate MnCI2x 4 H2O 1.810 g/l
Zinc sulphate heptahydrate ZnSO4x7 H2O 0.220 g/l
Copper sulphate pentahydrate CuSO4X 5 H2O 0.080 g/l
Sodium molybdate dihydrate NaMoO4 x 2 H2O 0.024 g/l
Cobalt chloride hexahydrate CoCl2x 6 H2O 0.040 g/l
No. 7 4 ml of the stock solution 6 is filled up with double distilled water in a delivery flask to 100 ml. The resulting solution is stock solution NO.7.
NO.8 Titriplex III Na2 EDTA x 2 H20 1.0 g/l
NO.9 Citric acid monohydrate 0.3 g/l
The solutions were filled up each to 1000 ml of double distilled water and were stable for three months in the refrigerator.

TEST MEDIUM / WATER PARAMETERS
The test culture medium was prepared one day before the beginning of the test out of three stock solutions:
1. Nutrient salts
Ammonium chloride, NH4CI: 1500 mg/l
Magnesium chloride hexahydrate, MgCI2x6 H20: 1200 mg/l
Calcium chloride dihydrate, CaCI2x2 H20: 1800 mg/l
Magnesium sulphate heptahydrate, MgSO4x 7 H20: 1500 mg/l
Potassium dihydrogen phosphate, KH2P04: 160 mg/l
Filled up to 1000 ml double distilled water.

2. Ferric-complex
Ferric-Ill-chloride hexahydrate, FeCI3x6 H20: 80 mg/l
Ethylenediaminetetraacetic acid dihydrate, Na2EDTA x 2 H20: 100 mg/l
Filled up to 1000 ml double distilled water.

3. Trace elements
The following trace elements were applied by an additional dilution step from a stock solution:
Zinc chloride, ZnCI2: 300 mg/l
Cobalt chloride hexahydrate, CoCI2 x 6 H20: 150 mg/ll
Copper-II-chloride dihydrate, CuCI2 x 2 H20: 1 mg/l
Sodium molybdate dihydrate, Na2MoO4 x 2 H20: 700 mg/l
10 ml from this stock solution were taken and the following trace elements were added:
Boric acid, H3BO4: 185 mg/l
Manganese-I I-chloride tetrahydrate, MnCI2 x 4 H20: 415 mg/l
This solution was then filled up to 1000 ml with double distilled water.
All solutions were autoclaved for 15 min. at 121 °C.

For 1000 ml culture medium 10 ml from stock solution 1, 1 ml from stock solution 2 and 1 ml from stock solution 3 were filled up to 1000 ml with double distilled water. Additionally 50 mg NaHC03 per litre were added.
All chemicals used for the media were classified as ''for analysis". The water used was double distilled.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: by photometry (A = 578 nm) or particle counting (Coulter Counter).
- Determination of algae titre: by using a spectrophotometer

Reference substance (positive control):

yes

Remarks:

K2Cr2O4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.28 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.59 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.65 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.14 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.96 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.65 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during the test, therefore a factor of 0.535 was used for multiplication

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.68 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during test - after re-evaluation of the study, a concentration reduction factor of 0.3793 was used

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.52 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during test - after re-evaluation of the study, a concentration reduction factor of 0.3793 was used

Details on results:

RESULTS NOMINAL
--BIOMASS
-The test substance inhibited significantly the increase of biomass.
- After 72 h the nominal EbC10and EbC50 values were 1.1 and 2.4 mg/l, respectively. The NOEC for biomass was 1.21 mg/l.
- A t-test was performed. At a = 0.05 = 95 % significant level the extinction values of the biomass for each replicate were compared to the values of the control.
- For the concentrations of 0.25; 0.55 and 1.21 mg/l no significant difference was observed however for the higher substance concentrations from 2.662 mg/l upwards there was a significant difference.

--GROWTH RATE
- There was a significant inhibition of growth rate after 72 hours.
- The nominal ErC10 and ErC50 values were 1.8 and 4.0 mg/l, respectively. The NOEC for growth rate was 1.21 mg/l.
- A t-test was performed. At a = 0.05 = 95 % significant level the extinction values of the growth rate for each replicate were compared to the values of the control.
- For the concentrations of 0.25; 0.55 and 1.21 mg/l no significant difference was observed however for the higher substance concentrations from 2.662 mg/l upwards there was a significant difference.

