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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-31 to 2011-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L
- Sampling method: The C12- and C14-fraction 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 once every 7days.
For the old media additionally prepared replicates with test media but without daphnids and algae were prepared and incubated under test conditions until sampling.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (40 mg/L test item was weighed out) was freshly prepared with dilution water.
- Eluate: Dilution water
- Differential loading: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 144 148 142 146 142 150
Control 146 146 141 171 151 142

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 21.7 20.9 20.8 21.4 21.0 20.4
Control 21.5 21.0 20.2 21.1 20.8 20.4

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 8.39 7.81 7.96 7.58 8.49 7.73
Control 8.46 7.68 8.50 7.75 8.53 7.86

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 9.05 8.15 8.86 7.53 8.61 8.38
Control 9.54 9.25 9.44 8.72 9.35 8.32

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 mg CaCO3/L.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: No range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

and mortality

Details on results:

- Mortality of parent animals:
The test item did not induced biologically substantial adult mortality of > 20 % at any of the tested concentration levels of 1.02 to 40.0 mg/L after 21 days.
All parental daphnids survived until the end of the study in the concentration levels of 1.02 to 16.0 mg/L and in the control. At the concentration level of 40.0 mg/L 1 of 10 parental daphnids died on day 20 and 9 of 10 parental daphnids survived until the end of the study.
An EC10- or EC50 for the adult mortality after 21 days could not be estimated because adult mortality > 10 % was not observed within the tested concentration range.

- No. of offspring produced per day per female:
Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L and the control. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods.
The average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was not statistically significant reduced in comparison to the control at any of the tested concentration levels (KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks, DUNN’S method, p = 0.05).
An EC10- or EC50-value for the reduction of the reproductive output could not be estimated, because no reduction of the reproductive output > 10 % was observed within the tested concentration range of 1.02 to 40.0 mg/L.

- Body length and weight of parent animals:
At the end of the test the total length of all surviving parental daphnids at each tested concentration level and the control were determined. The mean body lengths of the surviving parental daphnids at the nominal test item concentrations 1.02 to 40.0 mg/L were determined to be in the range of 5.19 to 5.60 mm per daphnid and 5.25 mm per daphnid at the control group, respectively.
The mean dry body weight was determined to be in the range of 0.47 to 0.72 mg per daphnid in the test groups and 0.66 mg per daphnid in the control, respectively.

- Type and number of morphological / behavioural abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % in the tested concentration levels of 1.02 to 40.0 mg/L

- Time to first brood release or time to hatch:
The first day of appearance of juveniles in all replicates juveniles of the tested concentration levels of 1.02 to 40.0 mg/L and the control was between day 8 and day 10.
Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods. There was no statistical significant difference at any of the tested concentration levels in comparison to the control (KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks, p = 0.05).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.70 (CI 1.59 - 1.86)

Reported statistics and error estimates:

The NOEC and the LOEC for the reproductive output and for the adult mortality were determined directly from the observation data. Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
For the determination of significant deviations of the reproductive output, the intrinsic rates of natural increase, the day of appearance of the first brood and the number of broods KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks (p = 0.05) was used. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal Test Item Conc. [mg/L]												No. of Parents prod. Juv.	Mean No. of Juveniles per Parent producing Juveniles			Comparison versus Control
	Number of Juveniles in Replicate No.										Total No.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
40.0	127	--	147	131	140	135	83	136	137	152	1188	9	132 ±	20	15	-0.763	no
16.0	135	140	147	147	143	140	133	135	132	131	1383	10	138 ±	6	4	-5.57	no
6.40	150	150	129	130	134	127	131	126	116	122	1315	10	132 ±	11	8	-0.382	no
2.56	121	123	121	125	115	126	116	132	128	97	1204	10	120 ±	10	8	8.09	no
1.02	123	132	127	129	123	129	128	127	121	132	1271	10	127 ±	4	3	2.98	no
Control	128	131	126	132	131	132	137	126	135	132	1310	10	131 ±	4	3	—	—

