Docosyltrimethylammonium methyl sulphate

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

2010-04-14 to 2010-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According OECD Guideline 211, GLP and analytical monitoring. Justification for read-across see chemical safety report chapter 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC Number:

271-756-9

EC Name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Cas Number:

68607-24-9

IUPAC Name:

68607-24-9

Constituent 2

Reference substance name:

C20/22 ATQ

IUPAC Name:

C20/22 ATQ

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels and the control were analysed at least once within 7 days in the fresh (0 h) and old media (48 h or 72 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 additionally prepared separate replicates without algae and test organisms were prepared and stored under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature before and after preparation.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 10 mg/L was freshly prepared with dilution water.
- Eluate: Natural river water
- Differential loading: 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 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: 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

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
Apr. 14th Apr. 16th Apr. 23rd Apr. 26th Apr. 28th Apr. 30th
2.00 94 105 n.a. n.a. n.a. n.a.
0.320 -- -- 103 102 129 130
Control 98 98 104 102 132 132

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
Apr. 14th Apr. 16th Apr. 23rd Apr. 26th Apr. 28th Apr. 30th
2.00 21.1 21.2 n.a. n.a. n.a. n.a.
0.320 -- -- 21.2 21.5 21.9 21.7
Control 19.6 20.6 20.0 21.4 19.2 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
Apr. 14th Apr. 16th Apr. 23rd Apr. 26th Apr. 28th Apr. 30th
2.00 7.94 8.00 n.a. n.a. n.a. n.a.
0.320 -- -- 7.85 7.66 7.60 7.79
Control 7.63 7.86 7.92 7.47 7.97 7.65

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
Apr. 14th Apr. 16th Apr. 23rd Apr. 26th Apr. 28th Apr. 30th
2.00 8.93 9.01 n.a. n.a. n.a. n.a.
0.320 -- -- 8.83 8.16 7.84 7.44
Control 9.63 8.82 9.39 7.17 9.26 6.45

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, Marienbergstraße, Germany (52° 10' 14.98'', 9° 46' 7.64'').
River: Leine
Location Marienbergstraße,
D-31171 Nordstemmen, Germany,
(52° 10' 14.98'', 9° 46' 7.64'')
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/L] 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
Immobilization Rates of the GLP Acute Immobilization Test with
C20/22 ATQ trocken
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal Concentration
of the Test Item
C20/22 ATQ trocken
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1. 2. 3. 4. MV 1. 2. 3. 4. MV
2.50 40 40 60 80 55 80 80 80 100 85
1.00 0 0 20 0 5 20 20 40 40 30
0.400 0 0 0 0 0 0 0 40 20 15
0.160 0 0 0 0 0 0 0 0 0 0
0.06402) 0 0 0 0 0 0 0 0 0 0
0.02562) 0 0 0 0 0 0 0 0 0 0
Control 0 0 0 0 0 0 0 0 0 0
NOEC (48h) 0.160 mg/L1)
LOEC (48h) 0.400 mg/L1)
EC50 (48 h)
with 95 % confidence limits (Cl) 1.32 mg/L (1.07 - 1.63 mg/L)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

128 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

320 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 320 µg/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 at the concentration levels 0.320 mg/L (90 %), 0.800 mg/L (100 %) and 2.00 mg/L (100 %). At the tested concentration levels 0.0512 and 0.128 mg/L and the control no adult mortality was observed within the exposure phase of 21 days. At the control all parental daphnids survived till the end of the test after 21 days. The EC50 for adult mortality after 21 days was calculated respectively to be 0.295 mg/L
(Cl: 0.128- 0.800 mg/L).

