D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-01-29 to 2014-02-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 62.5 – 125 – 250 – 500 – 1000 µg test item/L (dilution factor 2).
Expressed as concentrations of Glucamides based on the nominal content of 80.4 % (w/w) the test concentrations were:
50.3 – 101 – 201 – 402 – 804 µg/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, 14 (0 h) and at the end of the exposure intervals on days 1, 8, 15 (24 h). At the start of the exposure intervals (0 h), sampling was carried out immediately after preparation of the test concentrations. At the end of the exposure intervals (24 h), samples were taken directly from the test vessels.
Sorption to the walls of the test vessel was checked as specified below. For this purpose, additional replicates without daphnids and food algae were prepared and incubated under test conditions for quantification of sorption to the glassware.

The sorption of the test item on glass was quantified at the concentration levels of 250 – 500 – 1000 µg test item/L (double determinations) at the end of the exposure intervals (24 h) at least once within 7 days. Two additional replicates per concentration level without daphnids were prepared. After sampling for the test medium analysis (aqueous phase), the glassware was emptied and an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration. Before this procedure, the test vessels were carefully rinsed with demineralised water to remove the remaining test media.


- Sample storage conditions before analysis: All original samples were stored at room temperature until preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (1.00 mg/L of the test item were weighed out) was freshly prepared with dilution water for each exposure interval.
- Eluate: Dilution water
- Differential loading: 62.5 – 125 – 250 – 500 – 1000 µg test item/L (dilution factor 2).
Expressed as concentrations of Glucamides based on the nominal content of 80.4 % (w/w) the test concentrations were:
50.3 – 101 – 201 – 402 – 804 µg/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 renewal of the test solutions


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
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2014-01-29 2014-01-30 2014-02-05 2014-02-06 2014-02-12 2014-02-13
1000 171 178 169 172 165 160
Control 177 175 172 172 166 163

Test temperature:

Temperature [°C]
Nominal
test item
concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2014-01-29 2014-01-30 2014-02-05 2014-02-06 2014-02-12 2014-02-13
1000 21.0 20.3 22.0 21.2 21.6 22.0
Control 19.0 20.5 20.5 21.0 21.1 22.0

pH:

pH-Values
Nominal
test item
concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2014-01-29 2014-01-30 2014-02-05 2014-02-06 2014-02-12 2014-02-13
1000 7.88 7.47 7.71 7.61 7.22 7.20
Control 7.84 7.50 7.58 7.69 7.54 7.16

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
test item
concentration
[µg/L] I F I F I F
Day 0 Day 1 Day 7 Day 8 Day 14 Day 15
2014-01-29 2014-01-30 2014-02-05 2014-02-06 2014-02-12 2014-02-13
1000 8.11 8.90 7.55 6.70 8.30 8.28
Control 8.98 8.52 8.27 8.08 8.65 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): daily
- 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
Based on the results of an acute immobilisation test performed at the test facility [Noack, M. (2014): C18/C18 unsatd.-Glucamide - Acute Immobilisation Test to Daphnia magna, Semi-static, 48 h (Unpublished Report: Study-No. DAI15740, Project-No. 131014CS), 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 62.5 to 1000 µg test item/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI (<0.06-0.44 mg/L)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: adult mortality (highest tested conc.)

Details on results:

- Mortality of parent animals:
No significant adult mortality (> 20 %) was observed at any of the tested concentration levels and the control during the test period of 21 days (FISHER’S Exact Test). Therefore, the No Observed Effect Concentration (NOEC) for the adult mortality after 21 days was assessed at 1000 µg test item/L, which corresponds to 804 µg Glucamides/L. In the concentration levels of 62.5 and 125 µg test item/L and in the control, all parental daphnids survived until the end of the test after 21 days. One of ten parental daphnids at the concentration levels of 250 and 500 µg test item/L and two of ten parental daphnids at the concentration level of 1000 µg test item/L died until the end of the test, which is regarded to be not biologically significant according to the quality criteria of the test guideline. An EC10 or EC50 for the adult mortality was not calculated, due to the lack of significant effects.

