N-[3-(dimethylamino)propyl] C6-9 alkyl amides

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:

2019-01-09 to 2019-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Chemical name Amides, C5-9, N-[3-(dimethylamino)propyl]
Batch number 2697-100-2
CAS number 1044764-00-2
Purity (certified) 100% UVCB

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analyzed via LC-MS/MS 3 times during the test (e.g. once within a period of 7 days) in fresh media at the start of an exposure interval (0 hours) and in old media at the end of an exposure interval (48 or 72 hours). All samples were preferably analyzed on the day of collection keeping storage time as low as possible. The samples were analyzed with a LC-MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated.

Sampling for the analytical monitoring
At the start of the exposure intervals, samples of the fresh media were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals (48 or 72 hours), samples of the old media were taken directly from the test vessels.
For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

not specified

Details on test solutions:

Definitive Test
Based on the results of the non-GLP preliminary range finding test mentioned in Annex II, the definitive reproduction test was carried out with five concentrations of the test item C-6410 in the range of 0.500 to 50.0 mg/L as specified below. The definitive test was performed under semi-static conditions with a renewal of the test solutions three times per week. In the preliminary test, all prepared test solutions (up to 100 mg/L) were clear and Tyndall negative. Therefore, in the definitive test no further observations on the appearance of the stock solution and test solutions were made.

Preparation of the stock solution
A stock solution of 50.0 mg/L of the test item was freshly prepared with test medium for each exposure-renewal interval. An appropriate volume of the test item (relative density: 0.904 was taken into account) was placed by pipette into an appropriate amount of the test medium. The test item solution was stirred for 1 hour at 1100 rpm at room temperature. The stock solution was used as highest concentration level and for the preparation of further test concentrations by dilution with the test medium.

Test item concentrations
Five concentrations of the test item were prepared by dilution of the stock solution of 50.0 mg/L with the test medium and tested at follows:
0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L (separation factor √10), which corresponds to the geometric mean measured test item concentrations of:
0.409 - 1.37 - 2.69 - 11.5 - 50.4 mg/L

Control
10 replicates of dilution water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system The test species is Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004) is used. The composition of the culture medium is presented below.

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8

Culture feeding
The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

other: Same composition as the culture medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

see any other information on materials and methods incl. tables

Test temperature:

see any other information on materials and methods incl. tables

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Salinity:

not measured

Conductivity:

see any other information on materials and methods incl. tables

Details on test conditions:

Control
10 replicates of dilution water without test item

Test method
The study was performed with a semi-static test design with a medium renewal 3 times per week (i.e. on Monday, Wednesday and Friday).

Renewal of the test solutions
The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette.

Test duration
21 days

Test vessels
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses

Test volume
50 mL

Test medium
Same composition as the culture medium

Number of daphnids and replicates
10 daphnids in 10 replicates were used for all concentration levels and the control with one daphnid per replicate, which was held individually.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Renewal of the test solutions
The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Application
50 g test solution per replicate was weighed out into each test vessel. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Feeding
Daily feeding per test vessel; Pseudokirchneriella subcapitata (0.427 – 0.881 mL) and Desmodesmus subspicatus (0.326 – 0.339 mL) suspension were provided as food corresponding to 0.2 mg C per Daphnia and day. Variation was according to the density of the algae suspension, but was the same for all test groups on each feeding day.

Test temperature (target) The temperature should be in the range of 18 – 22 °C and should not vary by more than 2°C

Light intensity (target) Max. 1500 lx

Photoperiod 16/8 hours light/dark cycle

Aeration Test vessels were not aerated during the test

Reference substance (positive control):

yes

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

9.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

11.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

2.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

11.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

10.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

13.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

50.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

50.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

11.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Details on results:

- Behavioural abnormalities: Accidental and inadvertent mortality as defined in the OECD test guideline and abnormalities were not observed.
- Observations on body length and weight:see "any other information on results incl. tables"
- Other biological observations: No
- Mortality of control: One parent animal in the control group died. Actual mortality of the parent animal in the control group was 10%.
The mean number of living offspring produced per survived parent animal at the end of the test was 103 in the control group (required: at least 60 juveniles per parental daphnid).
- Other adverse effects control:No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:No
- Effect concentrations exceeding solubility of substance in test medium:No

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2019 01-23 to 2019-01-24. For results of the most recent of the monthly performed reference tests, see Table 12.

