D-Glucitol, 1-deoxy-1-(methylamino)-, N-C8-10 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:

2013-11-06 to 2013-11-29

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

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-[C8-10(even numbered) acyl] derivs.

EC Number:

940-284-1

Cas Number:

1591782-62-5

Molecular formula:

C15H31NO6 C17H35NO6

IUPAC Name:

D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-10(even numbered) acyl] derivs.

Constituent 2

Reference substance name:

Glucamide 810

IUPAC Name:

Glucamide 810

Details on test material:

- Physical state: Colourless powder
- Analytical purity: 93.9 % (w/w)
- Composition (certified):
C6-Glucamide / 0.2 % (w/w)
C8-Glucamide / 51.6 % (w/w)
C10-Glucamide / 41.4 % (w/w)
C12-Glucamide / 0.7 % (w/w)
- Sum formula: C15H31NO6 (C8-Glucamide) C17H35NO6 (C10-Glucamide)
- Molecular weight:
C8-Glucamide: 321.42 g/Mol
C10-Glucamide 349.47 g/Mol
- Purity test date: 2013-09-03
- Lot/batch No.:AC 93/13
- Expiration date of the lot/batch: 2015-07-25
- Storage condition of test material: Room temperature, protected from light, in original container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:25.0 – 50.0 – 100 – 200 – 400 mg/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, 9, 14, 19 (0 h) and at the end of the exposure intervals on days 2, 12, 16, 21 (48 or 72 h).
For the analyses at the end of the exposure intervals, samples were taken from additional replicates with test media, but without daphnids and algal food. The additional replicates were incubated under test conditions until sampling.
- Sample storage conditions before analysis: All original samples were stored at room temperature until sample preparation. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (400 mg/L of the test item were weighed out) was freshly prepared with dilution water for each exposure interval. Dispersion treatment was agitation.
- Eluate: Dilution water
- Differential loading: 25.0 – 50.0 – 100 – 200 – 400 mg/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: renewals 3 times per week


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 9 Day 12 Day 14 Day 16
2013-11-06 2013-11-08 2013-11-15 2013-11-18 2013-11-20 2013-11-22
400 164 203 Not determined, due to 100 % adult mortality
200 Not determined 219 151 174 182
Control 160 158 180 196 169 141

Test temperature:

Temperature [°C]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-11-06 2013-11-08 2013-11-15 2013-11-18 2013-11-20 2013-11-22
400 20.4 22.0 Not determined, due to 100 % adult mortality
200 Not determined 20.5 21.2 21.5 21.1
Control 21.4 22.0 20.5 21.0 21.2 21.2

pH:

pH-Values
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-11-06 2013-11-08 2013-11-15 2013-11-18 2013-11-20 2013-11-22
400 7.80 7.43 Not determined, due to 100 % adult mortality
200 Not determined 7.48 7.93 7.96 7.38
Control 7.58 7.33 7.84 7.69 7.54 7.44

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item
concentration
[mg/L] I F I F I F
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
2013-11-06 2013-11-08 2013-11-15 2013-11-18 2013-11-20 2013-11-22
400 8.57 6.57 Not determined, due to 100 % adult mortality
200 Not determined 8.64 9.97 8.25 6.98
Control 8.44 7.01 8.80 7.84 8.41 7.85

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 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 160 to 180 mg CaCO3/L.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark per day
- Light intensity: Not exceeding 15 - 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
A non GLP preliminary range finding test, conducted under semi-static conditions with a renewal of the test solutions three times per week (i.e. after 48 and 72 h) over a period of 14 days was conducted at the test facility. Test conditions of the range finding test were equivalent to the test conditions of the definitive test.