- No. of offspring produced per day per female:
No stillborn juveniles or aborted eggs were observed in the control and in the concentration level of 62.5 µg test item/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 levels of 125 to 250 µg test item/L and 5 % in the concentration level of 500 µg test item/L. At the concentration level of 1000 µg test item/L, the percentage of dead juveniles was 56 % in relation to the total number of produced juveniles (dead + alive), which is statistically significant in comparison to the control (KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNN’S Method, P = 0.05).

- 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 62.5 to 1000 µg test item/L were determined to be in the range of 4.53 to 5.05 mm per daphnid and 5.08 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 0.35 to 0.83 mg per daphnid in the concentration levels of 62.5 to 1000 µg test item/L and 0.65 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:
At the end of the test after 21 days, the number of broods with living juveniles produced by the surviving parental daphnids was 4 in the tested concentration levels of 62.5 to 500 mg/L and in the control. At the concentration level of 1000 µg test item/L, less than 4 broods with living juveniles or only dead juveniles and aborted eggs were produced by the surviving parental daphnids. The reduced number of broods with living juveniles at the concentration level of 1000 µg test item/L is statistically significant in comparison to the control (KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNN’S Method, P = 0.05).
The first appearance of living juveniles was observed until day 12 by all parental daphnids of the control and the concentration levels of 62.5 to 500 µg test item/L. At the concentration level of 1000 µg test item/L, the first appearance of living juveniles was observed between days 9 and 20.

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) = 1.74 (CI 1.64 -> 2.56) mg/L

Reported statistics and error estimates:

The NOEC and the LOEC based on the statistically significant reduction of the reproductive output and the NOEC for the 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 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 number of living juveniles per parental daphnid, the intrinsic rates of natural increase, the number of broods with living juveniles and the number of stillborn juveniles and aborted eggs, the P-value was < 0.05 and therefore the normality and/or 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 effects mentioned above KRUSKAL-WALLIS One Way Analysis of Variance on Ranks, DUNN’S Method (P = 0.05) was used. 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 reduction of the reproductive output was calculated by sigmoidal dose-response regression with the software GraphPad prism5. The confidence interval for EC-values were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The EC50-value for the reference item and its confidence limits was calculated accordingly. An EC10 or EC50 for the adult mortality was not calculated, due to the lack of significant effects.

Adsorption to the glass walls of the test vessels was determined exemplarily at the concentration levels of 250 – 500 – 1000 µg test item/L (double determinations) in separate replicates without daphnids at the end of the exposure intervals (24 h) on days 1, 8 and 15.
Two additional replicates per concentration level without daphnids were prepared. After sampling for the test medium analysis (aqueous phase), the glassware was emptied and rinsed carefully with demineralised water to remove the remaining test media. Thereafter, an appropriate amount of a suitable solvent was added. The concentration of the test item in this solution was measured and the adsorbed test item amount was calculated from this concentration.

Adsorption to the glass walls was in the range of 3 – 23 % of the nominal concentrationsofthe unsatd.C18 fraction of the test item C18/C18 unsatd.-Glucamide.

1^stExposure Interval (Day 1):
Measured Concentrations and Percent of the Nominal Concentrations ofthe Unsatd.C18 Fraction of the Test Item C18/C18 unsatd.-Glucamide in Aqueous Phase and at Glass Walls of Test Vessels without Daphnids after 24 hours

Unsatd.C18 fraction of the test itemC18/C18 unsatd.-Glucamide
Nominal concentration of the		Aqueous Phase (50 mL)				Adsorbed to Glass
		End of the 1^stexposure interval (24 h)				after exposure of 24 hours
		Replicate 1		Replicate 2		Replicate 1		Replicate 2
test item   [µg/L]	unsatd.C18 fraction of the test item [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
1000	743	582	78	562	76	70.7	10	49.7	7
500	372	305	82	268	72	29.7	8	17.8	5
250	186	93.6*	50	141*	76	28.3	15	9.88	5

*                    = reanalysed on 14-02-05 (2 replicates, 4 injections)

  

