Reaction products of fatty acid chlorides, C8-12 (even numbered) with glycine and sodium hydroxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-06-15 to 2011-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The C12- and C14 fractions of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low and the guideline quality criteria are no longer fulfilled. To overcome this testing issue Ca2+ ions are complexed by Na2EDTA. Na2EDTA has a very low acute toxicity to Daphnia magna which is confirmed by a Na2EDTA control in the preliminary range finding test. As no effects were expected in the ISO test water containing 800 mg/L of Na2EDTA, no additional control without Na2EDTA was necessary.
- Method: The stock solution (160 mg/L test item was weighed out) was freshly prepared with dilution water which contained Na2EDTA. Disppersion treatment: Agitation. For preparation of the further test concentrations of 1.64 to 64.0 mg/L aliquots of the stock solution were taken while stirring with approx. 600 rpm.
- Eluate: ISO Test water (following OECD 202, Annex 3) as described below. By addition of 800 mg/L of Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA * 2 H2O) the Ca2+ ions were completely complexed and therefore most of the test item was still in the aqueous solution.
The pH of the dilution water was adjusted to pH 7.0 ± 0.2 by addition of 1 M NaOH.
- Differential loading: 6 test item concentrations were tested, set up in a geometrical series with a dilution factor of 2.5:
1.64 - 4.10 - 10.2 - 25.6 - 64.0 - 160 mg/L, prepared with dilution water. 20 g of the test solution each was weighed out into the test vessels. The daphnids were inserted with a small amount of dilution water by using a pipette.
- Controls: Na2EDTA control: Dilution water containing 800 mg/L Na2EDTA without test item was tested under the same conditions as the test groups. As no Na2EDTA related effects were observed in the preliminary test no separate control without Na2EDTA was included.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water: 21.4 (mg CaCO3/L), This corresponds to 14 mg Ca2+/L. Ca2+ ions were complexed by Na2EDTA * 2 H2O.

Test temperature:

19 - 20 °C.

pH:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per loading rate, pH-control and control)
Hostapon SG
Nominal Test Item Concentration
[mg/L] pH-Value
160 6.95
64.0 6.86
25.6 6.83
10.2 6.85
4.10 6.91
1.64 6.91
Na2EDTA Control 7.08

Water Quality Parameters at Test End (48 h, Old Media) (measured in all replicates)
Hostapon SG
Nominal Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
160 6.92 6.91 6.89 6.86
64.0 6.93 6.89 6.92 6.88
25.6 6.81 6.80 6.81 6.87
10.2 6.80 6.83 6.85 6.85
4.10 6.81 6.81 6.81 6.84
1.64 6.96 6.95 6.95 6.93
Na2EDTA Control 7.08 7.04 6.99 6.98

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h, Fresh Media) (measured in one replicate per loading rate, pH-control and control)
Hostapon SG
Nominal Test Itemn Concentration [mg/L]
Dissolved O2-Concentration[mg/L]
160 8.91
64.0 8.92
25.6 8.92
10.2 8.91
4.10 8.95
1.64 8.96
Na2EDTA Control 9.10
Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item Concentration [mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
160 8.83 8.80 8.88 8.86
64.0 8.74 8.81 8.84
25.6 8.82 8.84 8.81 8.86
10.2 8.87 8.84 8.86 8.88
4.10 8.88 8.86 8.87 8.91
1.64 8.87 8.90 8.93 8.90
Na2EDTA Control 8.88 8.71 8.85 8.86