- No. of offspring produced per day per female:
The first brood was released till day 8 by all parent animals of the control and the concentration levels 0.0512 and 0.128 mg/L. Four to five broods were observed from the surviving daphnids at the concentration levels 0.0512 to 0.320 mg/L and control during the course of the study.
At the concentration level 0.320 mg/L the surviving parental daphnid released its first brood biologically significantly delayed on day 11. Release of the first brood after day 9 is regarded to be too late and biologically significant. The first brood was not statistically significant delayed at.
The average number of juveniles per parent alive at the end of the test in the control group was 124 after 21 days. There was a statistically significant reduction of the reproductive output determined at the tested concentration level 0.320 mg/L when compared to the control by One Way Analysis of Variance, HOLM-SIDAK method, p = 0.5
The NOEC was assessed to be 0.128 mg/L (the highest test concentration with no significant reduction of the reproductive output). The EC50- for the reduction of the reproductive output was estimated directly from the observation data to be > 0.320 mg/L, because effects > 50 % did not occur within the tested concentration range and the effects were not concentration related. An EC10- value for the reduction of the reproductive output could not estimated, because the effects were not concentration related. Due to mortality of all parental daphnids at the concentration levels 0.800 and 2.00 mg/L no reproduction was observed.
- Body length and weight of parent animals:
The mean values of the body length of the surviving parental daphnids at the test groups in the range of 5.00 to 5.45 mm per daphnid were comparable to the control group with 5.33 mm per daphnid.
The mean dry body weight was determined to be in the range of 0.30 to 0.80 mg per daphnid at the test groups and 0.92 mg per daphnid at the control, respectively.
- Type and number of morphological abnormalities:
No stillborn juveniles and aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles came to a maximum of < 1 % at the test concentrations of 0.0512 to 0.320 mg/L, which is not biologically significant.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.78 (CI 1.46 - 2.18)

Reported statistics and error estimates:

The NOEC and the LOEC for the reproductive output as the most sensitive effect were determined directly from the observation data. Significant deviations were determined in comparison to control using statistical standard procedures as Normality Test, Equal Variance Test and One Way Analysis of Variance.
Statistical evaluation of the reproduction rates was carried out using One Way Analysis of Variance, HOLM-SIDAK 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 EC50-value of the reference test and the adult mortality was calculated using sigmoidal dose-response regression. The confidence interval for the EC50-value was calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism.
The estimation of an EC50- or EC10-value for the reduction of the reproductive output was not applicable, because no effect > 50 % was observed within the tested concentration range and the effects were not concentration related.
The EC50 adult mortality was estimated (using sigmoidal dose-response regression) and the highest concentration causing no effect and the lowest concentration producing 100 % were used as confidence limits.

Any other information on results incl. tables

Number of 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 Juveniles in Replicate No.										Total No.				CV
																Red.	Stat.
	1	2	3	4	5	6	7	8	9	10	å	N	MV ± SD		[%]	[%]
2.00	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
0.800	--	--	--	--	--	--	--	--	--	--	--	0	--		--	--	--
0.320	--	--	--	94	--	--	--	--	--	--	94	1	94 ±	0	0	24.2	yes
0.128	133	124	139	108	106	145	123	140	113	121	1252	10	125 ±	14	11	-0.81	no
0.0512	126	123	100	123	108	126	122	131	113	122	1194	10	119 ±	9	8	4.03	no
Control	124	117	122	124	132	125	124	133	119	117	1237	10	124 ±	6	4	¿	¿

First Appearance of Living Juveniles in the Individual Groups

Nominal Test Item Concentration [mg/L]	Day of First Appearance of Living Juveniles										First
	in Replicate No.										Appearance
	1	2	3	4	5	6	7	8	9	10	Mean Day
2.00	--	--	--	--	--	--	--	--	--	--	--
0.800	--	--	--	--	--	--	--	--	--	--	--
0.320	--	--	--	11	--	--	--	--	--	--	11.0*
0.128	8	7	7	8	8	7	7	7	8	8	7.5
0.0512	8	7	7	8	8	8	8	8	7	8	7.7
Control	8	8	8	8	8	8	7	8	8	8	7.9

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
2.00	100^1)	100^1)	100^1)
0.800	100^1)	100^1)	100^1)
0.320	90^1)	90^1)	90^1)
0.128	0	0	20
0.0512	0	0	10
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
2.00	--	--	--	--	--	--	--	--	--	--	--	0	--	--
0.800	--	--	--	--	--	--	--	--	--	--	--	0	--	--
0.320	--	--	--	5.00	--	--	--	--	--	--	5.00	1	0.30	0.30
0.128	5.00	5.75	5.50	5.50	5.50	5.75	5.25	5.50	5.25	5.50	5.45	10	7.80	0.78
0.0512	5.50	5.25	5.25	5.00	5.25	5.00	5.25	5.50	5.50	5.50	5.25	10	8.00	0.80
Control	5.00	5.00	5.25	5.50	5.75	5.25	5.00	5.50	5.50	5.50	5.33	10	9.20	0.92

