Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

From March 24, 2010 to April 15, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA study

Justification for type of information:

Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Two concentration levels 30.0 and 270 µg/L and the control were analysed once within 7 d in the fresh (0 h) and old media (48 h).
- Sampling method: For the longest exposure interval of 72 h samples were taken from the fresh (0 h) and old media (72 h) once within the test period, respectively. For the analyses of the old media additional replicates without algae and test organisms were prepared and stored under test conditions.
The sorption of the test substance to the glassware was quantified once during the test period after the first sampling interval. Therefore, one test vessel of the test concentration 30.0 µg/L, containing daphnia and algae, was emptied after 72 h of exposure and rinsed twice with water. Thereafter an extraction of the test vessel was carried out.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10.0 mg/L was freshly prepared with dilution water and treated with ultrasound for 5 min at room temperature.
- Eluate: Natural river water
- Differential loading: 10.0 - 30.0 - 90.0 - 270 - 810 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


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
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 16 Day 19
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr.09th Apr. 12th
810 129 118 n.a. n.a. n.a. n.a.
270 -- -- 111 131 120 104
Control 121 115 111 126 94 111

Test temperature:

Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 16 Day 19
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr.09th Apr. 12th
810 19.9 21.2 n.a. n.a. n.a. n.a.
270 -- -- 20.3 21.3 21.1 21.0
Control 19.0 21.2 19.2 21.2 19.8 20.8

pH:

pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 16 Day 19
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr.09th Apr. 12th
810 8.13 8.16 n.a. n.a. n.a. n.a.
270 -- -- 8.13 7.76 7.76 7.75
Control 8.16 8.05 7.95 7.61 7.67 7.71

Dissolved oxygen:

Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 2 Day 5 Day 7 Day 16 Day 19
Mar. 24th Mar. 26th Mar. 29th Mar. 31st Apr.09th Apr. 12th
810 9.18 9.30 n.a. n.a. n.a. n.a.
270 -- -- 8.94 7.88 9.22 8.50
Control 9.53 9.44 9.19 6.17 9.79 7.85

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information in materials and methods incl. tables

Details on test conditions:

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


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water of the river Leine was used. This river is located near D-31171 Nordstemmen, Germany. Specifications of the natural river water are given in the table below.
River: Leine
Location D-31171 Nordstemmen
Sampling Date 2009-12-15
Weather on Day of Sampling cloudy, ca. -1 °C
Colour Yellowish, clear
pH 7.97
Conductivity [µS/cm] 386
Dissolved Oxygen [mg O2] 8.62
DOC [mg C/L] 3.9
TOC [mg C/L] 3.9
Ammonium-N [mg N/L] 0.042
Nitrate-N [mg N/L] 2.62
Total Nitrogen [mg N/L] 3.53
o-Phosphate-p [mg P/L] 0.062
Total Phosphate [mg P/L] 0.053
Suspended Matter [mg/L] 16.2
Total Hardness [mg CO3/L] 154
Storage conditions < 10 °C *



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


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


RANGE-FINDING STUDY
- Test concentrations: 10 - 1 - 0.1 mg/L
Immobilization Rates [%] of the Preliminary Acute Immobilization Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicate Replicate
1 2 MV 1 2 MV
10 100 100 100 100 100 100
1 10 30 20 50 70 60
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

173 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Adults

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

163.4 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Adults

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

252 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Adults

Remarks on result:

other: (90 - 810)

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

238 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

(94.5%)

Basis for effect:

mortality

Remarks:

adults

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

270 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

255.5 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals:
The test item induced significant adult mortality at the concentration levels 270 µg/L (60 %) and 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no mortality of parent animals was observed. The EC50 for adult mortality after 21 days was calculated respectively to be 252 µg/L (95 % confidence limits: 90 - 810 µg/L).
- No. of offspring produced per day per female:
Five broods were released by all surviving animals of the test concentration levels and the control groups.
The coefficient of variation of the mean number of living offspring produced per parent alive at the control group was calculated to be 7 %. At the concentration levels 10.0 to 90.0 µg/L the calculated coefficients of variation were in a range of 7 to 8 % and comparable to the control. At the concentration level 270 µg/L the coefficient of variation was 15 %.
The average number of juveniles per parent in the control group was 135 after 21 days. The reproductive output was not statistically significant reduced or increased at the concentration levels 10 to 270 µg/L when compared to the control (One Way Analysis of Variance, p = 0.05). Due to mortality of all parental daphnids at the concentration level 810 µg/L no reproduction was observed.

- Body length and weight of parent animals: The mean dry body weight at the concentration levels 10.0 to 90.0 µg/L and the total body lengths at the concentration levels 10.0 to 270 µg/L of the surviving parental daphnids, determined at the end of the study, were comparable to the control group.
The mean dry body weight at the concentration levels 10.0 to 90.0 µg/L was determined to be in the range of 0.83 to 0.89 mg per daphnid and 0.91 mg per daphnid at the control group. At the concentration level 270 µg/L the dry body weight per daphnid was 0.38 mg.
The mean values of the total body length at the concentration levels 10.0 to 270 µg/L were in the range of 4.94 to 5.45 mm per daphnid and comparable to the mean value of 5.48 mm per daphnid at the control group.