RESULTS MEASURED
- The results of the HPLC analysis by Henkel KGaA show a decrease of the test substance concentrations during the test, so that a value change based on the actual concentrations of the test substance. Because the nominal NOEC-values were 1.21 mg/l, the analytical results of nominal 0.25 mg/l and 0.55 mg/l were not included.
- For evaluation all analytical results for the monoester and diester (0 h- to 72 h) for the nominal concentrations of 1.21; 2.662 and 5.856 mg/l were added and the mean value was calculated. The resulting factor of 0.535 was used for multiplication of the already calculated EC- and NOEC-values:
--BIOMASS
- After 72 h the re-calculated EbC10 and EbC50 values were 0.59 and 1.28 mg/l, respectively. The NOEC for biomass was 0.65 mg/l
- The re-calculated ErC10 and ErC50 values were 0.96 and 2.14 mg/l, respectively. The NOEC for growth rate was 0.65 mg/l.

RE-EVALUATION OF STUDY DATA
a concentration reduction factor was calculated using the geometric mean of all measured concentrations: 0.3793

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EbC10: 0.45 mg/l
- EbC50: 0.73 mg/l

Reported statistics and error estimates:

- Statistical calculations were carried out with the t-test.

Table 1: Results (72 h-values) of statistical calculation for biomass and growth rates by t-test, alpha=0.05 for the nominal test concentrations

	Control	0.25 mg/l	0.55 mg/l	1.21 mg/l	2.662 mg/l	5.856 mg/l
Biomass	2.73	2.94	2.66	2.30	1.17	0.21
Reduction in %	0	- 8	3	15	57	92
t-test		-	-	-	+	+
Growth rate	0.07032	0.07137	0.07061	0.06876	0.05969	0.02132
t-test		-	-	-	+	+
Reduction in %	0	-2	0	2	15	68

- = no significant difference

+= significant difference

Validity criteria fulfilled:

yes

Conclusions:

In this study the test substance fully saturated TEA-Esterquat inhibited significantly the increase of biomass of the alga Scenedesmus subspicatus from >/= 2.662 mg/l. There was also a significant inhibition of the growth rate from >/= 2.662 mg/l. After 72 h the estimated EbC10 and EbC50 values were 0.59 and 1.28 mg/l, respectively. The NOEC for biomass was 0.65 mg/l. The estimated ErC10 and ErC50 values were 0.95 and 2.14 mg/l, respectively. The NOEC for growth rate was 0.65 mg/l. The results of the HPLC analysis by Henkel KGaA show a decrease of the test substance concentrations during the test, so that the above stated values change based on the actual concentrations of the test substance. For evaluation all analytical results for the monoester and diester (0 h- to 72 h) for the nominal concentrations of 1.21; 2.662 and 5.856 mg/l were added and the mean value was calculated. The resulting factor of 0.535 was used for multiplication of the already calculated EC- and NOEC-values: • After 72 h the re-calculated EbC10 and EbC50values were 0.59 and 1.28 mg/L, respectively. The NOEC for biomass was 0.65 mg/L. • The re-calculated ErC10and ErC50values were 0.96 and 2.14 mg/L, respectively. The NOEC for growth rate was 0.65 mg/L. The study data were re-evaluated. All analytical results for the monoester and diester (0 h- to 72 h) were used to calculate the concentration reduction factor as geometric mean resulting in 0.3793. The recalculated results: 72h-ErC10 = 0.68 mg/l 72h-ErC50 = 1.52 mg/l

Executive summary:

In a 72 hour acute toxicity study, the cultures of the alga Scenedesmus subspicatus were exposed to the test substance fully saturated TEA-Esterquat at nominal concentrations of 0.25, 0.55, 1.21, 2.662, 5.856 mg test mat./L under static conditions in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test) 

After 72 h the estimated EbC10 and EbC50 values were 0.59 and 1.28 mg/l, respectively. The NOEC for biomass was 0.65 mg/l.

The estimated ErC10 and ErC50 values were 0.95 and 2.14 mg/l, respectively. The NOEC for growth rate was 0.65 mg/l.

The results of the HPLC analysis by Henkel KGaA show a decrease of the test substance concentrations during the test, so that the above stated values change based on the actual concentrations of the test substance.

