D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-11 to 2014-07-02

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.

EC Number:

940-422-0

Cas Number:

1591783-13-9

Molecular formula:

C15H31NO6 (C8 derivative) C17H35NO6 (C10 derivative) C19H39NO6 (C12 derivative) C21H43NO6 (C14 derivative) C23H47NO6 (C16 derivative) C25H51NO6 (C18 derivative) C25H49NO6 (C18 unsatd. derivative)

IUPAC Name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.

Test material form:

other: waxy

Details on test material:

- Molecular formula: C19H39NO6 (C12-Glucamide) and C21H43NO6 (C14-Glucamide)
- Molecular weight: 377.52 – 405.57 g/mol for C12- and C14-Glucamide
- Substance type: UVCB
- Physical state: waxy solid
- Stability under test conditions: Stable (in water and light at room temperature)
- Storage condition of test material: Room temperature, protected from light, in the tightly closed original container
- Other:
- Solubility (in water at RT): Slightly soluble
- pH value (in aqueous solution): Ca. 8-10 at concentration of 10.000 [mg/L]
- Melting point: 70 – 71 °C
- Boiling point: No boiling point, substance decomposes before boiling
- Vapour pressure: < 0.1 Pa

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg test item/L (dilution factor 2).
Expressed as concentrations of Glucamides based on the nominal content of 81.0 % (w/w) the test concentrations were:
0.405 – 0.810 – 1.62 – 3.24 – 6.48 mg Glucamides/L


- Sampling method: All concentration levels of the test item and the control were analysed via LC-MS/MS at the start of the exposure intervals on days 0, 7, 16 (0 h) and at the end of the exposure intervals on days 2, 9 (48 h), 19 (72 h). Samples of the longest exposure interval of 72 h were taken on day 16 (start of the exposure interval, 0 h) and day 19 (end of the exposure interval, 72 h) and analysed.
At the start of the exposure intervals (0 h), sampling was carried out after preparation of the test concentrations.
At the end of the exposure intervals (48 and 72 h), samples were taken from additional replicates without daphnids and food algae. The additional replicates were incubated under test conditions until sampling.


Recoveries of the test item should be within ± 20 % of the nominal concentrations. The effect levels are given based on the nominal concentrations of the test item and the sum of Glucamides (active content of 81.0% was taken into account), because the test item is an UVCB substance (i.e. substances of Unknown or Variable composition, Complex reaction products or Biological materials).




- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored at room temperature until analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (8.00 mg/L of the test item were weighed out) was freshly prepared with dilution water for each exposure interval.
The stock solution was treated with ultrasound for 30 minutes at 40 °C.
- Eluate: Dilution water
- Differential loading: Five test item concentrations, prepared by diluting the stock solution of 8.00 mg/L with dilution water, were tested as follows:
0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg test item/L (dilution factor 2).
Expressed as concentrations of Glucamides based on the nominal content of 81.0 % (w/w) the test concentrations were:
0.405 – 0.810 – 1.62 – 3.24 – 6.48 mg Glucamides/L

- Controls: 10 replicates of dilution water without test item.

Test organisms

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: The test solutions were renewed 3 times per week (every 48 or 72 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by pipette.


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

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not observed

Test conditions

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 16 Day 19
2014-06-11 2014-06-13 2014-06-18 2014-06-20 2014-06-27 2014-06-30
8.00 182 178 Not determined, due to 100 % adult mortality
4.00 Not determined 178 185 168 178
Control 173 187 161 185 171 173

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 16 Day 19
2014-06-11 2014-06-13 2014-06-18 2014-06-20 2014-06-27 2014-06-30
8.00 22.0 21.4 Not determined, due to 100 % adult mortality
4.00 Not determined 21.3 21.8 21.8 21.9
Control 21.7 21.6 21.1 21.7 21.8 22.0

pH:

pH-Values
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 7 Day 9 Day 16 Day 19
2014-06-11 2014-06-13 2014-06-18 2014-06-20 2014-06-27 2014-06-30
8.00 7.03 7.11 Not determined, due to 100 % adult mortality
4.00 Not determined 6.95 7.31 7.06 7.38
Control 7.48 6.93 7.53 7.27 7.43 7.16

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 16 Day 19
2014-06-11 2014-06-13 2014-06-18 2014-06-20 2014-06-27 2014-06-30
8.00 8.11 8.63 Not determined, due to 100 % adult mortality
4.00 Not determined 8.41 8.12 8.46 8.42
Control 8.86 7.85 8.61 7.98 8.59 7.97