- Type and number of morphological abnormalities: Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles (sum of stillborn juvenile and aborted eggs) was 1 %, which is not biologically significant.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.44 (CI 1.35 - 1.54)

Reported statistics and error estimates:

Significant deviations were evaluated in comparison to the control using statistical standard procedures as Normality Test, Equal Variance Test and Analysis of Variance.
Statistical evaluation of the reproduction rates was carried out using One Way Analysis of Variance (p = 0.05) and the statistical evaluation of the intrinsic rates of natural increase was carried out using KRUSKAL-WALLIS, One Way Analysis of Variance On Ranks (p = 0.05), because normality test failed. The coefficients of variation around the mean number of living offspring produced per parent in the control and the test groups were evaluated.
The EC50-value of the reference test and mortality was calculated by sigmoidal dose-response regression. The EC50-value was estimated using the geometric mean of the highest concentration causing no effect and the lowest concentration producing 100 % effect. The highest concentration causing no effect and the lowest concentration producing 100 % were used as confidence limits for mortality. The estimation of an EC10- or EC50-value for the reduction or increase of the reproductive output could not be performed due to the absence of effects on reproduction.

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

Nominal conc. [µg/L]												No.	Mean No. of Juveniles alive			Comparison
	Number of Juveniles alive in Replicate No.										Total	of			CV	versus
											No.	Parents	per Parent			Control
												prod. Juv.	producing Juveniles			Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
810	--	--	--	--	--	--	--	--	--	--	--	0	n.a.		n.a.	n.a.	n.a.
270	--	--	147	155	--	--	--	109	--	126	537	4	134 ±	21	15	0.74	no
90.0	128	146	132	130	153	123	143	130	149	142	1376	10	138 ±	10	7	-2.22	no
30.0	126	128	142	131	142	141	150	118	125	118	1321	10	132 ±	11	8	2.22	no
10.0	133	137	115	138	134	139	121	146	120	124	1307	10	131 ±	10	8	2.96	no
Control	129	137	139	135	146	126	135	143	117	142	1349	10	135 ±	9	7	—	—

Stillborn Juveniles and Aborted Eggs after 21 Days

Nominal concentration	Number of		Total No.		Mean Stillborn Juveniles and Aborted Eggs per Parent alive
	Stillborn	Aborted
[µg/L]	Juveniles	Eggs	å	N	MV ± SD
810	--	--	--	0	--
270	2	3	5	4	1.25 ± 1.26
90.0	2	15	17	10	1.70 ± 1.77
30.0	7	12	19	10	1.90 ± 3.11
10.0	8	6	14	10	1.40 ± 1.08
Control	0	0	0	10	0 ± 0

First Appearance of Living Juveniles at the Individual Groups

Nominal concentration	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
[µg/L]	1	2	3	4	5	6	7	8	9	10	Mean Day
810	--	--	--	--	--	--	--	--	--	--	--
270	--	--	8	8	--	--	--	8	--	8	8.0
90.0	9	8	8	8	8	8	8	8	8	9	8.2
30.0	8	8	8	8	8	8	8	8	8	8	8.0
10.0	8	8	8	8	8	8	8	8	9	8	8.1
Control	8	8	8	8	8	9	8	8	8	8	8.1

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

                 (n = 10)

Nominal concentration	Adult Mortality [%]
[µg/L]	7 days	14 days	21 days
810	100*	100*	100*
270	0	0	60*
90.0	0	0	0
30.0	0	0	0
10.0	0	0	0
Control	0	0	0

Total Body Length and Dry Weight of the Parent Animals

Nominal 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
810	--	--	--	--	--	--	--	--	--	--	--	0	--	--
270	--	--	5.00	5.00	--	--	--	5.00	--	4.75	4.94	4	1.5	0.38
90.0	5.25	5.75	5.75	5.25	5.25	5.25	5.00	5.50	5.50	5.25	5.38	10	8.3	0.83
30.0	5.75	5.50	5.75	5.25	5.25	5.00	5.75	5.50	5.50	5.25	5.45	10	8.7	0.87
10.0	5.50	5.50	5.50	5.25	5.25	5.50	5.75	5.25	5.00	5.50	5.40	10	8.9	0.89
Control	5.50	5.50	5.75	5.25	5.25	5.50	5.25	5.50	5.50	5.75	5.48	10	9.1	0.91

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 21d NOEC (reproduction), EC10 and EC50 (mortality) of the test substance to Daphnia magna was found to be 270 μg/L (i.e., equivalent 255.5 μg/L), 173 μg/L (i.e., equivalent 163.5 μg/L) and 252 µg/L (i.e., equivalent to 238 µg/L) respectively.