For evaluation all analytical results for the monoester and diester (0 h- to 72 h) for the nominal concentrations of 1.21; 2.662 and 5.856 mg/l were added and the mean value was calculated. The resulting factor of 0.535 was used for multiplication of the already calculated EC- and NOEC-values:

• After 72 h the re-calculated EbC10 and EbC50values were 0.59 and 1.28 mg/L, respectively. The NOEC for biomass was 0.65 mg/L.

• The re-calculated ErC10and ErC50values were 0.96 and 2.14 mg/L, respectively. The NOEC for growth rate was 0.65 mg/L.

This toxicity study is classified as reliable without restrictions and satisfies the guideline requirements for Alga, Growth Inhibition study.

RE-EVALUATION

The study data were re-evaluated. All analytical results for the monoester and diester (0 h- to 72 h) were used to calculate the concentration reduction factor as geometric mean resulting in 0.3793.

The recalculated values: 72h-ErC10 = 0.68 mg/l, 72h-ErC50 = 1.52 mg/l.

 

Results Synopsis

 

Test Organism: Scenedesmus subspicatus

Test Type (Static): OECD Guideline 201

 

72hr EbC₀₅:  1.28 mg a.i./L

72hr ErC₅₀: 2.14mg a.i./L

72 hr NOEC: 0.65mg a.i./L ( biomass and growth rate)

Endpoint(s) Effected: Biomass, Growth rate

Recalculated 72h-ErC10 = 0.68 mg/l

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Analytical method is not suitable for the test substance.Therefore test concentrations and effect concentrations are based on nominal concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

At the beginning and at termination of the preliminary test and the main test analytical control measurements were performed by means of photometric analysis.

Vehicle:

not specified

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

20 - 24 °C

pH:

8.05 - 9.86

Nominal and measured concentrations:

The main test was performed with the following five concentrations (serial dilution factor: 3.16):
control, 100; 31.6; 10.0; 3.16; 1.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
Light quality: universal white light
Light intensity: around 10 000 Lux
Test vessels: 250 mL Erlenmeyer flasks
initial cell concentration: 10E4 cells/mL
- Control end cells density: 50.5x10E4 (mean)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6

The test cultures were inoculated with exponentially growing cultures of Scenedesmus
subspicatus and incubated for at least 72 h. The test was performed in a phytotron (RUMED Apparatebau)
under standardized conditions.The test vessels were placed on a shaking plate and shaken continuously
over the entire test period.

GROWTH MEDIUM
The stock solutions of the culture media for the algae were prepared according to
OECD guideline 201. For the performance of the algae growth inhibition test, 1-fold and 2-
fold concentrated culture media were prepared.


PREPARATION OF THE TEST SOLUTIONS
A stock dispersion containing 200 mg/L of the test article was prepared as by adding the required amount of the test article to warm test medium (ca. 30°C). This dispersion was agitated for 15 min in an ultrasonic water bath and subsequently on a magnetic stirrer. The actual test article concentrations were achieved by dilution of the stock dispersion.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.78 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during test - after re-evaluation of the study, a concentration reduction factor of 0.5844 was calculated

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.53 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: decrease of the test substance concentrations during test - after re-evaluation of the study, a concentration reduction factor of 0.5844 was calculated

Details on results:

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

Number of viable cells as a function of test article concentration (number of cells x10E4, mean value of 4 (test concentration) or 6 (control) replicates)

Concentration (nominal) [mg/L]	0 h	24 h	48 h	72 h
100	1.0	0.4	0.1	0.0
31.6	1.0	0.8	0.9	1.5
10.0	1.0	1.1	3.1	8.6
3.16	1.0	2.2	5.9	83.2
1.0	1.3	3.4	5.8	84.4
control	1.3	2.4	8.3	91.5

The results of the main test show a complete inhibition of the algae growth at concentration levels at or above 31.6 mg/L (nominal). At or below 3.16 mg/L (nominal), no significant reduction was observed. Over the entire concentration range, no obvious abnormal effects of the algae were found in comparison to the control groups.

The inhibition (%) based on the area under the growth curve and based on the growth rate were calculated for the main test on the basis of nominal concentrations as follows:

nominal conc.[mg/L]	inhibition (%) area under the curve	inhibition (%) growth rate
100	103.8	208.3
31.6	100.1	90.5
10.0	88.7	49.4
3.16	11.3	-3.9
1.0	9.5	1.9

Analytical control measurements were performed by means of photometric analysis at the

beginning (0 h) and end (72 h) of the preliminary and the main test. The analytical system gives

a linear response between 1.788 mg/L and 10 mg/L.