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water (following OECD 202, Annex 3) as described below. By addition of 800 mg/L of Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA * 2 H2O) the Ca2+ ions were completely complexed and therefore most of the test item was still in the aqueous solution. The pH of the dilution water was adjusted to pH 7.0 ± 0.2 by addition of 1 M NaOH.
- Conductivity: Dilution water at test start: 886 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.64 - 4.10 - 10.2 - 25.6 - 64.0 - 160 mg/L (dilution factor 2.5)
- Range finding study
Immobilization Rates of the Preliminary Test after 24 and 48 h of Exposure
(n = 20, divided into 2 replicates with 10 daphnids each)
Hostapon SG
IMMOBILIZATION [%]
Nominal Test Item Concentration
[mg/L] 24 h 48 h
Replicates Replicates
1. 2. MV 1. 2. MV
1001) 2) 20 50 35 80 90 85
10.01) 2) 10 0 5 30 0 15
1.001) 2) 0 0 0 20 0 10
Na2ETA Control 1) 2) 0 0 0 0 0 0
Control 3) 0 0 0 0 0 0
1) = Prepared with ISO test water (acc. to OECD 202), containing 800 mg/L of Ethylenediaminetetraacetic acid
disodium salt dihydrate (Na2EDTA * 2 H2O)
2) = The pH was adjusted with 1 M NaOH from pH = 3.9 to pH = 7
3) = Pure ISO test water acc. to OECD 202

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 6.0-7.4 (95% CI)

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 2.8-4.0 (95% CI)

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: n.a.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: n.a.

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.44 mg/L (CI 1.31 - 1.55 mg/L)

Reported statistics and error estimates:

EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were estimated directly from the observation data, without mathematical calculation. EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Hostapon SG	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
160	80	80	80	100	85	100	100	100	100	100
64.0	20	0	40	60	30	80	80	100	100	90
25.6	0	0	0	0	0	20	20	40	40	30
10.2	0	0	0	0	0	0	0	0	0	0
4.10	0	0	0	0	0	0	0	0	0	0
1.64	0	0	0	0	0	0	0	0	0	0
Na2EDTA Control	0	0	0	0	0	0	0	0	0	0

MV    = Mean value

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 33.0 mg/L (95 % confidence limits: 30.2 - 37.2 mg/L) or 6.5 mg/L based on active ingredient.
The NOEC after 48 h was determined to be 10.2 mg/L as the highest tested concentration level with no biologically significant effect. The LOEC after 48 h was determined to be 25.6 mg/L as the lowest tested concentration level with a biologically significant effect.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Hostapon SG (batch number: ESD 0006364) were determined according to OECD 202 (2004) from 2011-06-15 to 2011-06-17 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low and the guideline quality criteria are no longer fulfilled. To overcome this testing issue Ca²⁺ ions were complexed by Na₂EDTA. Na₂EDTA has a very low acute toxicity to Daphnia magna which is confirmed by a Na₂EDTA control in the preliminary range finding test. As no effects were expected in the ISO test water containing 800 mg/L of Na₂EDTA, no additional control without Na₂EDTA was necessary.

The study was conducted under static conditions over a period of 48 h with 6concentrations of the test item Hostapon SG ranging from 1.64 to 160 mg/Lset up in a geometric series with a dilution factor of 2.5. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh (0 h) and old media (48 h) of all concentration levels and the control.
The recoveries of the C12-fraction were in the range of 94 to 99 % of the nominal values in the fresh media (0 h) and 83 to 109 % in the old media (0 h). Recoveries of the C14-fraction were in the range of 89 to 98 % of the nominal values in the fresh media (0 h) and 66 to 94 % in the old media (0 h).

All effect values are based on the nominal concentrations of the test item Hostapon SG as it contains different C-chains of the N-Cocoyl glycine sodium salt, and toxicity cannot be attributed to any single compound.  

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

              (based on the nominal concentrations of the test item Hostapon SG)

	Test Duration [h]	Nominal Concentration of the Test Item Hostapon SG [mg/L]	Confidence Interval p = 95% [mg/L]
EC10	24	40.7	31.4 - 53.5
	48	17.3	14.3 - 20.4
EC50	24	86.7	74.6 - 103
	48	33.0	30.2 - 37.2
EC100	24	> 160	---
	48	160	---
NOEC	48	10.2	---
LOEC	48	25.6	---

