Reaction mass of sodium hydrogen N,N-dibutyl-10-(sulphonatooxy)octadecanamidate and sodium sulphooctadecanoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-11 to 2013-01-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted October 03, 2008.

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

May 30, 2008

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling: One sample from the freshly prepared stock solution and duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the first treatment period (Day 0) and at a treatment period in the second and third week (Days 10 and 17). For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the renewal periods, duplicate samples of the respective aged test media and controls were sampled at Day 3, 12 and 19. One of these three stability control treatments lasted for 72 hours (weekend), two for 48 hours, corresponding to the different test medium renewal periods.
Analyses: The concentration of the test item Humectol C liq hc was only measured in the highest test concentration of nominal 2 mg/L in all duplicate test medium samples from all sampling times. The other test media were not analysed, since they were below the NOEC determined in this test. From the control samples only one of the duplicate samples was analysed from each sampling date.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution was prepared by dissolving 22.2, 23.3, 20.1, 20.8, 20.6, 21.0, 21.8, 20.3 and 20.0 mg test item into 555, 582.5, 502.5, 520, 515, 525, 545, 507.5 and 500 mL test water respectively by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test medium. The appearance of the test item in the test media was determined at each test medium renewal period in the freshly prepared and aged test media of all test concentrations.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus / Clone 5
- Justification for species other than prescribed by test guideline: according to guideline
- Stage and instar at study initiation: 4.25 to 21.75 hours old
- Source: IBACON in-house laboratory culture
- Feeding during test: yes
- Food type: green algae (Desmodesmus subspicatus)
- Amount: based on the concentration of total organic carbon (TOC) in the algal food suspension. The approximate daily amounts of algal TOC/Daphnia were as follows:
Days 0-3: 0.1 mg
Days 4-7: 0.15 mg
Days 8-20: 0.2 mg

ACCLIMATION
- Acclimation conditions (same as test or not): same

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

19 - 21 °C in the freshly prepared media;
19 - 21 °C in the aged media

pH:

7.4 to 8.3 in the freshly prepared media;
7.4 to 8.5 in the aged media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

7.7 to 9.7 mg/L in the freshly prepared media;
8.1 to 10.8 mg/L in the aged media

Nominal and measured concentrations:

Nominal: 2.0, 0.633, 0.2, 0.063 and 0.02 mg test item/L and a control.
Measured: Within ± 20 % of the nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Material, size, fill volume: glass beaker, 100 mL, 60 mL
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: reconstituted water (Elendt "M4")

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark.
- Light intensity: 1050 - 1360 lux.

EFFECT PARAMETERS MEASURED
Mortality of Adults and Number of Young: The mortality of the test animals and the number of young daphnids were recorded for each day. Dead animals and offspring were removed at the same times.

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Survival

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Survival

Details on results:

Biological Results

Reproduction:

21-day NOEC 2.0 mg test item/L
21-day LOEC > 2.0 mg test item/L
21-day EC50 (95 % CI) > 2.0 mg test item/L (n.d.)
21-day EC20 (95 % CI) > 2.0 mg test item/L (n.d.)
21-day EC10 (95 % CI) > 2.0 mg test item/L (n.d.)

CI: Confidence interval
n.d.: Not determinable

Survival:
21-day NOEC 2.0 mg test item/L
21-day LOEC > 2.0 mg test item/L

Survival of Adults: At the test item concentrations of 0.02 and 0.063 mg test item/L, all Daphnia survived until the end of the test. In the control and the test item concentrations of 0.2 to 2.0 mg test item/L, one Daphnia died until the end of the test. Thus, the survival rate of Daphnia magna after 21 days was not significantly reduced in any test concentration.

Reproduction: The total numbers of live offspring produced by all surviving adults for each treatment group and the reproduction is given in Table 1. The reproduction of those Daphnia in the control was 147 ± 30 (mean ± SD) live offspring per adult. The coefficient of variation around the mean number of living offspring produced per parent animal in the control was 20.4 %. According to the results of a Williams-test (one-sided,  = 0.05), the lowest concentration showed a significant inhibition when compared to the control. However, since all test concentrations above the LOEC must have a harmful effect equal to or greater than those observed at the LOEC, and all higher test concentration showed no significant effect, this significance was deemed not to be treatment related. Therefore, no significant toxic effect of the test item on the mean reproduction was determined up to and including the highest nominal test concentration of 2.0 mg test item/L. The EC50 of the reproduction was determined to be > 2.0 mg test item/L.