For both tests, at least three representative concentration levels were analysed (main test: 100 mg/L, 10 mg/L, and 1 mg/L).

Results for main test:

nominal concentrations:      1.0 mg/L      10 mg/L       100 mg/L

0h                                         4.0 mg/L      6.0 mg/L       72 mg/L

48h                                       0.6 mg/L      5.0 mg/L       54 mg/L

ANALYTICAL CONTROL MEASUREMENTS

At the initial time of test measured concentrations were lower than the nominal concentrations. After 72 h the actual concentrations of the two higher concentration levels decreased to 75 and 83 % of the initial concentration. At the low concentration level the actual concentration decreased to less than 15% of the initial concentration.

No explanation is given for the discrepancies in nominal and analytical measured concentration at test initiation. Even though HPLC measurements were identified as suitable analytical methods in one part of the document photometric analysis was performed. Probably this method is not suitable to identify the test substance concentrations. Therefore no calculation of real concentrations

based on the analytical results were made. Test concentrations and effect concentrations are based on nominal concentrations.

RE-EVALUATION OF STUDY DATA

A concentration reduction factor was calculated using the geometric mean of measured concentrations: 0.5844

Validity criteria fulfilled:

yes

Conclusions:

In a 72 hour toxicity study with cultures of Desmodesmus subspicatus (current scientific name) The EC10 and EC50 values based on growth rate were determined for 2.61 and 6.46 mg/L nominal, respectively. This study is classified as reliable with restriction. Real test concentrations could not be determined. REEVALUATION OF STUDY DATA: using a calcuated concentration reduction factor (0.5844, geometric mean of measured concentration reduction) the following results were determined: 72h-ErC50 3.78 mg/L, 72h-ErC10 1.53 mg/L

Executive summary:

In a 72 hour toxicity study, the cultures of Desmodesmus subspicatus (current scientific name) were exposed to partially unsaturated TEA-Esterquat at nominal concentrations of 0, 1.0; 3.16; 10.0; 31.6; 100 mg/L under static conditions in accordance with the OECD Guideline 201. The EC10 and EC50 values based on growth rate were 2.61 and 6.46 mg/L, respectively. 
This study is classified as reliable with restriction and satisfies the guideline requirements for toxicity study with algae.

REEVALUATION OF STUDY DATA:

using a calcuated concentration reduction factor (0.5844, geometric mean of measured concentration reduction) the following results were determined: 72h-ErC50 3.78 mg/L 72h-ErC10 1.53 mg/L

Results Synopsis
Test Organism: Desmodesmus subspicatus
Test Type: Static
Growth rate:
72 hr ErC10: 2.61 mg a.i./L
72 hr ErC50: 6.46 mg a.i./L

Re-evaluation, using concentration reduction factor

72h-ErC50 3.78 mg a.i./L

72h-ErC10 1.53 mg a.i./L

Biomass:
72 hr EbC10: 1.12 mg a.i./L
72 hr EbC50: 5.91 mg a.i./L

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993-05-04 to 1993-05-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Method of preparation:
Direct dispersion in culture medium. 360 mg of test material was dispersed in culture medium and the volume adjusted to 1 litre to give a 360 mg/l
stock solution. 50 ml of this stock solution was dispensed in culture medium and the volume adjusted to 500 ml to give a 36 mg/l stock solution from which serial dilutions were made to prepare 18, 9.0, 4.5 and 2.25 mg/l stock solutions. 250 ml of the 2.2S, 4.S, 9.0, 18 and 36 mg/l stock solutions were each separately added to 250 ml of algal suspension to give the test series.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

24 °C

pH:

0 h: 8.3 - 8.4
96 h: 9.0 - 9.8

Nominal and measured concentrations:

Nominal concentrations: control, 1.125, 2.25, 4.5, 9.0, 18 mg/l

Details on test conditions:

Pre-culture:
- Sterile culture medium was inoculated from a master culture of Scenedesmus subspicatus and incubated under continuous illumination (approximately 7000 lux) and aeration at 21°C to give an algal suspension in log phase growth characterised by an absorbance of 0.874 (665nm). The suspension was diluted to an absorbance of 0.029 prior to use. This suspension had a mean cell density of 4.01 x 10E4 cells/ml.