Results on acitive ingredient can be seen in the result table above.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-12 to 2011-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test item Hostapon SG was analytically verified based on the C12- and the C14 fraction of the test item by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For each test concentration, a separate stock solution was freshly prepared in dilution water. For details see table
Preparation of the test concentrations
Stock solution /
Test concentration
[mg/L] 18.75 37.5 75.0 150 300
Volume of
stock solution
[mL] 2000 1000 500 500 500
Weighing of
test item
[mg] 37.5 37.5 37.5 75.0 150.0

- Eluate: Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low. To overcome this testing issue Elendt M4 medium, according to ELENDT (1990), modified to a total hardness of approximately 140 mg CaCO3/L was used.
- Differential loading: 18.75 - 37.5 - 75.0 - 150 - 300 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water: 141 (mg CaCO3/L)

Test temperature:

20.5 °C.

pH:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration level and control)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] pH-Value



300 8.07
150 8.01
75.0 7.99
37.5 7.97
18.75 7.97
Control 8.36

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
300 7.46 7.50 7.61 7.55
150 7.41 7.47 7.38 7.47
75.0 7.44 7.26 7.33 7.39
37.5 7.77 7.72 7.69 7.30
18.75 7.65 7.74 7.71 7.79
Control 7.79 7.78 7.90 7.86

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration level and control)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] Dissolved
O2-Concentration

[mg/L]
300 9.20
150 9.20
75.0 9.25
37.5 9.26
18.75 9.27
Control 9.38

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
300 5.99 6.16 6.07 6.14
150 5.08 5.99 5.55 5.68
75.0 6.40 4.55 4.67 5.46
37.5 8.09 8.02 8.01 6.64
18.75 8.69 8.55 8.54 8.54
Control 8.84 8.95 8.81 8.80

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2 +/- 0.8




- Culture medium different from test medium: No
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study: none

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 2.5-3.4 (95% CI)

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

7.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 : 1.47 mg/L (CI 1.34 - 1.61 mg/L)

Reported statistics and error estimates:

EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Hostapon SG		IMMOBILIZATION [%]
Nominal Test Item Concentration	Half of the Nominal Test Item Concentration	24 h					48 h
		Replicates					Replicates
[mg/L]	[mg/L]	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
300	150	100	100	100	100	100	100	100	100	100	100
150	75.0	100	100	100	100	100	100	100	100	100	100
75.0	37.5	100	80	100	80	90	100	100	100	100	100
37.5	18.75	40	60	60	100	65	40	60	80	100	70
18.75	9.375	0	0	0	20	5	0	0	0	20	5
Control		0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Based on half of the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 14.3 mg/L (95 % confidence limits: 12.5 – 16.9 mg/L), which corresponds to 2.8 mg a.i./L (95 % confidence limits: 2.48 – 3.35 mg a.i./L), nominal content of 19.8 % taken into account.
The NOEC after 48 h was determined to be 9.4 mg/L, which corresponds to 1.9 mg a.i./L. The LOEC after 48 h was determined to be 18.8 mg/L, which corresponds to 3.7 mg a.i./L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Hostapon SG (batch number: ESD 0006364) were determined according to OECD 202 (2004) from 2011-10-12 to 2011-10-18, with the definitive exposure phase from 2011-10-14 to 2011-10-16, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h with 5concentrations of the liquid test item Hostapon SG (19.8 % w/w active ingredient) ranging from 18.75 to 300 mg/L set up in a geometric series with a dilution factor of 2. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh (0 h) and old media (48 h) of all concentration levels and the control.

Hostapon SG is a long chain carboxylic acid similar to fatty acids forming calcium salt with very low solubility removing most of the test item from the aqueous solution by precipitation. The recovery of the test item at the end of the test (48 h) is very low and the guideline quality criteria for the analytical recovery are no longer fulfilled. To reduce the precipitation of Calcium salts, the total hardness in the Elendt M4 medium (B.P. Elendt, 1990), was set to a total hardness of approximately 140 mg CaCO₃/L still acceptable to achieve valid results.