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal Test Item Concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[mg/L]	Juveniles	Eggs	å	N	MV ± SD
40.0	16	5	21	9	2.33 ± 2.45
16.0	7	9	16	10	1.60 ± 1.78
6.40	10	16	26	10	2.60 ± 2.46
2.56	17	21	38	10	3.80 ± 1.99
1.02	2	10	12	10	1.20 ± 1.32
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal Test Item Concentration	Day of First Appearance of Living Juveniles*										First	Mean Number
	in Replicate No.										Appearance	of
[mg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day	Broods*
40.0	8	--	8	8	8	8	8	8	8	10	8.2	5.0
16.0	8	9	8	8	8	8	8	9	9	8	8.3	5.0
6.40	8	8	8	8	8	9	8	8	8	8	8.1	5.0
2.56	8	8	8	8	8	8	8	8	8	9	8.1	4.9
1.02	8	8	8	8	8	8	8	8	8	8	8.0	5.0
Control	9	8	8	8	8	8	8	8	9	8	8.2	5.0

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal Test Item Concentration	Adult Mortality [%]
[mg/L]	7 days	14 days	21 days
40.0	0	0	10
16.0	0	0	0
6.40	0	0	0
2.56	0	0	0
1.02	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
40.0	5.00	--	5.25	5.75	5.50	5.00	4.75	5.75	5.00	4.75	5.19	9	5.9	0.66
16.0	5.75	5.50	5.50	5.50	5.75	5.75	5.50	5.75	5.25	5.75	5.60	10	6.5	0.65
6.40	5.50	5.75	5.25	5.50	5.25	5.25	5.50	5.50	5.50	5.50	5.45	10	7.2	0.72
2.56	5.50	5.75	5.25	5.25	5.75	5.50	5.25	5.50	5.75	5.00	5.45	10	4.7	0.47
1.02	5.25	5.25	5.25	5.25	5.25	5.00	5.25	5.25	5.00	5.50	5.23	10	4.7	0.47
Control	5.00	5.25	5.25	5.50	5.50	5.50	5.00	5.25	5.00	5.25	5.25	10	6.6	0.66

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) for the reproductive output were determined directly from reduction of the reproductive output. EC10- and EC50- values for the reproductive output and the adult mortality could not be estimated because effects > 10 % were not observed.
As the concentrations in the old media (48 h) were below LOQ, the endpoints were assessed based on the nominal test item concentrations corrected with a factor of 2. 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. Therefore, it was concluded that an evaluation on half of the nominal concentrations sufficiently describes a worst case scenario. Additionally the endpoints are given based on half of nominal concentrations of the active ingredient (a.i.) taking its content of 19.8 % into account.

NOECReproduction : 20 mg/L, corresponding to 3.96 mg a.i./L
LOECReproduction : > 20 mg/L, corresponding to > 3.96 mg a.i./L

NOECAdult Mortality : 20 mg/L, corresponding to 3.96 mg a.i./L
LOECAdult Mortality : > 20 mg/L, corresponding to > 3.96 mg a.i./L

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item Hostapon SG(batch No. ESD 0006364) was conducted according to OECD 211 (2008) from 2011-08-31 to 2011-09-22 with the definitive exposure phase from 2011-08-31 to 2011-09-21 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany .

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with renewal of the test solutions three times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of the liquid test item Hostapon SG (19.8 % w/w active ingredient) were selected based on the results of an acute immobilization test performed at the test facility [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project No. DAI14343/ 100719CL), Dr.U.Noack-Laboratorien ] as follows: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L.

There was no effect observed in any of the tested concentration levels and in the control.

Testing above this concentration range was considered not to be useful, because significant mortality (> 50 %) was observed in the acute immobilization test. This was confirmed in a second acute immobilization test [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project DAI14343/111006CL), Dr.U.Noack-Laboratorien, Sarstedt, Germany] under the same test conditions of the Daphnia magna Reproduction Test giving an LOEC of 18.75 mg/L and an EC₅₀ of 14.3 mg/L after 48 hours.

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 an exposure interval (48 or 72 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 concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS of samples on days 0, 7, 14 (fresh media, 0 hours) and on days 2, 9, 16 (old media, 48 hours) at all concentration levelsand the control.
The recoveries in the fresh media (0 h) were in the range of 91 to 103 % of the nominal values for the C12-fraction and 87 to 106 % for the C14-fraction. Recoveries in the old media (48 h) were below the limit of quantification (LOQ) for both analytes.