Method of initiation:
- 250 ml algal pre-culture were added to 250 ml of sterile nutrient medium containing appropriate amounts of test material. This 500 ml total volume was divided into 3 x 100 ml replicates and the remainder discarded.

TEST SYSTEM
- Test vessel:
- Type: conical flask loosely stoppered to reduce evaporation
- Material, size, headspace, fill volume: 250 ml conical flasks containing 100 ml test solution
- All flasks were incubated and shaken (apprx. 100 rpm) in an orbital shaker
- Aeration: none. Gaseous exchange and suspension of algal cells maintained by orbital shaker
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: mean cell density of control: 0 h 4.01 x 10E4 cells/ml
- mean cell density of control, 72h 9.07 x 10E5 cells/ml
- Control end cells density: mean cell density of control: 96h 1.45 x 10E6 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, according to OECD


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous (approximately 7000 lux)
- Light intensity and quality: no data

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study over 96 h
- Test concentrations: 0.01, 0.10, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: the results showed significant impairment of growth at 10 and 100 mg/l indicating the choice of test concentrations employed


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Samples were taken at 0, 24, 48, 72 and 96 hours and the absorbance measured at 665 nm.
- The cell densities of the control cultures at initiation and at termination were determined by direct counting with the aid of a haemacytometer.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.264 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.832 - 3.694 mg/L recalculated from raw data after study finalisation

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.26 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: use of concentration reduction factor 38.71% as derived from three comparable algae studies

Details on results:

Nominal Effect concentrations:
72 h EbC50 = 5.8 mg/L test mat.
96 h EbC50 = 5.3 mg/L test mat.
24 h ErC50 = 8.8 mg/L test mat.
NOEC = 1.125 mg/L test mat.

72 h ErC10 = 3.3 mg/L test mat. nominal (recalculated from raw data)
72 h ErC10 = 1.26 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)

NOEC:
- There were no statistically significant differences (P > 0.05) between the control and the 1.125 mg/l test concentration and therefore the "No Observed Effect Concentration" (NOEC) is given as 1.125 mg/l.
Observations:
- There were no abnormalities detected in any of the control or test cultures at 1.125, 2.25, 4.5 and 9.0 mg/l. However, at the test concentration of 18 mg/l the cells were observed to be smaller in size than those in the control cultures, colourless and clumped together.

Table 1: Mean Cell Densities from the Exposure of Scenedesmus subspicatus to partially unsaturated TEA-Esterquat for a Period of 96 hours

 

Conc mg/l	Mean cell densities* (cells/ml)
	0 h	24h	48h	72h	96h
Control	4.01 x 10E4	1.33 x 10E5	4.45 x 10E5	9.07 x 10E5	1.45 x 10E6
1.125	3.70 x 10E4	1.30 x 10E5	4.63 x 10E5	9.44 x 10E5	1.43 x 10E6
2.25	4.63 x 10E4	1.33 x 10E5	4.54 x 10E5	8.33 x 10E5	1.10 x 10E6
4.5	4.01 x 10E4	1.11 x 10E5	2.19 x 10E5	4.45 x 10E5	5.17 x 10E5
9.0	3.70 x 10E4	7.09 x 10E4	1.52 x 10E5	2.64 x 10E5	3.50 x 10E5
18.0	4.32 x 10E4	3.39 x 10E4	5.56 x 10E5	6.18 x 10E4	1.02 x 10E5

 

Mean of 3 replicates

*Mean cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks.

Table 2: Inhibition growth from exposure of Scenedesmus subspicatus to partially unsaturated TEA-Esterquat for a period of 72 and 96 hours

Conc mg/l	Area under curve, 72 h	% inhibition	Area under curve, 96 h	% inhibition	Growth rate (0-24 h)	% inhibition
control	2.24 x 10E7	-	4.97 x 10E7	-	0.050	-
1.125	2.35 x 10E7	(5) increase	5.11 x 10E7	(3) increase	0.054	(9) increase
2.25	2.13 x 10E7	5	4.34 x 10E7	13	0.044	12
4.5	1.09 x 10E7	52	2.14 x 10E7	57	0.042	15
9.0	6.29 x 10E6	72	1.28 x 10E7	74	0.027	46
18.0	2.94 x 10E5	99	1.22 x 10E6	98	-0.010	120

Validity criteria fulfilled:

yes

Conclusions:

In this study with test substance partially unsaturated TEA-Esterquat the lowest EC50 is the EbC50 (72 h, biomass): 5.8 mg/l (nominal). The ErC50 (24 h, growth rate) is 8.8 mg/l (nominal). The reliability was assigned 2 because no analytical control was performed. EbC50 (72 h) = 5.8 mg/l ErC50 (24 h) = 8.8 mg/l NOEC = 1.125 mg/l 72 h ErC10 = 3.3 mg/L test mat. nominal (recalculated from raw data) 72 h ErC10 = 1.26 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1993-05-04 to 1993-05-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

no analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- The concentration, homogeneity and stability of the test material preparations were not determined by analysis. Data relating to the identity, purity and stability of the supplied test material are the responsibility of the sponsor.

Vehicle:

no

Details on test solutions:

Media preparation:
Direct dispersion in culture medium. 360 mg of test material was dispersed in culture medium and the volume adjusted to 1 litre to give a 360 mg/l stock solution. 50 ml of this stock solution was dispensed in culture medium and the volume adjusted to 500 ml to give a 36 mg/l stock solution from which serial dilutions were made to prepare 18, 9.0, 4.5 and 2.25 mg/l stock solutions. 250 ml of the 2.25, 4.5, 9.0, 18 and 36 mg/l stock solutions were each separately added to 250 ml of algal suspension to give the test series.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

24 °C

pH:

0 h: 8.3 - 8.5
96 h: 9.0 - 9.9

Dissolved oxygen:

no data

Salinity:

-

Nominal and measured concentrations:

Nominal concentrations: control, 1.125, 2.25, 4.5, 9.0, 18 mg/l

Details on test conditions:

Pre-culture:
- Sterile culture medium was inoculated from a master culture of Scenedesmus subspicatus and incubated under continuous illumination (approximately 7000 lux) and aeration at 21°C to give an algal suspension in log phase growth characterised by an absorbance of 0.874 (665nm). The suspension was diluted to an absorbance of 0.029 prior to use. This suspension had a mean cell density of 4.01 x 10E4 cells/ml.

Method of initiation:
- 250 ml algal pre-culture were added to 250 ml of sterile nutrient medium containing appropriate amounts of test material. This 500 ml total volume was divided into 3 x 100 ml replicates and the remainder discarded.

TEST SYSTEM
- Test vessel:
- Type: conical flask loosely stoppered to reduce evaporation
- Material, size, headspace, fill volume: 250 ml conical flasks containing 100 ml test solution
- All flasks were incubated and shaken (apprx. 100 rpm) in an orbital shaker
- Aeration: none. Gaseous exchange and suspension of algal cells maintained by orbital shaker
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: mean cell density of control: 0 h 4.01 x 10E4 cells/ml
- mean cell density of control, 72h 9.07 x 10E5 cells/ml
- Control end cells density: mean cell density of control: 96h 1.45 x 10E6 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, according to OECD


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous (approximately 7000 lux)
- Light intensity and quality: no data

TEST CONCENTRATIONS
- Range finding study over 96 h
- Test concentrations: 0.01, 0.10, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: the results showed significant impairment of growth at 10 and 100 mg/l indicating the choice of test concentrations employed

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.125 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 2.153 - 3.256 mg/L recalculated from raw data after study finalisation

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.05 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: use of concentration reduction factor 38.71% as derived from three comparable algae studies

Details on results:

Nominal Effect concentrations:

72 h EbC50 = 5.8 mg test mat./L
96 h EbC50 = 5 mg test mat./L
24 h ErC50 = 9 mg test mat./L
NOEC = 1.125 mg test mat./L

72 h ErC10 = 2.72 mg/L test mat. nominal (recalculated from raw data)
72 h ErC10 = 1.05 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)

NOEC:
- There were no statistically significant differences (P > 0.05) between the control and the 1.125 mg/l test concentration and therefore the "No Observed Effect Concentration" (NOEC) is given as 1.125 mg/l.
Observations:
- There were no abnormalities detected in any of the control or test cultures at 1.125, 2.25, 4.5 and 9.0 mg/l. However, at the test concentration of 18 mg/l the cells were observed to be smaller in size than those in the control cultures, colourless and clumped together.