The recoveries of the C12-fraction were in the range of 84 to 115 % of the nominal values in the fresh media (0 h) and 1 to 3 % in the old media (48 h). Recoveries of the C14-fraction were in the range of 84 to 114 % of the nominal values in the fresh media (0 h) and 2 to 7 % in the old media (48 h).

The test item concentrations at the beginning of the test were in the range of 84 to 115 % and at test end after 48 h below 80 % of the nominal concentrations even below 1 mg a.i./L for C12-and C14-fraction. It is assumed that the test item concentration at the test end is 0 mg/L and the test item concentration over the full 48h test period is the average concentration at test start and end (half nominal concentration).The results derived with this approach can be compared with the results from a 48h daphnia study with EDTA where the Calcium ions were complexed and no precipitation of Calcium salts were observed. As the test item was not removed by precipitation and the recoveries were in the acceptable range at test start and test end, the exposure over the test period can be regarded as maximum (Clariant, 2011). The EC₅₀(48h) of the study with EDTA is 6.5 mg/L active ingredient which is a factor of 2.3 higher than the EC₅₀ (48h) in this study without EDTA. It can therefore be concluded that the evaluation of this study without EDTA using a half nominal approach can be regarded as a conservative.

The endpoint assessment based on mean measured concentrations is not possible, because the toxicity cannot be attributed to any single compound of the test item Hostapon SG containing different C-chains of the N-Cocoyl glycine sodium salt. The endpoints are also given as active ingredient based on the amount of 19.8 % in the aqueous solution.

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1:    EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

                   (based on half of the nominal test item concentrations of Hostapon SG)

Endpoints (with 95 % confidence limits)	Test Duration [h]	Half of the Nominal Concentration of the
		Test Item Hostapon SG [mg/L]	Active Substance (19.8 % w/w) [mg a.i./L]
EC10	24	9.8 (Cl: n.a.)	1.9 (Cl: n.a.)
	48	9.8 (Cl: n.a.)	1.9 (Cl: n.a.)
EC50	24	15.1 (Cl: 12.9 - 18.1)	3.0 (Cl: 2.55 - 3.58)
	48	14.3 (Cl: 12.5 - 16.9)	2.8 (Cl: 2.48 - 3.35)
EC100	24	75.0	14.9
	48	37.5	7.4
NOEC	48	9.4	1.9
LOEC	48	18.8	3.7

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2011-06-15 to 2011-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Sodium Lauroyl Glycinate (SLG) (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The C12- and C14 fractions of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low and the guideline quality criteria are no longer fulfilled. To overcome this testing issue Ca2+ ions are complexed by Na2EDTA. Na2EDTA has a very low acute toxicity to Daphnia magna which is confirmed by a Na2EDTA control in the preliminary range finding test. As no effects were expected in the ISO test water containing 800 mg/L of Na2EDTA, no additional control without Na2EDTA was necessary.
- Method: The stock solution (160 mg/L test item was weighed out) was freshly prepared with dilution water which contained Na2EDTA. Disppersion treatment: Agitation. For preparation of the further test concentrations of 1.64 to 64.0 mg/L aliquots of the stock solution were taken while stirring with approx. 600 rpm.
- Eluate: ISO Test water (following OECD 202, Annex 3) as described below. By addition of 800 mg/L of Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA * 2 H2O) the Ca2+ ions were completely complexed and therefore most of the test item was still in the aqueous solution.
The pH of the dilution water was adjusted to pH 7.0 ± 0.2 by addition of 1 M NaOH.
- Differential loading: 6 test item concentrations were tested, set up in a geometrical series with a dilution factor of 2.5:
1.64 - 4.10 - 10.2 - 25.6 - 64.0 - 160 mg/L, prepared with dilution water. 20 g of the test solution each was weighed out into the test vessels. The daphnids were inserted with a small amount of dilution water by using a pipette.
- Controls: Na2EDTA control: Dilution water containing 800 mg/L Na2EDTA without test item was tested under the same conditions as the test groups. As no Na2EDTA related effects were observed in the preliminary test no separate control without Na2EDTA was included.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water: 21.4 (mg CaCO3/L), This corresponds to 14 mg Ca2+/L. Ca2+ ions were complexed by Na2EDTA * 2 H2O.