EC50-Value (with Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.06 mg/L
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
Confidence limits 1.00 - 4.00 mg/L

Reported statistics and error estimates:

Statistical evaluation
Significant deviations of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test) and a multiple test (Welsh-t-test after Bonferroni-Holm).
Prior to running the multiple test, a normality test and a variance homogeneity test were performed. P values of the normality and the variance homogeneity test were 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.01 for the normality test and the variance homogeneity test.
For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were calculated. Since no accidental and inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output. For the determination of significant deviations of the adult mortality, Step-down Cochran-Armitage test procedure was used.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.
All effect levels (NOEC, LOEC, EC10 / 50 and LC20 / 50 / 100) given are based on the geometric mean measured test item concentrations, since the measured test item concentrations were not within ± 20% of the nominal concentrations.

Determination of the EC-/LC-values
The EC10 / 50-values for the reproductive output and their 95% confidence limits were calculated by point estimates from the 3 parametric normal CDF with the software ToxRat Professional. The LC20 / 50-values for the adult mortality and their 95% confidence limits were calculated by probit analysis with ToxRat Professional. The LC100 was empirically derived from the observed data.

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. Results concerning the number of living juveniles of all concentration levels and the control for every test day are presented in Table 13 to Table 18.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 103 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels 11.5 and 50.4 mg/L (multiple sequentially-rejective Welsh-t-test procedure, α = 0.05). The determination of the EC10- and the EC50-value and their 95% confidence limits was carried out by point estimates from the 3 parametric normal CDF. The EC10 Reproduction for the test item C-6410 was 9.18 mg/L (confidence limits: not applicable). The EC50 Reproduction was 11.3 mg/L (confidence limits: not applicable). The LOEC Reproduction was 11.5 mg/L. The NOEC Reproduction was 2.69 mg/L.

Table3:      Effects on Reproduction for all Introduced and for Surviving Parents

(based on the geometric mean measuredconcentrations of the test item)

Geometric mean measured test item concentrations [mg/L]	Mean number of offspring per introduced parental daphnid			Mean number of offspring per survived parental daphnid
	Mean	SD	CV	Mean	SD	CV
50.4	0	Not applicable		0	Not applicable
11.5	47.2	25.5	54.0	58.1	19.5	33.6
2.69	113	11.8	10.5	113	11.8	10.5
1.37	104	10.3	9.9	104	10.3	9.9
0.409	99.5	7.59	7.6	99.5	7.59	7.6
Control	97.7	20.6	21.1	103	10.3	10.0

 

 Adult Mortality

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed.

The test item induced statistically significant adult mortality of 100% after 21 days in the concentration level 50.4 mg/L (Step down Cochran-Armitage Test Procedure, α = 0.05). In the concentration level 11.5 mg/L, a mortality rate of 30% of the introduced adults was observed, which is not statistically significant. In the concentration levels of 0.409 – 1.37 – 2.69 mg/L all parental daphnids survived until the end of the test (21 days). In the control a mortality rate of 10% of the introduced adults was observed. The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. Therefore, the LC20- and LC50-values were calculated by probit analysis.

The LC20 for the test item C-6410 was 10.6 mg/L (confidence limits: not applicable). The LC50 was 13.1 mg/L (confidence limits: not applicable). The LOEC was 50.4 mg/L. The NOEC was 11.5 mg/L.