Date of First Brood: The first young Daphnia released from their parent animals were recorded in the nominal test concentrations of 0.633 and 2.0 mg test item/L at the observation on day 8. In the control and the nominal test item concentrations up to and including 0.2 mg test item/L, the first offspring was recorded at the observation on day 9.
Signs of Intoxication: No particular signs of intoxication were observed at the test animals during the test.

Appearance of the Test Item in the Test Media: No remarkable observations.

Reported statistics and error estimates:

The NOEC and the LOEC for the reproduction and survival were evaluated by the Williams Multiple Sequential t-test and the Fisher's exact, respectively. The EC50, EC20 and EC10 of the reproduction could not be calculated due to absence of toxicity of the test item. The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat® Solutions GmbH.

Table 1: Number of Surviving Adult Daphnia magna Exposed to Test Item

Exposure Day	Nominal concentration [mg test item/L]
	Control	0.02	0.063	0.2	0.633	2.0
0	10	10	10	10	10	10
1	10	10	10	10	10	10
2	10	10	10	10	10	10
3	10	10	10	9	10	10
4	10	10	10	9	10	10
5	10	10	10	9	10	10
6	10	10	10	9	10	10
7	10	10	10	9	10	10
8	10	10	10	9	10	10
9	10	10	10	9	10	10
10	10	10	10	9	10	10
11	9	10	10	9	10	9
12	9	10	10	9	10	9
13	9	10	10	9	10	9
14	9	10	10	9	10	9
15	9	10	10	9	9	9
16	9	10	10	9	9	9
17	9	10	10	9	9	9
18	9	10	10	9	9	9
19	9	10	10	9	9	9
20	9	10	10	9	9	9
21	9	10	10	9	9	9
% surviving	-	111	111	100	100	100

Table 2: Number of Live Offspring Produced by all Surviving Adults within 21 Days of Exposure (Cumulative Values)

Exposure Day	Nominal concentration [mg test item/L]
	Control	0.02	0.063	0.2	0.633	2.0
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	0	0	0	0
4	0	0	0	0	0	0
5	0	0	0	0	0	0
6	0	0	0	0	0	0
7	0	0	0	0	0	0
8	0	0	0	0	31	18
9	129	120	115	43	108	142
10	129	120	115	135	142	142
11	129	120	137	135	241	239
12	376	387	352	223	436	405
13	376	387	380	379	436	441
14	376	387	380	423	638	605
15	624	729	734	469	881	778
16	624	729	773	606	935	778
17	636	729	773	705	1026	967
18	984	1059	1133	837	1306	1197
19	984	1059	1133	912	1306	1260
20	984	1059	1178	1095	1360	1424
21	1321	1213	1387	1183	1694	1581
No. of surviving adults	9	10	10	9	9	9
Average number of offspring per surviving adult	146.8	121.3	138.7	131.4	188.2	175.7

Table 3: Total Number of Live Immature Daphnia Produced by All Adults (Cumulative Values)

Exposure Day	Nominal concentration [mg test item/L]
	Control	0.02	0.063	0.2	0.633	2.0
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	0	0	0	0
4	0	0	0	0	0	0
5	0	0	0	0	0	0
6	0	0	0	0	0	0
7	0	0	0	0	0	0
8	0	0	0	0	31	18
9	143	120	115	43	122	155
10	143	120	115	135	156	155
11	143	120	137	135	292	252
12	390	387	352	223	487	418
13	390	387	380	379	487	454
14	390	387	380	423	736	618
15	638	729	734	469	979	791
16	638	729	773	606	1033	791
17	650	729	773	705	1124	980
18	998	1059	1133	837	1404	1210
19	998	1059	1133	912	1404	1273
20	998	1059	1178	1095	1458	1437
21	1335	1213	1387	1183	1792	1594
% of control	-	90.9	103.9	88.6	134.2	119.4

Table 4: Number of Live Immature Daphnia produced by each surviving Adult after 21 Days