Test temperature:

19 - 20 °C.

pH:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per loading rate, pH-control and control)
Hostapon SG
Nominal Test Item Concentration
[mg/L] pH-Value
160 6.95
64.0 6.86
25.6 6.83
10.2 6.85
4.10 6.91
1.64 6.91
Na2EDTA Control 7.08

Water Quality Parameters at Test End (48 h, Old Media) (measured in all replicates)
Hostapon SG
Nominal Test Item Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
160 6.92 6.91 6.89 6.86
64.0 6.93 6.89 6.92 6.88
25.6 6.81 6.80 6.81 6.87
10.2 6.80 6.83 6.85 6.85
4.10 6.81 6.81 6.81 6.84
1.64 6.96 6.95 6.95 6.93
Na2EDTA Control 7.08 7.04 6.99 6.98

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h, Fresh Media) (measured in one replicate per loading rate, pH-control and control)
Hostapon SG
Nominal Test Itemn Concentration [mg/L]
Dissolved O2-Concentration[mg/L]
160 8.91
64.0 8.92
25.6 8.92
10.2 8.91
4.10 8.95
1.64 8.96
Na2EDTA Control 9.10
Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item Concentration [mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
160 8.83 8.80 8.88 8.86
64.0 8.74 8.81 8.84
25.6 8.82 8.84 8.81 8.86
10.2 8.87 8.84 8.86 8.88
4.10 8.88 8.86 8.87 8.91
1.64 8.87 8.90 8.93 8.90
Na2EDTA Control 8.88 8.71 8.85 8.86

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water (following OECD 202, Annex 3) as described below. By addition of 800 mg/L of Ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA * 2 H2O) the Ca2+ ions were completely complexed and therefore most of the test item was still in the aqueous solution. The pH of the dilution water was adjusted to pH 7.0 ± 0.2 by addition of 1 M NaOH.
- Conductivity: Dilution water at test start: 886 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.64 - 4.10 - 10.2 - 25.6 - 64.0 - 160 mg/L (dilution factor 2.5)
- Range finding study
Immobilization Rates of the Preliminary Test after 24 and 48 h of Exposure
(n = 20, divided into 2 replicates with 10 daphnids each)
Hostapon SG
IMMOBILIZATION [%]
Nominal Test Item Concentration
[mg/L] 24 h 48 h
Replicates Replicates
1. 2. MV 1. 2. MV
1001) 2) 20 50 35 80 90 85
10.01) 2) 10 0 5 30 0 15
1.001) 2) 0 0 0 20 0 10
Na2ETA Control 1) 2) 0 0 0 0 0 0
Control 3) 0 0 0 0 0 0
1) = Prepared with ISO test water (acc. to OECD 202), containing 800 mg/L of Ethylenediaminetetraacetic acid
disodium salt dihydrate (Na2EDTA * 2 H2O)
2) = The pH was adjusted with 1 M NaOH from pH = 3.9 to pH = 7
3) = Pure ISO test water acc. to OECD 202

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 6.0-7.4 (95% CI)

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 2.8-4.0 (95% CI)

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: n.a.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: n.a.