Executive summary:

A study was conducted to determine the long-term toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3 -propanediammonium chloride, to aquatic invertebrates, according to OECD Guideline 211 in compliance with GLP. Daphnia magna were exposed to five concentrations of the test substance at 10, 30, 90, 270 and 810 µg/L for 21 d under semi- static conditions. The test solutions were renewed 3 times per week. The test substance was analytically verified in the concentration levels 30.0 and 270 µg/L and in the control on Days 0, 7, 16 (fresh media) and on Days 2, 9 (old media, 48 h) and 19 (old media, 72 h). The recoveries in the fresh media were in the range of 84 to 116% of the nominal values. Similar recoveries were determined in the old media (after 48 h and 72 h). The recoveries of the old media were in the range of 86 to 119%, demonstrating the stability of the test substance under test conditions throughout exposure. The adsorbed fraction of the test substance was determined to be 10 % of the nominal concentration at the concentration level 30.0 µg/L. Therefore, all effect values given are based on the nominal test substance concentrations. The average number of juveniles per parent in the control group was 135 after 21 d. The reproductive output was not statistically significant reduced at 10 to 270 µg/L when compared to the control. Due to mortality of all parental daphnids at 810 µg/L no reproduction was observed. The coefficient of variation (CV) of the mean number of living offspring produced per parent at the control group was calculated to be 7% and at 10.0 to 90.0 µg/L it was in a range of 7 to 8% and comparable to the control. At 270 µg/L the CV was 16%. The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. There is no statistically significant difference determined when the IR of the surviving daphnids of the treatment groups 10 to 270 µg/L compared to the control and no IR was calculated at 810 µg/L due to mortality of all parental daphnids. The test substance induced significant adult mortality at 270 µg/L (60%) and 810 µg/L (100%) after 21 d. At the other concentration levels and in the control no mortality of parent animals was observed. The mean dry body weight at 10 to 90 µg/L (0.83 to 0.89 mg) and the total body length at 10 to 270 µg/L (4.94 to 5.45 mm) of the surviving parental daphnids, determined at the end of the study, were comparable to the control group. At 270 µg/L the mean dry body weight per daphnid was 0.38 mg. Water quality parameters such as pH, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits. Under the test conditions, the 21d NOEC and EC10 of the test substance for reproduction in Daphnia magna was determined to be 270 μg/L (equivalent 255.5 μg/L) and 173 μg/L (equivalent 163.5 μg/L) respectively (Akzo nobel, 2010).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

21 day

Effect concentration:

163.5 µg/L

Additional information

A study was conducted to determine the long-term toxicity of the read across substance, N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3 -propanediammonium chloride, to aquatic invertebrates, according to OECD Guideline 211 in compliance with GLP. Daphnia magna were exposed to five concentrations of the test substance at 10, 30, 90, 270 and 810 µg/L for 21 d under semi- static conditions. The test solutions were renewed 3 times per week. The test substance was analytically verified in the concentration levels 30.0 and 270 µg/L and in the control on Days 0, 7, 16 (fresh media) and on Days 2, 9 (old media, 48 h) and 19 (old media, 72 h). The recoveries in the fresh media were in the range of 84 to 116% of the nominal values. Similar recoveries were determined in the old media (after 48 h and 72 h). The recoveries of the old media were in the range of 86 to 119%, demonstrating the stability of the test substance under test conditions throughout exposure. The adsorbed fraction of the test substance was determined to be 10 % of the nominal concentration at the concentration level 30.0 µg/L. Therefore, all effect values given are based on the nominal test substance concentrations. The average number of juveniles per parent in the control group was 135 after 21 d. The reproductive output was not statistically significant reduced at 10 to 270 µg/L when compared to the control. Due to mortality of all parental daphnids at 810 µg/L no reproduction was observed. The coefficient of variation (CV) of the mean number of living offspring produced per parent at the control group was calculated to be 7% and at 10.0 to 90.0 µg/L it was in a range of 7 to 8% and comparable to the control. At 270 µg/L the CV was 16%. The intrinsic rates of natural increase (IR) of the surviving parent animals accounting for generation time and number of offspring were used for calculation of population growth and maintenance. There is no statistically significant difference determined when the IR of the surviving daphnids of the treatment groups 10 to 270 µg/L compared to the control and no IR was calculated at 810 µg/L due to mortality of all parental daphnids. The test substance induced significant adult mortality at 270 µg/L (60%) and 810 µg/L (100%) after 21 d. At the other concentration levels and in the control no mortality of parent animals was observed. The mean dry body weight at 10 to 90 µg/L (0.83 to 0.89 mg) and the total body length at 10 to 270 µg/L (4.94 to 5.45 mm) of the surviving parental daphnids, determined at the end of the study, were comparable to the control group. At 270 µg/L the mean dry body weight per daphnid was 0.38 mg. Water quality parameters such as pH, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits. Under the test conditions, the 21d NOEC and EC10 of the test substance for reproduction in Daphnia magna was determined to be 270 μg/L (equivalent 255.5 μg/L) and 173 μg/L (equivalent 163.5 μg/L) respectively (Akzo nobel, 2010).