 

Table 4:   Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

             (n = 10)

Geometric mean measured test item concentrations [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
50.4	100	100	100^1)
11.5	10	10	30
2.69	0	0	0
1.37	0	0	0
0.409	0	0	0
Control	0	10	10

¹⁾= statistically significant (Step‑down Cochran-Armitage Test Procedure,α = 0.05)

 Intrinsic Rates of Natural Increase (IR)

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

Table 5:   Intrinsic Rates of Natural Increase

Geometric mean measured test item concentrations [mg/L]	Intrinsic rate of natural increase in replicate no.										Mean IR		CV
	1	2	3	4	5	6	7	8	9	10	MV±SD		[%]
50.4	--	--	--	--	--	--	--	--	--	--	--
11.5	--	0.200	0.265	0.220	0.282	--	--	0.254	0.245	0.245	0.244±	0.0274	11.2
2.69	0.381	0.376	0.364	0.369	0.352	0.383	0.395	0.356	0.365	0.358	0.370±	0.0137	3.7
1.37	0.371	0.385	0.369	0.390	0.388	0.372	0.374	0.364	0.380	0.364	0.376±	0.0095	2.5
0.409	0.354	0.374	0.377	0.331	0.337	0.360	0.377	0.351	0.360	0.338	0.356±	0.0170	4.8
Control	0.344	--	0.341	0.372	0.363	0.369	0.376	0.359	0.352	0.369	0.361±	0.0124	3.4

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

First Appearance of living Juveniles

The first appearance of living juveniles was observed between days 8 and 15 at the introduced parental daphnids of the control and the concentration levels 0.500 to 15.8 mg/L. However, some daphnids died before producing juveniles. For details, see Table 6.

Table 6:  First Appearance of Living Juveniles in the Individual Groups

Geometric mean measured test item concentrations [mg/L]	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First
											appearance
	1	2	3	4	5	6	7	8	9	10	mean day
50.4	--	--	--	--	--	--	--	--	--	--	--
11.5	--	11	15	12	12	12	12	12	10	13	12.1
2.69	8	8	8	8	9	8	8	8	9	8	8.2
1.37	8	8	8	8	8	8	8	8	8	8	8.0
0.409	8	8	8	8	8	8	8	8	8	10	8.2
Control	8	8	8	8	8	8	8	8	8	8	8.0

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

 

Stillborn Juveniles and Aborted Eggs

No stillborn juveniles or aborted eggs were observed in the control and in the concentration levels 0.409 – 2.69 – 11.5 – 50.4 mg/L during the exposure period of 21 days (see Table 7 and Table 8). Only in the concentration level 1.37 mg/L on day 18 of the exposure one aborted egg was observed.

Table 7:  Stillborn Juvenilesand Aborted Eggs produced by the Survived Parental Daphnids after
                  21 Days

Geometric mean measured test item concentrations [mg/L]	Number of		Total no.   sum	Number of survived adults N
	stillborn juveniles	aborted eggs
50.4	0	0	0	0
11.5	0	0	0	7
2.69	0	0	0	10
1.37	0	1	1	10
0.409	0	0	0	10
Control	0	0	0	9

N   = number of the survived parental daphnids

Table8:      Relative Number of Dead (Stillborn + Aborted Eggs)to Total Number of Juvenilesper Survived Parental Daphnids

Geometric mean measured test item concentrations [mg/L]	Number of juveniles			Percentage of dead juveniles^#) [%]
	Dead	Alive	Total
50.4	0	0	0	0
11.5	0	472	472	0
2.69	0	1130	1130	0
1.37	1	1043	1044	0.1
0.409	0	995	995	0
Control	0	977	977	0

Dead   = aborted eggs + stillborn juveniles

Total  = dead + alive juveniles

^#)          = related to the total number of juveniles

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested concentration levels 0.409 to 11.5 mg/L were in the range of 3.64 to 4.50 mm per daphnid and 4.56 mm per daphnid in the control group.

The mean dry weight of the survived parental daphnids was in the range of 0.21 to 0.53 mg per daphnid in the concentration levels 0.409 to 11.5 mg/L and 0.44 mg per daphnid in the control.