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.44 mg/L (CI 1.31 - 1.55 mg/L)

Reported statistics and error estimates:

EC100-values after 24 and 48 h and the NOEC and LOEC after 48 h were estimated directly from the observation data, without mathematical calculation. EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Hostapon SG	IMMOBILIZATION [%]
Nominal Test Item Concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
160	80	80	80	100	85	100	100	100	100	100
64.0	20	0	40	60	30	80	80	100	100	90
25.6	0	0	0	0	0	20	20	40	40	30
10.2	0	0	0	0	0	0	0	0	0	0
4.10	0	0	0	0	0	0	0	0	0	0
1.64	0	0	0	0	0	0	0	0	0	0
Na2EDTA Control	0	0	0	0	0	0	0	0	0	0

MV    = Mean value

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 33.0 mg/L (95 % confidence limits: 30.2 - 37.2 mg/L) or 6.5 mg/L based on active ingredient.
The NOEC after 48 h was determined to be 10.2 mg/L as the highest tested concentration level with no biologically significant effect. The LOEC after 48 h was determined to be 25.6 mg/L as the lowest tested concentration level with a biologically significant effect.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Hostapon SG (batch number: ESD 0006364) were determined according to OECD 202 (2004) from 2011-06-15 to 2011-06-17 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low and the guideline quality criteria are no longer fulfilled. To overcome this testing issue Ca²⁺ ions were complexed by Na₂EDTA. Na₂EDTA has a very low acute toxicity to Daphnia magna which is confirmed by a Na₂EDTA control in the preliminary range finding test. As no effects were expected in the ISO test water containing 800 mg/L of Na₂EDTA, no additional control without Na₂EDTA was necessary.

The study was conducted under static conditions over a period of 48 h with 6concentrations of the test item Hostapon SG ranging from 1.64 to 160 mg/Lset up in a geometric series with a dilution factor of 2.5. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh (0 h) and old media (48 h) of all concentration levels and the control.
The recoveries of the C12-fraction were in the range of 94 to 99 % of the nominal values in the fresh media (0 h) and 83 to 109 % in the old media (0 h). Recoveries of the C14-fraction were in the range of 89 to 98 % of the nominal values in the fresh media (0 h) and 66 to 94 % in the old media (0 h).

All effect values are based on the nominal concentrations of the test item Hostapon SG as it contains different C-chains of the N-Cocoyl glycine sodium salt, and toxicity cannot be attributed to any single compound.  

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

              (based on the nominal concentrations of the test item Hostapon SG)

	Test Duration [h]	Nominal Concentration of the Test Item Hostapon SG [mg/L]	Confidence Interval p = 95% [mg/L]
EC10	24	40.7	31.4 - 53.5
	48	17.3	14.3 - 20.4
EC50	24	86.7	74.6 - 103
	48	33.0	30.2 - 37.2
EC100	24	> 160	---
	48	160	---
NOEC	48	10.2	---
LOEC	48	25.6	---

Results on acitive ingredient can be seen in the result table above.

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2011-10-12 to 2011-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Sodium Lauroyl Glycinate (SLG) (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test item Hostapon SG was analytically verified based on the C12- and the C14 fraction of the test item by LC-MS/MS in the fresh media (0 h) and old media (48 h) of all concentration levels and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For each test concentration, a separate stock solution was freshly prepared in dilution water. For details see table
Preparation of the test concentrations
Stock solution /
Test concentration
[mg/L] 18.75 37.5 75.0 150 300
Volume of
stock solution
[mL] 2000 1000 500 500 500
Weighing of
test item
[mg] 37.5 37.5 37.5 75.0 150.0

- Eluate: Hostapon SG is a long chain carboxylic acid forming Calcium salt with very low solubility removing most of the test item from the aqueous solution. The recovery of the test item at the end of the test (48 h) is therefore very low. To overcome this testing issue Elendt M4 medium, according to ELENDT (1990), modified to a total hardness of approximately 140 mg CaCO3/L was used.
- Differential loading: 18.75 - 37.5 - 75.0 - 150 - 300 mg/L
- Controls: Dilution water without test item tested under the same conditions as the test groups.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20+/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µE*m-2*s-1. Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L. Culture Feeding: At least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata, Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 106 cells/mL.
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

Dilution water: 141 (mg CaCO3/L)

Test temperature:

20.5 °C.

pH:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration level and control)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] pH-Value