Number of Juveniles in the Control and the Concentration Level of 100 mg/L after 14 Days in the non GLP Preliminary Range Finding Test

Nominal No. of Mean no. of
test item Number of living juveniles in replicate no. Total parents juveniles per Comparison
concentration no. prod. parent CV versus
[mg/L] juveniles producing control juveniles Inc. Stat. 1 2 3 4 5 6 7 8 9 10  N MV ± SD [%] [%]
100 19 66 -- 83 70 70 63 71 82 77 601 9 67 ± 19 29* 16 yes
Control 58 65 54 53 53 70 50 64 55 54 576 10 58 ± 7 11 — —




N = number of parental daphnids producing living juveniles
MV ± SD = mean value ± standard deviation
CV = coefficient of variation
Inc. = increase of the reproductive output
Stat. = statistical significance (MANN-WHITNEY Rank Sum Test, p = 0.05)
yes = statistically significant
* = biologically significant variation between the test replicates (coefficient of variation > 25 %)
-- = not applicable, due to the mortality of the parental daphnid

At the nominal test item concentration of 100 mg/L, the number of living juveniles was significant increased in comparison to the control (MANN-WHITNEY Rank Sum Test, p = 0.05).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
The test item induced biologically significant adult mortality of 40 % at the concentration level of 200 mg/L and 100 % at the concentration level of 400 mg/L during the test period of 21 days. In the concentration levels of 25.0 to 100 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days. The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 190 mg/L (95 % confidence limits: Not applicable). The EC50 for the adult mortality was calculated accordingly to be 202 mg/L (95 % confidence limits: 100 – 400 mg/L). The EC100 for the adult mortality was 400 mg/L.

- No. of offspring produced per day per female:
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 89 in the control group. The reproductive output was statistically significant increased at the concentration level of 100 mg/L and statistically significant reduced at the concentration level of 200 mg/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 400 mg/L, all daphnids died before producing juveniles. At all other concentration levels, the reproductive output was comparable to the reproductive output at the control.

The coefficient of variation of the number of living offspring produced per parental daphnid was 11 % in the control. At the concentration levels of 25.0 to 100 mg/L, the coefficient of variation was in the range of 10 to 15 %. The coefficient of variation of 46 % at the concentration level of 200 mg/L is regarded to be biologically significant. Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %.

Based on the significant increase and reduction of the reproductive output and the significant adult mortality at the nominal concentration of 100, 200 and 400 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 50.0 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 100 mg/L.

The EC10 for the reduction of the reproductive output was assessed to be in the range of 100 – 200 mg/L. The EC50 was assessed to be in the range of 200 – 400 mg/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 25.0 to 200 mg/L were determined to be in the range of 5.23 to 5.40 mm per daphnid and 5.13 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 0.57 to 0.81 mg per daphnid in the concentration levels of 25.0 to 200 mg/L and 0.63 mg per daphnid in the control.


- Type and number of morphological / behavioural abnormalities: please refer to the respective table
- Time to first brood release or time to hatch:
The first brood was released between days 8 and 11 by all parental daphnids of the control and the concentration levels of 25.0 to 100 mg/L. At the concentration level of 200 mg/L, the first brood was released between days 11 and 16, which is delayed in comparison to the control group.
At the tested concentration levels of 25.0 to 50.0 mg/L and in the control, 4 broods were produced by the surviving parental daphnids. At the concentration level of 100 mg/L, one of ten surviving parental daphnids produced five broods and nine of ten surviving parental daphnids produced four broods. At the concentration level of 200 mg/L, one of six surviving parental daphnids produced only three broods and five of six surviving parental daphnids produced four broods. At the concentration level of 400 mg/L, all daphnids died before producing 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) = 2.1 (CI 1.8 - 2.4) mg/L

Reported statistics and error estimates:

The NOEC and the LOEC based on the statistical significance of the reproductive output (increase and reduction) 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 and analysis of variance. 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 stillborn juveniles and aborted eggs, the P-value was < 0.05 and therefore the normality and the equal variance test failed. The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05. For the determination of significant deviations of the reproductive output, One Way Analysis of Variance, DUNNETT’S method (p = 0.05) was used. Significant deviations of the number of stillborn juveniles and aborted eggs were determined by KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNN’S method (p = 0.05). 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 reduction of the reproductive output and the EC100-value for the adult mortality after 21 days was deduced directly from the observation data. The EC10- and the EC50-value for the adult mortality was calculated by sigmoidal dose-response regression with the software GraphPad prism5. The lowest concentration level causing 100 % adult mortality and the highest concentration level causing no adult mortality were used as confidence limits for the EC50-value, because only one partial effect was observed.