Table 1: Mean Cell Densities from the Exposure of Scenedesmus subspicatus to partially unsaturated TEA-Esterquat for a Period of 96 hours

 

Conc mg/l	Mean cell densities* (cells/ml)
	0 h	24h	48h	72h	96h
Control	4.01 x 10E4	1.33 x 10E5	4.49 x 10E5	9.07 x 10E5	1.45 x 10E6
1.125	4.32 x 10E4	1.33 x 10E5	4.91 x 10E5	1.03 x 10E6	1.34 x 10E6
2.25	4.63 x 10E4	1.36 x 10E5	4.86 x 10E5	7.87 x 10E5	1.10 x 10E6
4.5	4.31 x 10E4	1.23 x 10E5	2.53 x 10E5	4.28 x 10E5	5.67 x 10E5
9.0	4.01 x 10E4	9.88 x 10E4	1.98 x 10E5	2.64 x 10E5	4.00 x 10E5
18.0	4.31 x 10E4	4.31 x 10E4	5.87 x 10E5	8.02 x 10E4	1.23 x 10E5

 

Mean of 3 replicates

*Mean cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 fields of view for each of the replicate flasks.

Table 2: Inhibition growth from exposure of Scenedesmus subspicatus to partially unsaturated TEA-Esterquat for a period of 72 and 96 hours

Conc mg/l	Area under curve, 72 h	% inhibition	Area under curve, 96 h	% inhibition	Growth rate (0-24 h)	% inhibition
control	2.24 x 10E7	-	4.97 x 10E7	-	0.050	-
1.125	2.47 x 10E7	(10) increase	5.21 x 10E7	(5) increase	0.047	6
2.25	2.16 x 10E7	4	4.31 x 10E7	13	0.045	10
4.5	1.16 x 10E7	48	2.25 x 10E7	55	0.044	12
9.0	7.88 x 10E6	65	1.49 x 10E7	70	0.038	25
18.0	8.19 x 10E5	96	2.23 x 10E6	96	0.00	100

 

Validity criteria fulfilled:

yes

Conclusions:

In this study with test substance partially unsaturated TEA-Esterquat the lowest EC50 is the EbC50 (72 h, biomass): 5.8 mg/l (nominal). The ErC50 (24 h, growth rate) is 9.0 mg/l (nominal). The reliability was assigned 2 because no analytical control was performed. EbC50 (72 h) = 5.8 mg/l ErC50 (24 h) = 9.0 mg/l NOEC = 1.125 mg/l 72 h ErC10 = 2.72 mg/L test mat. nominal (recalculated from raw data) 72 h ErC10 = 1.05 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)

Description of key information

72 h ErC50 = 2.14 mg/L (OECD TG 201, Scenedesmus subspicatus); GLP, RL1; read-across: partially unsaturated TEA-Esterquat
72 h ErC10 = 1.48 mg/L (WoE, geometric mean of five 72 h ErC10 values) (OECD TG 201); GLP, RL1-2; read-across: partially unsaturated TEA-Esterquat

Key value for chemical safety assessment

EC50 for freshwater algae:

2.14 mg/L

EC10 or NOEC for freshwater algae:

1.48 mg/L

Additional information

Reliable and relevant data are available for partially unsaturated TEA-Esterquat and fully saturated TEA-Esterquat.

 

Summary

Acute value 72 h ErC50:

The lowest effect concentration of all valid tests is used. The test was made with fully saturated TEA-Esterquat which inhibited significantly the increase of biomass of the alga   Scenedesmus subspicatus in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test). The 72h-ErC50 was 2.14 mg/L.

 

Long-term value 72 h EC10:

As long-term effect value, instead of the NOEC, the EC10 is used. The current OECD guideline 201 as well as ECHA Guidance R.10, 2008 (Table R.10-1) as well as ECHA CLP Guidance, Nov. 2012 (4.1.3.1.1) recommend the use of the ErC10 instead of NOEC for classification as well as starting point for PNEC derivation.

For the long-term effect value 72 h ErC10, relevant studies are considered in a Weight of Evidence (WoE) approach: For the endpoint “Toxicity to aquatic algae and cyanobacteria”, there are five valid and comparable studies for the same taxonomic group available. For each study there is a full endpoint study record (EPR) in the dossier.