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 test item concentrations in the fresh media were in the range of 87 to 106 % and in the old media after 48 h below the LOQ of the analytical method. It is assumed that the test item concentration at the end of every exposure interval of 48 or 72 h is 0 mg/L and the test item concentration over the full 48 or 72 h exposure interval is the average concentration of the initially and finally measured concentration (half nominal concentration). The results derived with this approach can be compared with the results from a 48h acute immobilization test with Daphnia magna using EDTA where the Calcium ions were complexed and no precipitation of Calcium salts were observed [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project DAI14343/111006CL), Dr.U.Noack-Laboratorien, Sarstedt, Germany]. 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. 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 average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was not statistically significant reduced in comparison to the control at any of the tested concentration levels (Kruskal-Wallis, One Way Analysis of Variance on Ranks, Dunn’s method, p = 0.05).
An EC₁₀- or EC₅₀-value for the reduction of the reproductive output could not be estimated, because no reduction of the reproductive output > 10 % was observed within the tested concentration range of 1.02 to 40.0 mg/L.

·   The coefficient of variation of the number of living offspring produced per parent was 3 % in the control.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by Kruskal-Wallis, One Way Analysis of Variance on Ranks (p = 0.05).There was no statistically significant difference at any of the tested concentration levels of 1.02 to 40.0 mg/L when compared to the control.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % in the tested concentration levels of 1.02 to 40.0 mg/L. In the control no dead juveniles or aborted eggs were observed.

·   Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L and the control. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods. There was no statistical significant difference at any of the tested concentration levels in comparison to the control (Kruskal-Wallis, One Way Analysis of Variance on Ranks, p = 0.05). The first brood was released till day 10 and by all surviving parent animals of the control and the nominal test item concentrations of 1.02 to 40.0 mg/L.

The test item did not induced biologically substantial adult mortality of > 20 % at any of the tested concentration levels of 1.02 to 40.0 mg/L after 21 days. All parental daphnids survived until the end of the study in the concentration levels of 1.02 to 16.0 mg/L and in the control. At the concentration level of 40.0 mg/L 1 of 10 parental daphnids died on day 20 and 9 of 10 parental daphnids survived until the end of the study. An EC₁₀- or EC₅₀for the adult mortality after 21 days could not be estimated because adult mortality > 10 % was not observed within the tested concentration range.

·   The mean body lengths of the surviving parental daphnids at the nominal test item concentrations 1.02 to 40.0 mg/L were determined to be in the range of 5.19 to 5.60 mm per daphnid and 5.25 mm per daphnid at the control group, respectively. The mean dry body weight was determined to be in the range of 0.47 to 0.72 mg per daphnid in the test groups and 0.66 mg per daphnid in the control, respectively.

·   The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC)after 21 days and the observation data mentioned above are summarized in Table 1.

Table 1:       Test Item Related Effects (NOEC and LOEC)

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

Effects		Hostapon SG Half of the Nominal Test Item Concentrations [mg/L] (Half of the Nominal Concentrations of the active ingredient [mg a.i./L])
		Control	0.510 (0.10)	1.28 (0.25)	3.20 (0.63)	8.00 (1.58)	20.0 (3.96)
Mean Number of Living Juveniles per Producing Parent (Reproduction Rate ± SD)		131 ± 4	127 ± 4	120 ± 10	132 ± 11	138 ± 6	132 ± 20
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]		3	3	8	8	4	15
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]		0	1	3	2	1	2
Mean Intrinsic Rates of Natural Increase		0.50	0.51	0.49	0.50	0.50	0.50
Appearance of First Brood [Mean Day]		8.2	8.0	8.1	8.1	8.3	8.2
Mean Number of Broods		5.00	5.00	4.90	5.00	5.00	5.00
NOECReproduction		20.0 mg/L (corresponding to    3.96 mg a.s./L)
LOECReproduction		> 20.0 mg/L (corresponding to > 3.96 mg a.s./L)
Adult Mortality after 21 Days [%]		0	0	0	0	0	10
Parent Animals:	Mean Dry Weight [mg]	0.66	0.47	0.47	0.72	0.65	0.66
	Mean Body Length [mm]	5.25	5.23	5.45	5.45	5.60	5.19
NOECAdult Mortality		20.0 mg/L (corresponding to    3.96 mg a.s./L)
LOECAdult Mortality		> 20.0 mg/L (corresponding to > 3.96 mg a.s./L)