Any other information on results incl. tables

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
																Inc.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
400	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
200	--	--	75	32	--	71	15	65	--	71	329	6	55 ±	25	46*	38	yes
100	122	115	107	135	106	132	90	96	109	90	1102	10	110 ±	16	15	-24	yes
50.0	98	109	97	104	95	114	93	94	120	116	1040	10	104 ±	10	10	-17	no
25.0	102	103	81	80	85	80	94	90	91	81	887	10	89 ±	9	10	0	no
Control	87	103	81	70	98	101	82	87	88	90	887	10	89 ±	10	11	—	—

Relative Number of Dead (Stillborn + Aborted Eggs) to Total Number of Juveniles

Nominal test item concentration [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
400	--	--	--	--
200	239	329	568	42
100	17	1102	1119	2
50.0	15	1040	1055	1
25.0	0	887	887	0
Control	0	887	887	0

First Appearance of Living Juveniles 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
400	--	--	--	--	--	--	--	--	--	--	--	--
200	--	--	11	11	--	12*	16*	11	--	11	12.0	3.8
100	10	9	9	10	10	9	9	9	10	9	9.4	4.1
50.0	9	10	8	10	8	11	8	9	11	11	9.5	4.0
25.0	10	10	9	8	8	9	8	8	9	8	8.7	4.0
Control	10	10	9	9	11	10	9	11	11	10	10.0	4.0

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

*    = noticeable delay of the occurrence of the first brood

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
400	100^1)	100^1)	100^1)
200	30^1)	30^1)	40^1)
100	0	0	0
50.0	0	0	0
25.0	0	0	0
Control	0	0	0

Total Body Length 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
400	--	--	--	--	--	--	--	--	--	--	--	0	--	--
200	--	--	5.50	5.00	--	5.75	5.25	5.25	--	5.25	5.33	6	3.4	0.57
100	5.50	5.25	5.25	5.50	5.75	5.25	5.50	5.25	5.25	5.50	5.40	10	7.3	0.73
50.0	5.75	5.50	5.50	5.75	5.00	5.25	5.00	5.25	5.00	5.25	5.33	10	8.1	0.81
25.0	4.75	5.50	5.00	5.25	5.25	5.00	5.25	5.50	5.50	5.25	5.23	10	7.9	0.79
Control	5.00	5.00	5.25	5.25	5.25	5.50	5.25	5.00	5.00	4.75	5.13	10	6.3	0.63

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the significant increase and reduction of the reproductive output at the nominal concentrations of 100, 200 and 400 mg/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 50.0 mg/L and the Lowest Observed Effect Concentration (LOEC) was assessed at 100 mg/L.
The EC10 for the reduction of the reproductive output was assessed to be in the range of 100 – 200 mg/L. The EC50 was assessed to be in the range of 200 – 400 mg/L.
The test item induced biologically significant adult mortality of 40 % at the concentration level of 200 mg/L and 100 % at the concentration level of 400 mg/L during the test period of 21 days. In the concentration levels of 25.0 to 100 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days.
The EC10 for the adult mortality was calculated by sigmoidal dose-response regression to be 190 mg/L (95 % confidence limits: Not applicable). The EC50 for the adult mortality was calculated accordingly to be 202 mg/L (95 % confidence limits: 100 – 400 mg/L). The EC100 for the adult mortality was 400 mg/L.