Table 9:  Total Body Length and Dry Weight of the Survived Parental Daphnids

Geometric mean measured test item concentrations [mg/L]	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
50.4	--	--	--	--	--	--	--	--	--	--	--	--	--	--
11.5	--	3.50	4.00	4.00	3.50	--	--	4.00	3.50	3.00	3.64	7	1.5	0.21
2.69	4.50	4.50	4.50	4.50	4.00	4.50	4.50	4.50	4.50	4.50	4.45	10	5.3	0.53
1.37	4.50	4.50	4.50	4.50	4.50	4.50	4.50	4.50	4.50	4.50	4.50	10	5.2	0.52
0.409	4.00	4.50	4.50	4.50	4.50	4.50	4.50	4.50	4.00	4.00	4.35	10	4.7	0.47
Control	4.50	--	4.50	4.50	4.50	4.50	4.50	4.50	4.50	5.00	4.56	9	4.0	0.44

N  = number of the survived parental daphnids

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

 

Presence of Males

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

Occurrence of Ephippia (Winter Eggs)

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

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.
A summary of the assessed effect levels based on the geometric mean measured concentrations of the test item C-6410 is given in the table below.
The overall effect threshold for effects of the test item under the test conditions was 2.69 mg/L (NOEC) and 11.5 mg/L (LOEC) based on the geometric mean measured concentrations of the test item C-6410.

Endpoints for Reproduction and Mortality
Effect values C-6410
Geometric mean measured test item concentrations [mg/L]
EC10 Reproduction 9.18 (Cl: Not applicable)
EC50 Reproduction 11.3 (Cl: Not applicable)
LOEC Reproduction 11.5
NOEC Reproduction 2.69
LC20 Adult mortality after 21 days 10.6 (Cl: Not applicable)
LC50 Adult mortality after 21 days 13.1 (Cl: Not applicable)
LC100 Adult mortality after 21 days 50.4
LOEC Adult mortality after 21 days 50.4
NOEC Adult mortality after 21 days 11.5

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) with the test item C-6410 (batch number: 2697-100-2) was conducted according to OECD 211 (2012) from 2019-01-09 to 2019-01-30.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item C-6410 is a clear, amber liquid. Nominal concentrations of the test item C-6410 were selected based on the results of a preliminary range finding test (non-GLP) as follows: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L.

A stock solution with a nominal concentration of 50.0 mg/L was prepared with dilution water and was used as the highest concentration level and for the preparation of further four concentration levels in the range of 0.500 to 15.8 mg/L in a geometric series with a separation factor of √10.

The test item concentrations of C-6410 were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 or 72 hours, respectively) in all concentration levels and the control.

The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 75 to 125% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 5 to 104% of the nominal concentrations.

Since the measured test item concentrations were not within ± 20% of the nominal concentrations, the geometric mean measured concentrations were calculated and used for evaluation of the effect-concentrations. The geometric mean measured concentrations are: 0.409 - 1.37 - 2.69 - 11.5 - 50.4 mg/L.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. To be valid, the maximum control mortality in females must not exceed 20% and the mean offspring number at day 21 must be at least 60.

In the present test the mortality was 10% and the mean offspring number was 103.

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.

A summary of all endpoints based on the geometric mean measured concentrations of the test item C-6410 is given

 

Copy of Table 1:  Endpoints for Reproduction and Mortality (based on the geometric mean measuredtest item concentrations)

Effect values	C-6410 Geometric mean measured test item concentrations [mg/L]
EC10 Reproduction (with 95%confidence limits)	9.18 (Cl: Not applicable)
EC50 Reproduction (with 95%confidence limits	11.3 (Cl: Not applicable)
LOECReproduction	11.5
NOECReproduction	2.69
LC20 Adult mortality after 21 days (with 95%confidence limits)	10.6 (Cl: Not applicable)
LC50 Adult mortality after 21 days (with 95%confidence limits)	13.1 (Cl: Not applicable)
LC100 Adult mortality after 21 days	50.4
LOECAdult mortality after 21 days	50.4
NOECAdult mortality after 21 days	11.5

 

Description of key information

A Daphnia magna reproduction test (semi-static, 21 d) with C-6410 was conducted according to OECD 211 (2012) under GLP conditions.