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed with a watch glasss
- Material, size, headspace, fill volume: 100 mL glass beaker, filled with 50 mL test solution
- Aeration: No
- Renewal rate of test solution (frequency): The test solutions were renewed 3 times per week (every 48 or 72 h. For this purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by pipette.
- 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 160 to 180 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
Based on the results of an acute immobilisation test performed at the test facility [Noack, M. (2014): Glucamide CC - Acute Immobilisation Test to Daphnia magna, Static, 48 h (Unpublished Report: Study-No. DAI15922, Project-No. 140326CS), DR. U. NOACK-LABORATORIEN, Sarstedt, Germany], the definitive reproduction test was carried out with 5 concentration levels of the test item in the range of 0.500 to 8.00 mg test item/L, prepared with dilution water as specified below.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 1.62 - 6.48 mg/L

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 1.62 - 6.48 mg/L

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.24 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: reproduction / mortality

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

6.48 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: reproduction / mortality

Effect concentrations 5

Duration:

21 d

Dose descriptor:

other: EC100

Effect conc.:

6.48 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality of 100 % at the concentration level of 8.00 mg/L during the test period of 21 days. In the concentration levels of 0.500 to 2.00 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. At the concentration level of 4.00 mg/L, an adult mortality of 20 % was observed, which is not statistically significant (FISHER’S Exact Test).
The adult mortality was the only effect in this study. Based on the significant adult mortality of 100 % at the concentration level of 8.00 mg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 4.00 mg test item/L, which corresponds to 3.24 mg Glucamides/L, and the Lowest Observed Effect Concentration (LOEC) was assessed at 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.
The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 3.82 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.10 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L). The EC50 for the adult mortality was calculated accordingly to be 4.32 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.50 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L). The EC100 for the adult mortality was 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.



- No. of offspring produced per day per female:
The number of stillborn juveniles and aborted eggs per surviving parental daphnid of the test and control groups are presented (please refer to the respective table). The fraction of dead juveniles (sum of stillborn juveniles and aborted eggs) expressed as a percentage, compared to the total number of produced juveniles (alive, stillborn juveniles and aborted eggs) of the surviving daphnids is presented in Table 7.
No stillborn juveniles or aborted eggs were observed in the control during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 3 % at the concentration levels of 0.500 to 4.00 mg test item/L.


- Body length and weight of parent animals:
The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.500 to 4.00 mg test item/L were determined to be in the range of 4.88 to 5.09 mm per daphnid and 5.15 mm per daphnid in the control group.
The mean dry body weight of the surviving parental daphnids was determined to be in the range of 0.68 to 0.85 mg per daphnid in the concentration levels of 0.500 to 4.00 mg test item/L and 0.74 mg per daphnid in the control.

- Reproductive Output:
The number of juveniles in all replicates of the control and the test groups was counted and recorded every day.
The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 110 in the control group. Effects concerning the reproductive output were not observed. There was no statistically significant difference at any of the tested concentration levels of 0.500 to 4.00 mg test item/L in comparison to the reproductive output in the control (One Way Analysis of Variance, DUNNETT’S method, p = 0.05).
At the concentration level of 8.00 mg test item/L, all daphnids died before producing juveniles.
The coefficient of variation of the number of living offspring produced per surviving parental daphnid was 7 % in the control. At the concentration levels of 0.500 to 4.00 mg test item/L, the coefficient of variation was in the range of 9 to 17 % . Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %.
EC-values were not calculated. The lack of significant effects combined with the lack of an effect level greater than 50 % makes the calculation of an EC10- or an EC50-value uncertain.


- Type and number of morphological / behavioural abnormalities: please refer to the respective table
- Time to first brood release or time to hatch:
The first appearance of living juveniles was observed between days 7 and 10 in all replicates of the control and the concentration levels of 0.500 to 4.00 mg test item/L. At the concentration level of 8.00 mg test item/L all parental daphnids died before appearance of the first brood.
At the tested concentration levels of 0.500 to 4.00 mg test item/L and the control, 5 broods with living juveniles were produced by the surviving parental daphnids. However, one parental daphnid at the concentration level of 0.500 mg test item/L produced 4 broods with living juveniles.

No males were observed in the control or in the test groups during the test.

No ephippia were observed in the control or in the test groups during the test.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.53 (CI 1.35 - 1.72) mg/L

Reported statistics and error estimates:

The NOEC and the LOEC based on the statistically significant adult mortality were deduced from the results of the statistical evaluation as described below.
Significant deviations were determined in comparison to the control using statistical standard procedures as normality test, equal variance test, analysis of variance and FISHER’S Exact Test. Prior to running the analysis of variance, a normality test and an equal variance test was performed. P-values of the normality and equal variance tests should be 0.05. For the determination of significant deviations of the number of living juveniles per parental daphnid, the P-values were > 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.
The determination of significant deviations of the number of living juveniles per parental daphnid was conducted with One Way Analysis of Variance, (P = 0.05). Additionally, statistical significance of the observed adult mortality was proven via FISHER’S Exact Test in comparison to the control.
The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.