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

2011-08-31 to 2011-09-21

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 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L
- Sampling method: The C12- and C14-fraction 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 once every 7days.
For the old media additionally prepared replicates with test media but without daphnids and algae were prepared and incubated under test conditions until sampling.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (40 mg/L test item was weighed out) was freshly prepared with dilution water.
- Eluate: Dilution water
- Differential loading: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L
- Controls: 10 replicates of dilution water without test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Strain/clone: Clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna STRAUS is recommended in the guideline
- Source: Own breeding (Origin: Institut fuer Wasser- Boden- und Lufthygiene)
- Age of parental stock (mean and range, SD): > 14 days
- Feeding during test
- Food type: Mix of Pseudokirchneriella subcapitata and Desmodesmus subspicatus
- Amount: 0.2 mg C/daphnia per day
- Frequency: daily


ACCLIMATION
- Acclimation period: 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: During acclimation the daphnids were not fed
- Feeding frequency: None
- Health during acclimation (any mortality observed): Healthy

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: The parent animals were removed from the culture medium and the juveniles collected over a sieve and flushed into fresh medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Hardness:

Total Hardness [mg/L] as CaCO3
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 144 148 142 146 142 150
Control 146 146 141 171 151 142

Test temperature:

Temperature [°C]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 21.7 20.9 20.8 21.4 21.0 20.4
Control 21.5 21.0 20.2 21.1 20.8 20.4

pH:

pH-Values
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 8.39 7.81 7.96 7.58 8.49 7.73
Control 8.46 7.68 8.50 7.75 8.53 7.86

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Test Item
Concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 14 Day 16
Aug. 31st Sept. 2nd Sept. 7th Sept. 9th Sept. 14th Sept. 16th
40.0 9.05 8.15 8.86 7.53 8.61 8.38
Control 9.54 9.25 9.44 8.72 9.35 8.32

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): 3 x per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control and pH-control (replicates): 10


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to ELENDT (1990), modified to a total hardness of > 140 mg CaCO3/L.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Max. 20 µE*m-2*s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Adult mortality: daily
- Number of juveniles: daily
- Stillborn juveniles and aborted eggs: daily
- Appearance of first brood
- Intrinsic rate of natural increase: test end
- Growth (total length and dry weight): test end


RANGE-FINDING STUDY
- Test concentrations: No range-finding study

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

and mortality

Details on results:

- Mortality of parent animals:
The test item did not induced biologically substantial adult mortality of > 20 % at any of the tested concentration levels of 1.02 to 40.0 mg/L after 21 days.
All parental daphnids survived until the end of the study in the concentration levels of 1.02 to 16.0 mg/L and in the control. At the concentration level of 40.0 mg/L 1 of 10 parental daphnids died on day 20 and 9 of 10 parental daphnids survived until the end of the study.
An EC10- or EC50 for the adult mortality after 21 days could not be estimated because adult mortality > 10 % was not observed within the tested concentration range.

- No. of offspring produced per day per female:
Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L and the control. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods.
The average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was not statistically significant reduced in comparison to the control at any of the tested concentration levels (KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks, DUNN’S method, p = 0.05).
An EC10- or EC50-value for the reduction of the reproductive output could not be estimated, because no reduction of the reproductive output > 10 % was observed within the tested concentration range of 1.02 to 40.0 mg/L.

- Body length and weight of parent animals:
At the end of the test the total length of all surviving parental daphnids at each tested concentration level and the control were determined. The mean body lengths of the surviving parental daphnids at the nominal test item concentrations 1.02 to 40.0 mg/L were determined to be in the range of 5.19 to 5.60 mm per daphnid and 5.25 mm per daphnid at the control group, respectively.
The mean dry body weight was determined to be in the range of 0.47 to 0.72 mg per daphnid in the test groups and 0.66 mg per daphnid in the control, respectively.