Endpoints assessed C8/C10-Glucamide AC93/13
Nominal test
item concentration
[mg/L]
EC10 Adult mortality (with 95 % confidence limits) 190 mg/L (Not applicable)*
EC50 Adult mortality (with 95 % confidence limits) 202 mg/L (100 – 400 mg/L)**
EC100 Adult mortality (with 95 % confidence limits) 400 mg/L
EC10 Reproduction 100 – 200 mg/L
EC50 Reproduction 200 – 400 mg/L
NOEC 50.0 mg/L
LOEC 100 mg/L

Executive summary:

The Daphnia magna Reproduction Test (semi-static, 21 d) of the test item C8/C10-Glucamide AC93/13 (batch number: AC93/13) was conducted according to OECD 211 (2012) from 2013-11-06 to 2013-11-29, with the definitive exposure phase from 2013-11-06 to 2013-11-27, at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

Test species wasDaphnia 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 undersemi-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 test item C8/C10-Glucamide AC93/13 were selected as follows: 25.0–50.0–100–200–400 mg/L.

The test item C8/C10-Glucamide AC93/13 was analytically verified via LC-MS/MS at the start of the exposure intervals on days 0, 9, 14 (0 h) and at the end of the exposure intervals on days 2, 12, 16 (48 or 72 h) in all concentration levels and the control.

The measured concentrations of the C8 fraction of the test item at the start of the exposure intervals (0 h) were in the range of 92 to 111 % of the nominal values. The measured concentration of the C10 fraction of the test item at the start of the exposure intervals (0 h) were in the range of 84 to 108 % of the nominal values.

The measured concentrations of theC8 fraction of the test item at the end of the exposure intervals (48 or 72 h) were in the range of 85 to 114 % of the nominal values. The measured concentration of the C10 fraction of the test item at the end of the exposure intervals (48 or 72 h) were in the range of 84 to 105 % of the nominal values.

The measured concentrations of the C8 and the C10 fraction of the test item C8/C10-Glucamide AC93/13 were within ± 20 % of the nominal values, indicating that the concentrations of the test item C8/C10-Glucamide AC93/13 were successfully maintained for the duration of the test. Therefore, the nominal concentrations of the test item C8/C10-Glucamide AC93/13 were used for assessment of the effect levels.

 

 

Based on the significant increase and reduction of the reproductive output

at the nominal concentration of 100, 200 and 400 mg/L,

the No Observed Effect Concentration (NOEC) after 21 days was assessed at 50.0 mg/L

and the Lowest Observed Effect Concentration (LOEC) was assessed at 100 mg/L.

 

 

 

·  The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 89 in the control group. The reproductive output was statistically significant increased at the concentration level of 100 mg/L and statistically significant reduced at the concentration level of 200 mg/L in comparison to the reproductive output in the control (One Way Analysis of Variance,Dunnett’smethod, p = 0.05).
At the concentration level of 400 mg/L, all daphnids died before producing juveniles. At all other concentration levels, the reproductive output was comparable to the reproductive output at the control.

    The EC₁₀for the reduction of the reproductive output was assessed to be in the range of 100 – 200 mg/L. The EC₅₀was assessed to be in the range of 200 – 400 mg/L.

 

·  The coefficient of variation of thenumber of living offspring produced per parental daphnid was 11 % in the control. At the concentration levels of 25.0 to 100 mg/L, the coefficient of variation was in the range of 10 to 15 %. The coefficient of variation of 46 % at the concentration level of 200 mg/L is regarded to be biologically significant .Generally in a well-run test, the coefficient of variation around the mean number of living offspring per parent animal should be ≤ 25 %.

 

·  The test item induced biologically significant adult mortality of 40 % at the concentration level of 200 mg/L and 100 % at the concentration level of 400 mg/L during the test period of 21 days.In the concentration levels of 25.0 to 100 mg/L and in the control, all parental daphnids survived until the end of the test after 21 days.
The EC₁₀for the adult mortality was calculated by sigmoidal dose-response regression to be 190 mg/L (95 % confidence limits: Not applicable). The EC₅₀for the adult mortality was calculated accordingly to be 202 mg/L (95 % confidence limits: 100 – 400 mg/L). The EC₁₀₀for the adult mortality was 400 mg/L.