300 8.07
150 8.01
75.0 7.99
37.5 7.97
18.75 7.97
Control 8.36

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] pH-Values
Replicates
1 2 3 4
300 7.46 7.50 7.61 7.55
150 7.41 7.47 7.38 7.47
75.0 7.44 7.26 7.33 7.39
37.5 7.77 7.72 7.69 7.30
18.75 7.65 7.74 7.71 7.79
Control 7.79 7.78 7.90 7.86

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h, Fresh Media)
(measured in one replicate per concentration level and control)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] Dissolved
O2-Concentration

[mg/L]
300 9.20
150 9.20
75.0 9.25
37.5 9.26
18.75 9.27
Control 9.38

Water Quality Parameters at Test End (48 h, Old Media)
(measured in all replicates)
Hostapon SG
Nominal
Test Item
Concentration
[mg/L] Dissolved O2 -Concentration [mg/L]
Replicates
1 2 3 4
300 5.99 6.16 6.07 6.14
150 5.08 5.99 5.55 5.68
75.0 6.40 4.55 4.67 5.46
37.5 8.09 8.02 8.01 6.64
18.75 8.69 8.55 8.54 8.54
Control 8.84 8.95 8.81 8.80

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
according to ELENDT (1990)
Component Concentration [mg/L]
CaCl2 x 2 H2O 147
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 4.30
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Na2EDTA x 2 H2O 5.00
FeSO4 x 7 H2O 1.99
H3BO3 0.29
MnCl2 x 4 H2O 0.36
LiCl 0.30
SrCl2 x 6 H2O 0.15
RbCl 0.071
NaBr 0.016
Na2MoO4 x 2 H2O 0.063
CuCl x 2 H2O 0.017
ZnCl2 0.013
CoCl2 x 6 H2O 0.010
KJ 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamine 0.0010
Biotin 0.00075
pH 8.2 +/- 0.8




- Culture medium different from test medium: No
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
IImmobility was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim within 15 sec after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study: none

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

3.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 2.5-3.4 (95% CI)

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

7.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 : 1.47 mg/L (CI 1.34 - 1.61 mg/L)

Reported statistics and error estimates:

EC10- and EC50-values were calculated by sigmoidal dose-response regression. The confidence intervals were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism. The concentration effect relationships are shown graphically.

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                 (n = 20, divided into 4 replicates with 5 daphnids each)

Hostapon SG		IMMOBILIZATION [%]
Nominal Test Item Concentration	Half of the Nominal Test Item Concentration	24 h					48 h
		Replicates					Replicates
[mg/L]	[mg/L]	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
300	150	100	100	100	100	100	100	100	100	100	100
150	75.0	100	100	100	100	100	100	100	100	100	100
75.0	37.5	100	80	100	80	90	100	100	100	100	100
37.5	18.75	40	60	60	100	65	40	60	80	100	70
18.75	9.375	0	0	0	20	5	0	0	0	20	5
Control		0	0	0	0	0	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

Based on half of the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 14.3 mg/L (95 % confidence limits: 12.5 – 16.9 mg/L), which corresponds to 2.8 mg a.i./L (95 % confidence limits: 2.48 – 3.35 mg a.i./L), nominal content of 19.8 % taken into account.
The NOEC after 48 h was determined to be 9.4 mg/L, which corresponds to 1.9 mg a.i./L. The LOEC after 48 h was determined to be 18.8 mg/L, which corresponds to 3.7 mg a.i./L.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS) the effects of the test item Hostapon SG (batch number: ESD 0006364) were determined according to OECD 202 (2004) from 2011-10-12 to 2011-10-18, with the definitive exposure phase from 2011-10-14 to 2011-10-16, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h with 5concentrations of the liquid test item Hostapon SG (19.8 % w/w active ingredient) ranging from 18.75 to 300 mg/L set up in a geometric series with a dilution factor of 2. Twenty daphnids were exposed to each concentration level and the control.

The concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS in the fresh (0 h) and old media (48 h) of all concentration levels and the control.