- Type and number of morphological / behavioural abnormalities:
Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % in the tested concentration levels of 1.02 to 40.0 mg/L

- Time to first brood release or time to hatch:
The first day of appearance of juveniles in all replicates juveniles of the tested concentration levels of 1.02 to 40.0 mg/L and the control was between day 8 and day 10.
Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods. There was no statistical significant difference at any of the tested concentration levels in comparison to the control (KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks, p = 0.05).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.70 (CI 1.59 - 1.86)

Reported statistics and error estimates:

The NOEC and the LOEC for the reproductive output and for the adult mortality were determined directly from the observation data. Significant deviations were determined in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
For the determination of significant deviations of the reproductive output, the intrinsic rates of natural increase, the day of appearance of the first brood and the number of broods KRUSKAL-WALLIS, One Way Analysis of Variance on Ranks (p = 0.05) was used. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

Number of Juveniles in the Control and Test Groups after 21 Days

Nominal Test Item Conc. [mg/L]												No. of Parents prod. Juv.	Mean No. of Juveniles per Parent producing Juveniles			Comparison versus Control
	Number of Juveniles in Replicate No.										Total No.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
40.0	127	--	147	131	140	135	83	136	137	152	1188	9	132 ±	20	15	-0.763	no
16.0	135	140	147	147	143	140	133	135	132	131	1383	10	138 ±	6	4	-5.57	no
6.40	150	150	129	130	134	127	131	126	116	122	1315	10	132 ±	11	8	-0.382	no
2.56	121	123	121	125	115	126	116	132	128	97	1204	10	120 ±	10	8	8.09	no
1.02	123	132	127	129	123	129	128	127	121	132	1271	10	127 ±	4	3	2.98	no
Control	128	131	126	132	131	132	137	126	135	132	1310	10	131 ±	4	3	—	—

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal Test Item Concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[mg/L]	Juveniles	Eggs	å	N	MV ± SD
40.0	16	5	21	9	2.33 ± 2.45
16.0	7	9	16	10	1.60 ± 1.78
6.40	10	16	26	10	2.60 ± 2.46
2.56	17	21	38	10	3.80 ± 1.99
1.02	2	10	12	10	1.20 ± 1.32
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal Test Item Concentration	Day of First Appearance of Living Juveniles*										First	Mean Number
	in Replicate No.										Appearance	of
[mg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day	Broods*
40.0	8	--	8	8	8	8	8	8	8	10	8.2	5.0
16.0	8	9	8	8	8	8	8	9	9	8	8.3	5.0
6.40	8	8	8	8	8	9	8	8	8	8	8.1	5.0
2.56	8	8	8	8	8	8	8	8	8	9	8.1	4.9
1.02	8	8	8	8	8	8	8	8	8	8	8.0	5.0
Control	9	8	8	8	8	8	8	8	9	8	8.2	5.0

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                 (n = 10)

Nominal Test Item Concentration	Adult Mortality [%]
[mg/L]	7 days	14 days	21 days
40.0	0	0	10
16.0	0	0	0
6.40	0	0	0
2.56	0	0	0
1.02	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parental Daphnids

Nominal Test Item Concentration [mg/L]	Total Length of the Parent Animals [mm]										MV	N	Dry Weight [mg]
	Replicate No.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
40.0	5.00	--	5.25	5.75	5.50	5.00	4.75	5.75	5.00	4.75	5.19	9	5.9	0.66
16.0	5.75	5.50	5.50	5.50	5.75	5.75	5.50	5.75	5.25	5.75	5.60	10	6.5	0.65
6.40	5.50	5.75	5.25	5.50	5.25	5.25	5.50	5.50	5.50	5.50	5.45	10	7.2	0.72
2.56	5.50	5.75	5.25	5.25	5.75	5.50	5.25	5.50	5.75	5.00	5.45	10	4.7	0.47
1.02	5.25	5.25	5.25	5.25	5.25	5.00	5.25	5.25	5.00	5.50	5.23	10	4.7	0.47
Control	5.00	5.25	5.25	5.50	5.50	5.50	5.00	5.25	5.00	5.25	5.25	10	6.6	0.66

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) for the reproductive output were determined directly from reduction of the reproductive output. EC10- and EC50- values for the reproductive output and the adult mortality could not be estimated because effects > 10 % were not observed.
As the concentrations in the old media (48 h) were below LOQ, the endpoints were assessed based on the nominal test item concentrations corrected with a factor of 2. 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. Therefore, it was concluded that an evaluation on half of the nominal concentrations sufficiently describes a worst case scenario. Additionally the endpoints are given based on half of nominal concentrations of the active ingredient (a.i.) taking its content of 19.8 % into account.