2^ndExposure Interval (Day 8):
Measured Concentrations and Percent of the Nominal Concentrations ofthe Unsatd. C18 Fraction of the Test Item C18/C18 unsatd.-Glucamide in Aqueous Phase and at Glass Walls of Test Vessels without Daphnids after 24 hours

Unsatd.C18 fraction of the test itemC18/C18 unsatd.-Glucamide
Nominal concentration of the		Aqueous Phase (50 mL)				Adsorbed to Glass
		End of the 2^ndexposure interval (24 h)				after exposure of 24 hours
		Replicate 1		Replicate 2		Replicate 1		Replicate 2
test item   [µg/L]	unsatd.C18 fraction of the test item [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
1000	743	704	95	708	95	37.3	5	24.7	3
500	372	270	73	276	74	28.6	8	29.6	8
250	186	122	66	91.4	49	13.4	7	19.4	10

Meas. conc. = measured concentrationof unsatd. C18 fractionof the test item

                        C18/C18 unsatd.-Glucamide, mean value of 2 injections, dilution factor taken into account

%                  = Percent of the nominal concentration of the unsatd. C18 fraction

LOQ_M    = Limit of quantification of theanalytical method(2.23 µg/L of the unsatd. C18 fraction)

3^rdExposure Interval (Day 15):
Measured Concentrations and Percent of the Nominal Concentrations of the Unsatd.C18 Fraction of the Test Item C18/C18 unsatd.-Glucamide in Aqueous Phase and at Glass Walls of Test Vessels without Daphnids after 24 hours

Unsatd.C18 fraction of the test itemC18/C18 unsatd.-Glucamide
Nominal concentration of the		Aqueous Phase(50 mL)				Adsorbed to Glass
		End of the 2^ndexposure interval (24 h)				after exposure of 24 hours
		Replicate 1		Replicate 2		Replicate 1		Replicate 2
test item   [µg/L]	unsatd.C18 fraction of the test item [µg/L]	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%	Meas. conc. [µg/L]	%
1000	743	599	81	458	62	133	18	90.6	12
500	372	201	54	272	73	44.3	12	56.9	15
250	186	64.3	35	80.2	43	27.5	15	43.3	23

Meas. conc. = measured concentrationof unsatd. C18 fractionof the test item

                        C18/C18 unsatd.-Glucamide, mean value of 2 injections, dilution factor taken into account

%                  = Percent of the nominal concentration of the unsatd. C18 fraction

LOQ_M          = Limit of quantification of theanalytical method(2.23 µg/L of the unsatd. C18 fraction)

Total Body Length of the Parental Daphnids

Nominal test item concentration [µg/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
1000	4.75	4.25	--	4.25	--	4.75	4.25	4.50	4.75	4.75	4.53	8	2.8	0.35
500	4.75	--	5.00	5.00	5.00	4.75	5.00	5.00	5.00	5.25	4.97	9	5.9	0.66
250	5.00	5.00	5.00	4.75	5.00	5.00	5.00	5.00	5.00	--	4.97	9	6.3	0.70
125	4.75	5.00	5.00	5.00	4.75	5.00	5.00	5.00	5.00	5.00	4.95	10	8.3	0.83
62.5	5.00	5.00	5.25	5.00	5.00	5.25	5.00	4.75	5.00	5.25	5.05	10	7.8	0.78
Control	5.25	5.00	5.00	5.00	5.00	5.00	5.00	5.25	5.00	5.25	5.08	10	6.5	0.65

Validity criteria fulfilled:

yes

Conclusions:

The reduction of the reproductive output was the most sensitive effect in this study. Based on the significant reduction of the reproductive output at the concentration levels of 500 and 1000 µg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 250 µg test item/L, which correspondes to 201 µg Glucamides/L, and the Lowest Observed Effect Concentration (LOEC) was assessed at 500 µg test item/L, which corresponds to 402 µg Glucamides/L.
The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 392 µg test item/L (95% confidence limits: < 62.5 – 438 µg test item/L), which corresponds to 315 µg Glucamides/L (95% confidence limits: < 50.3 – 352 µg Glucamides/L).
The EC50 was calculated accordingly to be 590 µg test item/L (95 % confidence limits: 568 – 613 mg test item/L), which corresponds to 474 µg Glucamides/L (95% confidence limits: 457 – 493 µg Glucamides/L).
No significant adult mortality (> 20 %) was observed at any of the tested concentration levels and the control during the test period of 21 days (FISHER’S Exact Test). Therefore, the No Observed Effect Concentration (NOEC) for the adult mortality after 21 days was assessed at 1000 µg test item/L, which corresponds to 804 µg Glucamides/L. An EC10 or EC50 for the adult mortality was not calculated, due to the lack of significant effects.