 

·  No stillborn juveniles or aborted eggs were observed in the control and in the concentration level of 25.0 mg/L during the exposure period of 21 days. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was 1 % in the concentration level of 50.0 mg/L and 2 % in the concentration level of 100 mg/L. At the concentration level of 200 mg/L, the percentage of dead juveniles was 42 % in relation to the total number of produced juveniles (dead + alive), which is statistically significant in comparison to the control (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’smethod, p = 0.05). At the concentration level of 400 mg/L, all daphnids died before producing juveniles.

 

·  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 brood was released between days 8 and 11 by all parental daphnids of the control and the concentration levels of 25.0 to 100 mg/L. At the concentration level of 200 mg/L, the first brood was released between days 11 and 16, which is delayed in comparison to the control group.
At the tested concentration levels of 25.0 to 50.0 mg/L and in the control, 4 broods were produced by the surviving parental daphnids. At the concentration level of 100 mg/L, one of ten surviving parental daphnids produced five broods and nine of ten surviving parental daphnids produced four broods. At the concentration level of 200 mg/L, one of six surviving parental daphnids produced only three broods and five of six surviving parental daphnids produced four broods. At the concentration level of 400 mg/L, all daphnids died before producing juveniles.

 

·  The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 25.0 to 200 mg/L were determined to be in the range of 5.23 to 5.40 mm per daphnid and 5.13 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 0.57 to 0.81 mg per daphnid in the concentration levels of 25.0 to 200 mg/L and 0.63 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 was 2.1 mg/L (95% confidence limits: 1.8 - 2.4 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.  

  

         Test Item Related Effects (NOEC, LOEC, EC₁₀, EC₅₀and EC₁₀₀)

           (based on the nominal concentrations of the test itemC8/C10-Glucamide AC93/13)

Effects		C8/C10-Glucamide AC93/13 Nominal test item concentrations[mg/L])
		Control		25.0		50.0		100		200		400
Mean Number of Living Juveniles per Producing Parent (Reproduction Rate±SD)		89±10		89±9		104±10		110±16^1)		55±25^1)		---
Coefficient of Variation of theMean Number of Juveniles per Producing Parent [%]		11		10		10		15		46^2)		---
Mean Intrinsic Rates of Natural Increase		0.41		0.44		0.43		0.43		0.33		---
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]		0		0		1		2		42^3)		---
Mean Number of Broods		4.0		4.0		4.0		4.1		3.8		---
Appearance of First Brood [Mean Day]		10.0		8.7		9.5		9.4		12.0		---
Parental Daphnids: Mean Dry Weight [mg]		0.63		0.79		0.81		0.73		0.57		---
Parental Daphnids: Mean Body Length [mm]		5.13		5.23		5.33		5.40		5.33		---
Adult Mortality after 21 Days [%]		0		0		0		0		40^4)		100^4)
EC10 Adult mortality(with 95 %confidence limits)		190 mg/L (Not applicable)*
EC50 Adult mortality(with 95 %confidence limits)		202 mg/L (100 – 400 mg/L)**
EC100 Adult mortality(with 95 %confidence limits)		400 mg/L
EC10 Reproduction		100 – 200 mg/L
EC50 Reproduction		200 – 400 mg/L
NOEC		50.0 mg/L
LOEC		100 mg/L

SD       = standard deviation

¹⁾          = statistical significance (One Way Analysis of Variance,Dunnett’smethod, p = 0.05)

²⁾          = noticeable variation between the test replicates (coefficient of variation > 25 %)

³⁾          = statistical significance (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’smethod, p = 0.05)

⁴⁾          = significant adult mortality (> 20 %)

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

*          = no confidence limits available, becauseonly one partial effect was observed

**         =The lowest concentration level causing 100 % adult mortality and the highest concentration level causing no
  adult mortality were used as confidence limits for the EC₅₀-value, because only one partial effect was observed.