NOECReproduction : 20 mg/L, corresponding to 3.96 mg a.i./L
LOECReproduction : > 20 mg/L, corresponding to > 3.96 mg a.i./L

NOECAdult Mortality : 20 mg/L, corresponding to 3.96 mg a.i./L
LOECAdult Mortality : > 20 mg/L, corresponding to > 3.96 mg a.i./L

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item Hostapon SG(batch No. ESD 0006364) was conducted according to OECD 211 (2008) from 2011-08-31 to 2011-09-22 with the definitive exposure phase from 2011-08-31 to 2011-09-21 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany .

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, individually held, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with renewal of the test solutions three times per week. Aim of the Daphnia Reproduction Test over 21 days was to assess effects on the reproduction capacity and other test item-related effects or parameters such as time of production of first brood, adult mortality, intrinsic rate of natural increase, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of the liquid test item Hostapon SG (19.8 % w/w active ingredient) were selected based on the results of an acute immobilization test performed at the test facility [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project No. DAI14343/ 100719CL), Dr.U.Noack-Laboratorien ] as follows: 1.02 - 2.56 - 6.40 - 16.0 - 40.0 mg/L.

There was no effect observed in any of the tested concentration levels and in the control.

Testing above this concentration range was considered not to be useful, because significant mortality (> 50 %) was observed in the acute immobilization test. This was confirmed in a second acute immobilization test [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project DAI14343/111006CL), Dr.U.Noack-Laboratorien, Sarstedt, Germany] under the same test conditions of the Daphnia magna Reproduction Test giving an LOEC of 18.75 mg/L and an EC₅₀ of 14.3 mg/L after 48 hours.

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 an exposure interval (48 or 72 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 concentrations of the C12- and C14-fraction of the test item Hostapon SG were analytically verified by LC-MS/MS of samples on days 0, 7, 14 (fresh media, 0 hours) and on days 2, 9, 16 (old media, 48 hours) at all concentration levelsand the control.
The recoveries in the fresh media (0 h) were in the range of 91 to 103 % of the nominal values for the C12-fraction and 87 to 106 % for the C14-fraction. Recoveries in the old media (48 h) were below the limit of quantification (LOQ) for both analytes.

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 test item concentrations in the fresh media were in the range of 87 to 106 % and in the old media after 48 h below the LOQ of the analytical method. It is assumed that the test item concentration at the end of every exposure interval of 48 or 72 h is 0 mg/L and the test item concentration over the full 48 or 72 h exposure interval is the average concentration of the initially and finally measured concentration (half nominal concentration). The results derived with this approach can be compared with the results from a 48h acute immobilization test with Daphnia magna using EDTA where the Calcium ions were complexed and no precipitation of Calcium salts were observed [Noack, M. (2011): Hostapon SG - Acute Immobilization Test to Daphnia magna, Static, 48 h (Study-No./Project DAI14343/111006CL), Dr.U.Noack-Laboratorien, Sarstedt, Germany]. 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. 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 average number of juveniles per parent in the control group was 131 after 21 days. The reproductive output was not statistically significant reduced in comparison to the control at any of the tested concentration levels (Kruskal-Wallis, One Way Analysis of Variance on Ranks, Dunn’s method, p = 0.05).
An EC₁₀- or EC₅₀-value for the reduction of the reproductive output could not be estimated, because no reduction of the reproductive output > 10 % was observed within the tested concentration range of 1.02 to 40.0 mg/L.

·   The coefficient of variation of the number of living offspring produced per parent was 3 % in the control.

·   The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. The mean IR of the surviving daphnids of the treatment groups were compared to the control by Kruskal-Wallis, One Way Analysis of Variance on Ranks (p = 0.05).There was no statistically significant difference at any of the tested concentration levels of 1.02 to 40.0 mg/L when compared to the control.

·   Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles and aborted eggs was ≤ 3 % in the tested concentration levels of 1.02 to 40.0 mg/L. In the control no dead juveniles or aborted eggs were observed.