Effect values Nominal concentrations of the Nominal concentrations of the
test item C18/C18 unsatd.-Glucamide active components
[µg test item/L] (sum of glucamides 80.4% (w/w)) [µg/L]

NOEC Adult mortality 1000 804
EC10 Reproduction
(with 95 % confidence limits) 392 (< 62.5 – 438) 315 (< 50.3 – 352)
EC50 Reproduction
(with 95 % confidence limits) 590 (568 – 613) 474 (457 – 493)
NOEC Reproduction 250 201
LOEC Reproduction 500 402

Executive summary:

TheDaphnia magna Reproduction Test (semi-static, 21 d) of the test item C18/C18 unsatd.-Glucamide (batch number:SN119/13/3-8) was conducted according to OECD 211 (2012) from 2014-01-29 to 2014-02-20, with the definitive exposure phase from2014-01-29 to 2014-02-19,at Dr.U.Noack-Laboratorien,Käthe-Paulus-Str.1, 31157Sarstedt, 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 a daily renewal of the test solutions. 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 intrinsic rate of natural increase, number of broods with living juveniles, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality and body length of the parental daphnids.

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

Nominal concentrations of the test item C18/C18 unsatd.-Glucamide were selected based on the results of an acute immobilisation test performed at the test facility [Noack, M. (2014):C18/C18 unsatd.-Glucamide-Acute Immobilisation Test to Daphnia magna, Semi-static, 48 h (Unpublished Report:Study-No. DAI15740, Project-No. 131014CS),Dr. U. Noack-Laboratorien, Sarstedt, Germany]. The chosen nominal test item concentrations areas follows: 62.5–125–250–500–1000 µg test item/L.
Based on the nominal content of 80.4 % (w/w) Glucamides in the UVCB substance, the test concentrations were: 50.3 – 101 – 201 – 402 – 804 µg/L.

The concentrations of the main component unsatd.C18 fraction of the test itemC18/C18 unsatd.-Glucamidewereanalytically verified via LC-MS/MS at the start of the exposure intervals on days 0, 7, 14 (0 h) and at the end of the exposure intervals on days 1, 8, 15 (24 h) in all concentration levels and the control.

The measured concentrations of the unsatd. C18 fraction of the test item C18/C18 unsatd.-Glucamide at the start of the exposure intervals were in the range of 90 to 119 % of the nominal values. The measured concentration of the unsatd. C18 fraction of the test item C18/C18 unsatd.-Glucamide at the end of the exposure intervals (24 h) were in the range of 40 to 109 % of the nominal values.

Adsorption to the glass walls of the test vessels was determined exemplarily at the concentration levels of 250 – 500 – 1000 µg test item/L (double determinations) in separate replicates without daphnids at the end of the respective exposure intervals (24 h) on days 1, 8 and 15.
Adsorption to the glass walls was in the range of 3 – 23 % of the nominal concentrationsofthe unsatd. C18 fraction of the test ItemC18/C18 unsatd.-Glucamide.

The effect levels given are based on the nominal concentrations of the test item C18/C18 unsatd.-Glucamide and the active components (sum of glucamides).

 

 

·  The average number of living juveniles per surviving parental daphnid at the end of the test after 21 days was 94 in the control group. Th ereproductive output was statistically significant reduced at the concentration levels of 500 and 1000 µg test item/L in comparison to the reproductive output in the control (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’sMethod, P = 0.05). At all other concentration levels, the reproductive output was comparable to the reproductive output in the control.