Nominal concentration [mg test item/L]
Replicate	Control	0.02	0.063	0.2	0.633	2.0
1	-	144	134	83	191	196
2	178	116	117	135	205	158
3	131	113	124	124	176	166
4	137	0	161	-	182	-
5	169	145	127	136	190	167
6	168	165	158	123	-	190
7	113	146	158	176	161	201
8	179	140	163	154	222	168
9	95	122	106	130	191	163
10	151	122	139	122	176	172
mean	146.8	121.3	138.7	131.4	188.2	175.7
SD	29.9	45.7	20.4	25.2	17.7	15.7
CV	20.4 %	37.6 %	14.7 %	19.2 %	9.4 %	8.9 %
% of control	-	82.6*	94.5	89.6	128.2	119.7

[Williams t_test, a = 0.05; * = significant]

_ = adult died in the course of the study

Table 5: Summary of Analytical Results

Sample description [mg test item/L]	Mass 492.3		Mass 394.6
	% of nominal^1	RSD [%]	% of nominal^1	RSD [%]
control	n.a.	n.a.	n.a.	n.a.
2	85	11	87	10

¹mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n.a.: not applicable

n: number of analysed samples

Validity criteria fulfilled:

yes

Conclusions:

The influence of the test item on the survival and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21 day NOEC was determined to be 2.0 mg test item/L for the reproduction and for the survival of the adult test animals. The 21-day EC50 for the reproduction was > 2.0 mg test item/L.

Executive summary:

The purpose of this study was to evaluate the toxicity of the test item on the survival and reproduction of Daphnia magna during an exposure period of 21 days according to OECD TG 211 and EU Method C.20. Young Daphnia (< 24 hours old) were exposed in a semi-static test to aqueous test media containing the test item at various concentrations. The study encompassed 5 test concentrations and one control with 10 replicates (2.0, 0.633, 0.2, 0.063 and 0.02 mg test item/L and a control), each containing one Daphnia. A semi-static test was performed. The mortality rate, the reproduction and symptoms of intoxication were compared with corresponding parameters in the control. The quantification of the test item was performed using liquid chromatography (LC-MS-method). In the freshly prepared test media 95 % of the nominal test concentration was found in case of measurement of masses 492.3 and 394.6. In the aged test media 78 % (mass 492.3) and 82 % (mass 394.6) was found (mean values of nominal 2 mg/L, respectively). Correct dosing of the test item could be demonstrated. Therefore, all biological endpoints were based on nominal test concentrations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. All validity criteria were met. The 21-day NOEC was determined to be 2.0 mg test item/L for the reproduction and for the survival of the adult test animals. The 21-day EC50 for the reproduction was > 2.0 mg test item/L.

Description of key information

The toxicity of the test item on the survival and reproduction of Daphnia magna was determined during an exposure period of 21 days. The 21 day NOEC was determined to be 2.0 mg test item/L for the reproduction and for the survival of the adult test animals. The 21-day EC₅₀ for the reproduction was > 2.0 mg test item/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2 mg/L

Additional information

The purpose of this study was to evaluate the toxicity of the test item on the survival and reproduction of Daphnia magna during an exposure period of 21 days according to OECD TG 211 and EU Method C.20. Young Daphnia (< 24 hours old) were exposed in a semi-static test to aqueous test media containing the test item at various concentrations. The study encompassed 5 test concentrations and one control with 10 replicates (2.0, 0.633, 0.2, 0.063 and 0.02 mg test item/L and a control), each containing one Daphnia. A semi-static test was performed. The mortality rate, the reproduction and symptoms of intoxication were compared with corresponding parameters in the control. The quantification of the test item was performed using liquid chromatography (LC-MS-method). In the freshly prepared test media 95 % of the nominal test concentration was found in case of measurement of masses 492.3 and 394.6. In the aged test media 78 % (mass 492.3) and 82 % (mass 394.6) was found (mean values of nominal 2 mg/L, respectively). Correct dosing of the test item could be demonstrated. Therefore, all biological endpoints were based on nominal test concentrations. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. All validity criteria were met. The 21-day NOEC was determined to be 2.0 mg test item/L for the reproduction and for the survival of the adult test animals. The 21-day EC50 for the reproduction was > 2.0 mg test item/L.

The reported effect concentrations refer to the commercial formulation containing 46.2% (w/w) of the REACH registration substance which reflects the test item used in the study.