The EC10- and the EC50-value for the adult mortality was calculated by sigmoidal dose-response regression with the software GraphPad prism5. The highest concentration level causing no adult mortality and the lowest concentration level producing 100% adult mortality were used as confidence limits, because only one partial effect was observed (only one concentration level with an effect > 0 % and < 100 %). An EC10 or EC50 for the reduction of the reproductive output was not calculated, due to the lack of significant effects. The EC50-value for the reference item was calculated accordingly. The confidence interval for the EC50-value of the reference item was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

Any other information on results incl. tables

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) of the control and the highest tested concentration level of 8.00 mg test item/L were measured in one replicate. Due to 100 % adult mortality at the concentration level of 8.00 mg/L on day 5, the water quality parameters were determined at the concentration level of 4.00 mg/L thereafter.

Significant deviations of ≥1.5 units between initial and final pH values were not observed.

The incubator temperature was in the range of 19 to 21 °C.

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
8.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
4.00	4.75	5.00	5.25	5.00	--	5.25	5.25	5.00	5.25	--	5.09	8	5.4	0.68
2.00	5.00	5.25	4.75	5.25	5.00	5.25	5.00	5.00	5.00	5.00	5.05	10	6.8	0.68
1.00	5.00	5.00	5.00	5.00	5.00	5.00	5.00	5.25	5.25	4.75	5.03	10	8.5	0.85
0.500	4.75	4.50	4.75	5.25	4.50	4.75	5.00	5.00	5.25	5.00	4.88	10	7.8	0.78
Control	5.00	5.00	5.25	5.25	5.25	5.25	5.25	5.50	4.75	5.00	5.15	10	7.4	0.74

MV      = mean value

N        = number of the surviving parental daphnids

--         = not applicable, due to the mortality of the parental daphnid(s)

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

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles										First	Mean
	in replicate no.										appearance	number
	1	2	3	4	5	6	7	8	9	10	mean day	of broods
8.00	--	--	--	--	--	--	--	--	--	--	--	--
4.00	9	9	8	9	--	8	9	9	8	--	8.6	5.0
2.00	8	7	8	8	7	8	8	8	8	8	7.8	5.0
1.00	7	8	8	7	7	8	9	8	7	8	7.7	5.0
0.500	8	10	8	8	8	8	8	8	8	8	8.2	4.9
Control	8	8	8	8	8	8	7	8	8	8	7.9	5.0

--   = not applicable, due to the mortality of the parental daphnid(s)

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

  (n = 10)

Nominal test item concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
8.00	100	100	100
4.00	0	10	20
2.00	0	0	0
1.00	0	0	0
0.500	0	0	0
Control	0	0	0

Number of living 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 living juveniles in replicate no.										Total no.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV±SD		[%]	[%]
8.00	--	--	--	--	--	--	--	--	--	--	0	0	--		--	--	--
4.00	70	110	120	94	--	115	86	111	105	--	811	8	101±	17	17	8	no
2.00	116	126	127	119	94	94	108	95	124	111	1114	10	111±	13	12	-1	no
1.00	113	112	100	122	100	96	110	89	107	101	1050	10	105±	10	9	5	no
0.500	105	79	100	107	115	108	109	113	113	105	1054	10	105±	10	10	5	no
Control	113	115	105	104	126	108	102	121	108	102	1104	10	110±	8	7	—	—

conc.        = concentration

N               = number of the surviving parental daphnids producing living juveniles

MV ± SD    = mean value ± standard deviation

CV             = coefficient of variation

Red.          = reduction of the reproductive output (a negative value means increase of the reproductive output)

Stat.           = statistical significance(One Way Analysis of Variance,Dunnett’sMethod,P = 0.05)

yes            = statistically significant

no            = statistically not significant

--               = not applicable, due to the mortality of the parental daphnid(s)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The adult mortality was the only effect in this study. Based on the significant adult mortality of 100 % at the concentration level of 8.00 mg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 4.00 mg test item/L, which corresponds to 3.24 mg Glucamides/L, and the Lowest Observed Effect Concentration (LOEC) was assessed at 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.
The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 3.82 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.10 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L).
The EC50 for the adult mortality was calculated accordingly to be 4.32 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.50 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L). The EC100 for the adult mortality was 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.
Effects concerning the reproductive output were not observed. EC-values were not calculated. The lack of significant effects combined with the lack of an effect level greater than 50 % makes the calculation of an EC10- or an EC50-value uncertain.