Hostapon SG is a long chain carboxylic acid similar to fatty acids forming calcium salt with very low solubility removing most of the test item from the aqueous solution by precipitation. The recovery of the test item at the end of the test (48 h) is very low and the guideline quality criteria for the analytical recovery are no longer fulfilled. To reduce the precipitation of Calcium salts, the total hardness in the Elendt M4 medium (B.P. Elendt, 1990), was set to a total hardness of approximately 140 mg CaCO₃/L still acceptable to achieve valid results.

The recoveries of the C12-fraction were in the range of 84 to 115 % of the nominal values in the fresh media (0 h) and 1 to 3 % in the old media (48 h). Recoveries of the C14-fraction were in the range of 84 to 114 % of the nominal values in the fresh media (0 h) and 2 to 7 % in the old media (48 h).

The test item concentrations at the beginning of the test were in the range of 84 to 115 % and at test end after 48 h below 80 % of the nominal concentrations even below 1 mg a.i./L for C12-and C14-fraction. It is assumed that the test item concentration at the test end is 0 mg/L and the test item concentration over the full 48h test period is the average concentration at test start and end (half nominal concentration).The results derived with this approach can be compared with the results from a 48h daphnia study with EDTA where the Calcium ions were complexed and no precipitation of Calcium salts were observed. As the test item was not removed by precipitation and the recoveries were in the acceptable range at test start and test end, the exposure over the test period can be regarded as maximum (Clariant, 2011). The EC₅₀(48h) of the study with EDTA is 6.5 mg/L active ingredient which is a factor of 2.3 higher than the EC₅₀ (48h) in this study without EDTA. It can therefore be concluded that the evaluation of this study without EDTA using a half nominal approach can be regarded as a conservative.

The endpoint assessment based on mean measured concentrations is not possible, because the toxicity cannot be attributed to any single compound of the test item Hostapon SG containing different C-chains of the N-Cocoyl glycine sodium salt. The endpoints are also given as active ingredient based on the amount of 19.8 % in the aqueous solution.

The water quality parameters (pH-value and dissolved oxygen concentration), measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1:    EC₁₀-, EC₅₀- (with Confidence Interval), EC₁₀₀ - Values, NOEC and LOEC

                   (based on half of the nominal test item concentrations of Hostapon SG)

Endpoints (with 95 % confidence limits)	Test Duration [h]	Half of the Nominal Concentration of the
		Test Item Hostapon SG [mg/L]	Active Substance (19.8 % w/w) [mg a.i./L]
EC10	24	9.8 (Cl: n.a.)	1.9 (Cl: n.a.)
	48	9.8 (Cl: n.a.)	1.9 (Cl: n.a.)
EC50	24	15.1 (Cl: 12.9 - 18.1)	3.0 (Cl: 2.55 - 3.58)
	48	14.3 (Cl: 12.5 - 16.9)	2.8 (Cl: 2.48 - 3.35)
EC100	24	75.0	14.9
	48	37.5	7.4
NOEC	48	9.4	1.9
LOEC	48	18.8	3.7

Description of key information

In the key study, based on the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 33.0 mg/L (95 % confidence limits: 30.2 - 37.2 mg/L) or 6.5 mg/L based on active ingredient.

The NOEC after 48 h was determined to be 10.2 mg/L as the highest tested concentration level with no biologically significant effect. The LOEC after 48 h was determined to be 25.6 mg/L as the lowest tested concentration level with a biologically significant effect.

There is a second non key study available. Based on half of the nominal concentrations of the test item Hostapon SG the 48-h EC50 for Daphnia magna was estimated to be 14.3 mg/L (95 % confidence limits: 12.5 – 16.9 mg/L), which corresponds to 2.8 mg a.i./L (95 % confidence limits: 2.48 – 3.35 mg a.i./L), nominal content of 19.8 % taken into account.

The NOEC after 48 h was determined to be 9.4 mg/L, which corresponds to 1.9 mg a.i./L. The LOEC after 48 h was determined to be 18.8 mg/L, which corresponds to 3.7 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6.5 mg/L