·   Five broods were observed during the test period at all surviving parental daphnids of the control and the concentration levels 1.02, 6.40, 16.0 and 40.0 mg/L and the control. At the concentration level of 2.56 mg/L 1 of 10 parental daphnids released four broods and 9 of 10 daphnids had five broods. There was no statistical significant difference at any of the tested concentration levels in comparison to the control (Kruskal-Wallis, One Way Analysis of Variance on Ranks, p = 0.05). The first brood was released till day 10 and by all surviving parent animals of the control and the nominal test item concentrations of 1.02 to 40.0 mg/L.

The test item did not induced biologically substantial adult mortality of > 20 % at any of the tested concentration levels of 1.02 to 40.0 mg/L after 21 days. All parental daphnids survived until the end of the study in the concentration levels of 1.02 to 16.0 mg/L and in the control. At the concentration level of 40.0 mg/L 1 of 10 parental daphnids died on day 20 and 9 of 10 parental daphnids survived until the end of the study. An EC₁₀- or EC₅₀for the adult mortality after 21 days could not be estimated because adult mortality > 10 % was not observed within the tested concentration range.

·   The mean body lengths of the surviving parental daphnids at the nominal test item concentrations 1.02 to 40.0 mg/L were determined to be in the range of 5.19 to 5.60 mm per daphnid and 5.25 mm per daphnid at the control group, respectively. The mean dry body weight was determined to be in the range of 0.47 to 0.72 mg per daphnid in the test groups and 0.66 mg per daphnid in the control, respectively.

·   The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC)after 21 days and the observation data mentioned above are summarized in Table 1.

Table 1:       Test Item Related Effects (NOEC and LOEC)

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

Effects		Hostapon SG Half of the Nominal Test Item Concentrations [mg/L] (Half of the Nominal Concentrations of the active ingredient [mg a.i./L])
		Control	0.510 (0.10)	1.28 (0.25)	3.20 (0.63)	8.00 (1.58)	20.0 (3.96)
Mean Number of Living Juveniles per Producing Parent (Reproduction Rate ± SD)		131 ± 4	127 ± 4	120 ± 10	132 ± 11	138 ± 6	132 ± 20
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]		3	3	8	8	4	15
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]		0	1	3	2	1	2
Mean Intrinsic Rates of Natural Increase		0.50	0.51	0.49	0.50	0.50	0.50
Appearance of First Brood [Mean Day]		8.2	8.0	8.1	8.1	8.3	8.2
Mean Number of Broods		5.00	5.00	4.90	5.00	5.00	5.00
NOECReproduction		20.0 mg/L (corresponding to    3.96 mg a.s./L)
LOECReproduction		> 20.0 mg/L (corresponding to > 3.96 mg a.s./L)
Adult Mortality after 21 Days [%]		0	0	0	0	0	10
Parent Animals:	Mean Dry Weight [mg]	0.66	0.47	0.47	0.72	0.65	0.66
	Mean Body Length [mm]	5.25	5.23	5.45	5.45	5.60	5.19
NOECAdult Mortality		20.0 mg/L (corresponding to    3.96 mg a.s./L)
LOECAdult Mortality		> 20.0 mg/L (corresponding to > 3.96 mg a.s./L)

Description of key information

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) for the reproductive output were determined directly from reduction of the reproductive output. EC10- and EC50- values for the reproductive output and the adult mortality could not be estimated because effects > 10 % were not observed.

As the concentrations in the old media (48 h) were below LOQ, the endpoints were assessed based on the nominal test item concentrations corrected with a factor of 2. 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. Therefore, it was concluded that an evaluation on half of the nominal concentrations sufficiently describes a worst case scenario. Additionally the endpoints are given based on half of nominal concentrations of the active ingredient (a.i.) taking its content of 19.8 % into account.

NOECReproduction       :       20 mg/L, corresponding to    3.96 mg a.i./L

LOECReproduction       :    > 20 mg/L, corresponding to > 3.96 mg a.i./L

NOECAdult Mortality       :       20 mg/L, corresponding to    3.96 mg a.i./L

LOECAdult Mortality       :    > 20 mg/L, corresponding to > 3.96 mg a.i./L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4 mg/L