    The reduction of the reproductive output was the most sensitive effect in this study. Based on the significant reduction of the reproductive output at the concentration levels of 500 and 1000 µg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 250 µg test item/L, which correspondes to 201 µg Glucamides/L, andtheLowest Observed Effect Concentration (LOEC) was assessed at 500 µg test item/L, which corresponds to 402 µg Glucamides/L.

    The EC₁₀for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 392 µg test item/L (95% confidence limits: < 62.5 – 438 µg test item/L), which corresponds to 315 µg Glucamides/L (95% confidence limits: < 50.3 – 352 µg Glucamides/L). The EC₅₀was calculated accordingly to be 590 µg test item/L (95 % confidence limits: 568 – 613 mg test item/L), which corresponds to 474 µg Glucamides/L (95% confidence limits: 457 – 493 µg Glucamides/L).

 

·  The coefficient of variation of thenumber of living offspring produced per parental daphnid was 5 % in the control. At the concentration levels of 62.5 to 500 µg test item/L, the coefficient of variation was in the range of 11 to 17 %. The coefficient of variation of 56 % at the concentration level of 1000 µg test item/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 %.

 

·  Theintrinsic 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 intrinsic rates of natural increase determined for the surviving daphnids at the concentration levels of 62.5 to 500 µg test item/L were comparable to the IR at the control (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’sMethod, P = 0.05). At the concentration level of 1000 µg test item/L, there was a statistical significance in comparison to the control.For details, seeTable1,Table4andFigure3.

 

·  At the end of the test after 21 days, thenumber of broods with living juvenilesproduced by the surviving parental daphnids was 4 in the tested concentration levels of 62.5 to 500 mg/L and in the control. At the concentration level of 1000 µg test item/L, less than 4 broods with living juveniles or only dead juveniles and aborted eggs were produced by the surviving parental daphnids. The reduced number of broods with living juveniles at the concentration level of 1000 µg test item/Lis statistically significant in comparison tothe control (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’sMethod, P = 0.05).
Thefirst appearance ofliving juvenileswas observed until day 12 by all parental daphnids of the control and the concentration levels of 62.5 to 500 µg test item/L. At the concentration level of 1000 µg test item/L, thefirst appearance ofliving juveniles was observed between days 9 and 20.For details, seeTable1andTable5.

 

·  Nostillborn juvenilesoraborted eggswere observed in the control and in the concentration level of 62.5 µg test item/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 levels of 125 to 250 µg test item/L and 5 % in the concentration level of 500 µg test item/L. At the concentration level of 1000 µg test item/L, the percentage of dead juveniles was 56 % 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). For details, seeTable6andTable7.

 

·  No significant adult mortality (> 20 %) was observed at any of the tested concentration levels and the control during the test period of 21 days (Fisher’sExact Test). Therefore, the No Observed Effect Concentration (NOEC) for the adult mortality after 21 days was assessed at 1000 µg test item/L,which corresponds to 804 µg Glucamides/L.
In the concentration levels of 62.5 and 125 µg test item/L and in the control, all parental daphnids survived until the end of the test after 21 days. One of ten parental daphnids at the concentration levels of 250 and 500 µg test item/L and two of ten parental daphnids at the concentration level of 1000 µg test item/L died until the end of the test, which is regarded to be not biologically significant according to the quality criteria of the test guideline. An EC₁₀or EC₅₀for the adult mortality was not calculated, due to the lack of significant effects.

 

·  The mean values of the body length of the surviving parental daphnids in the tested concentration levels of 62.5 to 1000 µg test item/L were determined to be in the range of 4.53 to 5.05 mm per daphnid and 5.08 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 0.35 to 0.83 mg per daphnid in the concentration levels of 62.5 to 1000 µg test item/L and 0.65 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 toAQS P 9/2 (02/2000) and OECD 202 is monthly carried out with potassium dichromate as reference item.
The EC₅₀of the reference item was1.74 mg/L (95% confidence limits: 1.64 - > 2.56 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 C18/C18 unsatd.-Glucamide 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 C18/C18 unsatd.-Glucamide and the active components (sum of glucamides))