Effect values Nominal concentrations of the Nominal concentrations
test item given as sum of glucamides
Glucamide CC (81.0% (w/w))
[mg test item/L] [mg Glucamides/L]

EC10 Adult mortality
(with 95 % confidence limits) 3.82 (CI: 2.00 – 8.00) 3.10 (CI: 1.62 – 6.48)
EC50 Adult mortality
(with 95 % confidence limits) 4.32 (Cl: 2.00 – 8.00) 3.50 (CI: 1.62 – 6.48)
EC100 Adult mortality 8.00 6.48
NOEC Reproduction / Adult mortality 4.00 3.24
LOEC Reproduction / Adult mortality 8.00 6.48

Executive summary:

TheDaphnia magna Reproduction Test (semi-static, 21 d) of the test item Glucamide CC (batch number:EK13/177) was conducted according to OECD 211 (2012) from2014-06-11 to 2014-07-02 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids were used, individually held in ten replicates per concentration level, and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions with a 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 adult mortality, intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, body length and dry weight of the surviving parental daphnids.

The test item is an UVCB substance (substances of Unknown or Variable composition, Complex reaction products or Biological materials) and is slightly soluble in water at room temperature.

Nominal concentrations of the test item Glucamide CC were selected based on the results of an acute immobilisation test performed at the test facility [Noack, M. (2014):Glucamide CC-Acute Immobilisation Test to Daphnia magna, Static, 48 h(Unpublished Report:Study-No. DAI15922, Project-No.140326CS), Dr.U.Noack-Laboratorien, Sarstedt, Germany]. The chosen nominal test item concentrations areas follows:0.500-1.00-2.00-4.00-8.00 mg test item/L.
Based on the nominal content of 81.0 % (w/w) Glucamides in the UVCB substance, the test concentrations were:0.405–0.810–1.62–3.24–6.48 mg Glucamides/L.

The concentrations of the main components C12-Glucamide and C14-Glucamide were analytically verified via LC-MS/MS at the start of the exposure intervals on days 0, 7, 16 (0 h) and at the end of the exposure intervals on days 2, 9 (48 h), 19 (72 h)in all concentration levels and the control.

The measured concentrations of the component C12-Glucamide at the start of the exposure intervals (0 h) were in the range of 81 to 118 % of the nominal values and 85 to 107 % of the nominal values at the end of the exposure intervals (48 and 72 h). The measured concentrations of the component C14-Glucamide at the start of the exposure intervals (0 h) were in the range of 82 to 115 % of the nominal values and 55 to 103 % of the nominal values at the end of the exposure intervals (48 and 72 h).

Nevertheless the effect levels are based on the nominal concentrations of the test itemGlucamide CC and the active components Glucamides (sum of glucamides).

 

The adult mortality was the only effect in this study. Based on the significant adult mortality of 100 % at the concentration level of 8.00 mg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 4.00 mg test item/L, which corresponds to 3.24 mg Glucamides/L, and the Lowest Observed Effect Concentration (LOEC) was assessed at 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.

 

 

·  The test item induced significant adult mortality of 100 % at the concentration level of 8.00 mg test item/L during the test period of 21 days.In the concentration levels of 0.500 to 2.00 mg test item/L and in the control, all parental daphnids survived until the end of the test after 21 days. At the concentration level of 4.00 mg test item/L, an adult mortality of 20 % was observed, which is not statistically significant (Fisher’sExact Test).
The EC₁₀for the adult mortality was calculated by sigmoidal dose-response regression to be 3.82 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.10 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L). The EC₅₀for the adult mortality was calculated accordingly to be 4.32 mg test item/L (95 % confidence limits: 2.00 – 8.00 mg test item/L), which corresponds to 3.50 mg Glucamides/L (95% confidence limits: 1.62 – 6.48 mg Glucamides/L). The EC₁₀₀for the adult mortality was 8.00 mg test item/L, which corresponds to 6.48 mg Glucamides/L.

 

·  The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 110 in the control group. Effects concerning the reproductive output were not observed. There was no statistically significant difference at any of the tested concentration levels of 0.500 to 4.00 mg test item/L in comparison to the reproductive output in the control (One Way Analysis of Variance, p = 0.05). At the concentration level of 8.00 mg test item/L, all daphnids died before producing juveniles.
EC-values were not calculated. The lack of significant effects combined with the lack of an effect level greater than 50 % makes the calculation of an EC₁₀- or an EC₅₀-value uncertain.