Nominal concentrations of the test item C-6410 were selected based on the results of a preliminary range finding test (non-GLP) as follows: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L.

The test concentrations of C-6410 were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 or 72 hours, respectively) in all concentration levels and the control.

The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 75 to 125% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 5 to 104% of the nominal concentrations.

Since the measured test item concentrations were not within ± 20% of the nominal concentrations, the geometric mean measured concentrations were calculated and used for evaluation of the effect-concentrations. The geometric mean measured concentrations are: 0.409 - 1.37 - 2.69 - 11.5 - 50.4 mg/L.

The environmental conditions were within the acceptable limits. All validity criteria of the test guideline were met.

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.

A summary of all endpoints based on the geometric mean measured concentrations of the test item C-6410 is given

 

Copy of Table 1:  Endpoints for Reproduction and Mortality(based on the geometric mean measuredtest item concentrations)

Effect values	C-6410 Geometric mean measured test item concentrations [mg/L]
EC10 Reproduction (with 95%confidence limits)	9.18 (Cl: Not applicable)
EC50 Reproduction (with 95%confidence limits	11.3 (Cl: Not applicable)
LOECReproduction	11.5
NOECReproduction	2.69
LC20 Adult mortality after 21 days (with 95%confidence limits)	10.6 (Cl: Not applicable)
LC50 Adult mortality after 21 days (with 95%confidence limits)	13.1 (Cl: Not applicable)
LC100 Adult mortality after 21 days	50.4
LOECAdult mortality after 21 days	50.4
NOECAdult mortality after 21 days	11.5

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

9.18 mg/L

Additional information

A Daphnia magna reproduction test (semi-static, 21 d) with the test item C-6410 (batch number: 2697-100-2) was conducted according to OECD 211 (2012) from 2019-01-09 to 2019-01-30.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item C-6410 is a clear, amber liquid. Nominal concentrations of the test item C-6410 were selected based on the results of a preliminary range finding test (non-GLP) as follows: 0.500 - 1.58 - 5.00 - 15.8 - 50.0 mg/L.

A stock solution with a nominal concentration of 50.0 mg/L was prepared with dilution water and was used as the highest concentration level and for the preparation of further four concentration levels in the range of 0.500 to 15.8 mg/L in a geometric series with a separation factor of √10.

The test item concentrations of C-6410 were analytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on days 0, 9, 14 (0 hours) and in old media at the end of the exposure intervals on days 2, 12, 16 (48 or 72 hours, respectively) in all concentration levels and the control.

The measured concentrations of the test item in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 75 to 125% of the nominal concentrations. At the end of the respective exposure intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 5 to 104% of the nominal concentrations.

Since the measured test item concentrations were not within ± 20% of the nominal concentrations, the geometric mean measured concentrations were calculated and used for evaluation of the effect-concentrations. The geometric mean measured concentrations are: 0.409 - 1.37 - 2.69 - 11.5 - 50.4 mg/L.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. To be valid, the maximum control mortality in females must not exceed 20% and the mean offspring number at day 21 must be at least 60.

In the present test the mortality was 10% and the mean offspring number was 103.

The effects on reproduction were evaluated based on the reproduction per introduced parent animal. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor.

A summary of all endpoints based on the geometric mean measured concentrations of the test item C-6410 is given

 

Copy of Table 1:  Endpoints for Reproduction and Mortality(based on the geometric mean measuredtest item concentrations)

Effect values	C-6410 Geometric mean measured test item concentrations [mg/L]
EC10 Reproduction (with 95%confidence limits)	9.18 (Cl: Not applicable)
EC50 Reproduction (with 95%confidence limits	11.3 (Cl: Not applicable)
LOECReproduction	11.5
NOECReproduction	2.69
LC20 Adult mortality after 21 days (with 95%confidence limits)	10.6 (Cl: Not applicable)
LC50 Adult mortality after 21 days (with 95%confidence limits)	13.1 (Cl: Not applicable)
LC100 Adult mortality after 21 days	50.4
LOECAdult mortality after 21 days	50.4
NOECAdult mortality after 21 days	11.5