Effects	Nominal concentrations of the test itemC18/C18 unsatd.-Glucamide (concenrations given assum of glucamides, content 80.4 % (w/w), are given in italic font)) [µg/L]
	Control	62.5 (50.3)	125 (101)	250 (201)	500 (402)	1000 (804)
Mean Number of Living Juveniles per Producing Parental Daphnid (Reproduction Rate±SD)	94±4	84±10	86±10	85±10	74±12^1)	11±6^1)
Coefficient of Variation of theMean Number of Juveniles per Producing Parent [%]	5	11	11	11	17	56^2)
Mean Intrinsic Rates of Natural Increase	0.39	0.38	0.39	0.39	0.38	0.24^1)
First Appearance of Living Juveniles [Mean Day]	11.0	10.8	10.7	10.7	10.4	12.4
Mean Number of Broods with Living Juveniles	4.0	4.0	4.0	4.0	4.0	2.4^1)
Percentage of Dead Juveniles Related to the Total Number of Juveniles [%]	0	0	< 1	< 1	5	56^1)
Parental Daphnids: Mean Dry Weight [mg]	0.65	0.78	0.83	0.70	0.66	0.35
Parental Daphnids: Mean Body Length [mm]	5.08	5.05	4.95	4.97	4.97	4.53
Adult Mortality^3)after 21 Days [%]	0	0	0	10	10	20
NOECAdult mortality	1000 µg test item/L 804 µg Glucamides/L
EC10 Reproduction(with 95 %confidence limits)	392 µg test item/L (< 62.5 – 438 µg test item/L) 315 µg Glucamides/L (< 50.3 – 352 µg Glucamides/L)
EC50 Reproduction(with 95 %confidence limits)	590 µg test item/L (568 – 613 µg test item/L) 474 µg Glucamides/L (457 – 493 µg Glucamides/L)
NOECReproduction	250 µg test item/L 201 µg Glucamides/L
LOECReproduction	500 µg test item/L 402 µg Glucamides/L

SD       = standard deviation

¹⁾          =statistical significance (Kruskal-WallisOne Way Analysis of Variance on Ranks,Dunn’sMethod, P = 0.05)

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

³⁾          = no statistically significant adult mortality observed (Fisher’sExact Test)

Description of key information

The reduction of the reproductive output was the most sensitive effect in this study. Based on the significant reduction of the reproductive output at the concentration levels of 500 and 1000 µg test item/L, the No Observed Effect Concentration (NOEC) after 21 days was assessed at 250 µg test item/L, which correspondes to 201 µg Glucamides/L, and the Lowest Observed Effect Concentration (LOEC) was assessed at 500 µg test item/L, which corresponds to 402 µg Glucamides/L.

The EC10 for the reduction of the reproductive output was calculated by sigmoidal dose-response regression to be 392 µg test item/L (95% confidence limits: < 62.5 – 438 µg test item/L), which corresponds to 315 µg Glucamides/L (95% confidence limits: < 50.3 – 352 µg Glucamides/L).

The EC50 was calculated accordingly to be 590 µg test item/L (95 % confidence limits: 568 – 613 mg test item/L), which corresponds to 474 µg Glucamides/L (95% confidence limits: 457 – 493 µg Glucamides/L).

No significant adult mortality (> 20 %) was observed at any of the tested concentration levels and the control during the test period of 21 days (FISHER’S Exact Test). Therefore, the No Observed Effect Concentration (NOEC) for the adult mortality after 21 days was assessed at 1000 µg test item/L, which corresponds to 804 µg Glucamides/L. An EC10 or EC50 for the adult mortality was not calculated, due to the lack of significant effects.

 

 

Effect values	Nominal concentrations of the test item C18/C18 unsatd.-Glucamide [µg test item/L]	Nominal concentrations of the active components (sum of glucamides 80.4% (w/w)) [µg/L]
NOEC Adult mortality	1000	804
EC10 Reproduction (with 95 % confidence limits)	392 (< 62.5 – 438)	315 (< 50.3 – 352)
EC50 Reproduction (with 95 % confidence limits)	590 (568 – 613)	474 (457 – 493)
NOEC Reproduction	250	201
LOEC Reproduction	500	402

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

392 µg/L