 

·  The coefficient of variation of the number of living offspring produced per surviving parental daphnid was 7 % in the control. At the concentration levels of 0.500 to 4.00 mg test item/L, the coefficient of variation was in the range of 9 to 17 % . Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %.

 

·  The intrinsic rates of natural increase (IR) of the surviving parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance.

 

·  The first appearance of living juveniles was observed between days 7 and 10 in all replicates of the control and the concentration levels of 0.500 to 4.00 mg test item/L. At the concentration level of 8.00 mg test item/L all parental daphnids died before appearance of the first brood.At the tested concentration levels of 0.500 to 4.00 mg test item/L and the control, 5 broods with living juveniles were produced by the surviving parental daphnids. However, one parental daphnid at the concentration level of 0.500 mg test item/L produced 4 broods with living juveniles.

 

·  No stillborn juveniles or aborted eggs were observed in the control during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was ≤ 3 % at the concentration levels of 0.500 to 4.00 mg test item/L.

 

·  The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 0.500 to 4.00 mg test item/L were determined to be in the range of 4.88 to 5.09 mm per daphnid and 5.15 mm per daphnid in the control group.

    The mean dry body weight of the surviving parental daphnids was determined to be in the range of 0.68 to 0.85 mg per daphnid in the concentration levels of 0.500 to 4.00 mg test item/L and 0.74 mg per daphnid in the control .

 

·      No males or ephippia (winter eggs) were observed in the control or in the test groups.

 

·  Water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and temperature) were within the acceptable limits.

·         In order to prove the validity of the test system and test conditions at the test facility, an acute immobilisation test according to AQS P 9/2 (02/2000) and OECD 202 is monthly carried out with potassium dichromate as reference item.
The EC₅₀of the reference item was1.53 mg/L (95% confidence limits: 1.35 - 1.72 mg/L) after 24 hours and therefore within the prescribed concentration range of 0.6 - 2.4 mg/L of quality criteria according to AQS P 9/2 (02/2000) for daphnids clone 5 cultured in Elendt M4 medium. The EC₅₀-value of the reference item is also within the recommended range of 0.6 - 2.1 mg/L according to OECD-Guideline 202.

 

A summary of all test item related effects and the assessed effect levels based on the nominal concentrations of the test item Glucamide CC and the active components (sum of glucamides) is given in the table below.

  

         

Test Item Related Effects, NOEC, LOEC, EC₁₀and EC₅₀

(based on the nominal concentrations of the test item Glucamide CC and the active components (sum of glucamides))

Effects	Nominal concentrations of the test item Glucamide CC (concentrations given assum of glucamides, content 81.0 % (w/w), are presented in italic font)) [mg/L]
	Control	0.500 (0.405)	1.00 (0.810)	2.00 (1.62)	4.00 (3.24)	8.00 (6.48)
Mean Number of Living Juveniles per Producing Parental Daphnid (Reproduction Rate±SD)	110±8	105±10	105±10	111±13	101±17	---
Coefficient of Variation of theMean Number of Juveniles per Producing Parent [%]	7	10	9	12	17	---
Mean Intrinsic Rates of Natural Increase	0.50	0.48	0.49	0.50	0.45	---
First Appearance of Living Juveniles [Mean Day]	7.9	8.2	7.7	7.8	8.6	---
Mean Number of Broods with Living Juveniles	5.0	4.9	5.0	5.0	5.0	---
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]	0	< 1	3	3	1	---
Parental Daphnids: Mean Dry Weight [mg]	0.74	0.78	0.85	0.68	0.68	---
Parental Daphnids: Mean Body Length [mm]	5.15	4.88	5.03	5.05	5.09	---
Adult Mortality after 21 Days [%]	0	0	0	0	20	100^1)
EC10 Adult mortality(with 95 %confidence limits)	3.82 mg test item/L (2.00 – 8.00 mg test item/L) 3.10 mg Glucamides/L (1.62 – 6.48 mg Glucamides/L)
EC50 Adult mortality(with 95 %confidence limits)	4.32 mg test item/L (2.00 – 8.00 mg test item/L) 3.50 mg Glucamides/L (1.62 – 6.48 mg Glucamides/L)
EC100 Adult mortality	8.00 mg test item/L 6.48 mg Glucamides/L
NOECReproduction / Adult mortality	4.00 mg test item/L 3.24 mg Glucamides/L
LOECReproduction / Adult mortality	8.00 mg test item/L 6.48 mg Glucamides/L

SD       = standard deviation

¹⁾          = statistically significant adult mortality observed (Fisher’sExact Test)

---        = not applicable, due to 100 % adult mortality