The 72 h ErC10 values for these studies are:

(a): 0.68 mg/L

(b): 1.53 mg/L

(c): 5.09 mg/L

(d): 1.26 mg/L

(e): 1.26 mg/L

The assessment of these comparable studies showed no study inherent reason for differences in results. All five studies were conducted following guidance OECD Guideline 201. All five EPR consider for effect calculation a concentration reduction factor to map the decrease of nominal concentration during the test duration. The values for the 72 h ErC10 range from 0.68 - 5.09 mg/L. For the WoE approach, the geometric mean of the five 72 h ErC10 values was calculated. According to this WoE approach, for this endpoint the 72 h ErC10 = 1.48 mg/L.

 

Studies in detail

(a)

In a 72 hour acute toxicity study, the cultures of the alga Scenedesmus subspicatus were exposed to the test substance fully saturated TEA-Esterquat at nominal concentrations of 0.25, 0.55, 1.21, 2.662, 5.856 mg test mat./L under static conditions in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test).  

After 72 h the estimated EbC10 and EbC50 values were 0.59 and 1.28 mg/L, respectively. The NOEC for biomass was 0.65 mg/L.

The estimated ErC10 and ErC50 values were 0.95 and 2.14 mg/l, respectively. The NOEC for growth rate was 0.65 mg/L.

The results of the HPLC analysis by Henkel KGaA show a decrease of the test substance concentrations during the test, so that the above stated values change based on the actual concentrations of the test substance.

For evaluation all analytical results for the monoester and diester (0 h - to 72 h) for the nominal concentrations of 1.21; 2.662 and 5.856 mg/L were added and the mean value was calculated. The resulting factor of 0.535 was used for multiplication of the already calculated EC- and NOEC-values:

• After 72 h the re-calculated EbC10 and EbC50 values were 0.59 and 1.28 mg/L, respectively. The NOEC for biomass was 0.65 mg/L.

• The re-calculated ErC10and ErC50 values were 0.96 and 2.14 mg/L, respectively. The NOEC for growth rate was 0.65 mg/L.

RE-EVALUATION OF STUDY DATA:

The study data were re-evaluated. All analytical results for the monoester and diester (0 h - to 72 h) were used to calculate the concentration reduction factor as geometric mean resulting in 0.3793.

The recalculated values:

72h-ErC10 = 0.68 mg/L

72h-ErC50 = 1.52 mg/L.

 

(b)

In a 72 hour toxicity study, the cultures of Desmodesmus subspicatus were exposed to partially unsaturated TEA-Esterquat at nominal concentrations of 0, 1.0; 3.16; 10.0; 31.6; 100 mg/L under static conditions in accordance with the OECD Guideline 201. The EC10 and EC50 values based on growth rate were 2.61 and 6.46 mg/L, respectively. 

RE-EVALUATION OF STUDY DATA:

using a calculated concentration reduction factor (0.5844, geometric mean of measured concentration reduction) the following results were determined:

72h-ErC50 3.78 mg/L

72h-ErC10 1.53 mg/L 

 

(c)

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test), the cultures of the green algae Desmodesmus subspicatus were exposed to partially unsaturated TEA-Esterquat (CAS No. 157905-74-3) at nominal concentrations of 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L under static conditions. The average mean (geometric mean) concentration during the test was about 37 % (19,75 %) of the nominal concentrations. The 72 h NOEC and 72 h EC50 values based on growth rate were 9.25 and 22.3 mg/L (mean measured concentrations), respectively.

After study finalisation, ErC10 was recalculated from raw data with probit analysis after study report finalisation.

The 72-h ErC10 was 25.8 mg/L (nominal) resp. 5.09 mg/L (geometric mean measured concentration).

 

(d)

In this study with partially unsaturated TEA-Esterquat the ErC50 (24 h, growth rate) was 8.8 mg/L (nominal). The reliability was assigned 2 because no analytical control was performed.

ErC50 (24 h) = 8.8 mg/L

NOEC = 1.125 mg/L

72 h ErC10 = 3.3 mg/L test mat. nominal (recalculated from raw data) 

72 h ErC10 = 1.26 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)

 

(e)

In this study with partially unsaturated TEA-Esterquat the ErC50 (24 h, growth rate) is 9.0 mg/L (nominal). The reliability was assigned 2 because no analytical control was performed.    

ErC50 (24 h) = 9.0 mg/L 

NOEC = 1.125 mg/L 

72 h ErC10 = 2.72 mg/L test mat. nominal (recalculated from raw data)   

72 h ErC10 = 1.05 mg/L test mat. (calculated from nominal with concentration reduction factor 38.71%)