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The Lowest Observed Effect Concentration (LOECReproduction) and the No Observed Effect Concentration (NOECReproduction) after 21 days based on the reduction of the reproductive output as the most sensitive effects in this study were determined directly from the observed reduction of the reproductive output. The EC50-values for the reduction of the reproductive output were estimated directly from the observation data to be > 0.320 mg/L, because effects > 50 % did not occur within the tested concentration range. An EC10- value for the reduction of the reproductive output could not estimated, because the effects were not concentration related. The EC50 for adult mortality after 21 days was calculated respectively to be 0.295 mg/L
(Cl: 0.128- 0.800 mg/L).

All effect values given are based on the nominal concentrations of C20/22 ATQ trocken.

Endpoints assessed

C20/22 ATQ trocken
Nominal Test Item Concentration
[mg/L]
NOECReproduction 0.128
LOECReproduction 0.320
EC50, Reproduction > 0.320
EC50, Adult Mortality 0.295 (Cl: 0.128- 0.800)

Executive summary:

The Daphnia magna Reproduction Test (Semi-Static, 21 d) of the test item C20/22 ATQ trocken was conducted according to OECD 211 (2008) from 2010-04-14 to 2010-05-28 with the definitive exposure phase between 2010-04-14 to 2010-05-05 at Dr.U.Noack-Laboratorien, Käthe-Paulus-str. 1, D-31157 Sarstedt, Germany .

Test system was Daphnia magna STRAUS (Clone 5). 10 test organisms, individually held, were used per concentration level and control. At test start they were 2 to 24 h old. The study was carried out under semi-static conditions with a three times per week 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 time of production of intrinsic rate of natural increase, first brood, adult mortality, occurrence of aborted eggs and stillborn juveniles and body length of the parental daphnids.

Nominal concentrations of C20/22 ATQ trocken were selected as follows: 0.0512 - 0.128 - 0.320 - 0.800 - 2.00 mg/L.

The tested concentration levels of the test item C20/22 ATQ trocken and the control were analytically verified via LC-MS of samples on days 0, 9, 14, 16 (fresh media, 0 h) and on days 2, 16 (old media, 48 h), 12 and 19 (old media, 72 h). The initially measured concentrations of the fresh media were close to the nominal concentrations. In the fresh media (0 h) the recovery rates were in the range of 64 to 114 % for the C22 fraction and 75 to 106 % for the C20-fraction at the test concentration levels of nominal 0.0512 to 2.00 mg/L.

The recovery rates in the old media (48 or 72 h) were in the range of 73 to 106 % for the C22 fraction and 51 to 97 % for the C20-fraction at the test concentration levels of C20/22 ATQ trocken.

Adsorption to the test vessels after 72 h of exposure were determined exemplarily at the dosage levels 0.0512 to 0.320 mg/L. The absorbed amount of the C20- and the C22-fraction was in the range of 22 to 39 % of the applied amount.

The test item has a low water solubility and sorbs to organic and inorganic materials by different mechanisms. The sorption processes are mostly non-linear, means are concentration dependent. Due to these properties the test item is difficult to test in synthetic water (e.g. sorption to the test organism and walls of the test vessel) and results from such tests depend from the test settings applied. Using natural river water which contains particulate as well as dissolved organic carbon to which the test item can sorb partially reduces the difficulties encountered in tests with synthetic water e.g. preventing that the test item settles onto surfaces. The sorbed fraction of the test item is difficult to extract from the test system which normally leads to low analytical recoveries. Due to the short exposure period these low recoveries cannot be associated to biodegradation. This means the test substance is present in the test system and therefore available for exposure (dissolved in water and sorbed also called bulk). Due to the properties of the test item nominal concentrations have to be used instead of measured ones (see Table 1). This so called Bulk Approach is described by ECETOC (2003). Sorption of the test item to the glass ware of the test system was monitored and found to be small.

The average number of juveniles per parent alive at the end of the test in the control group was 124 after 21 days. There was a statistically significant reduction of the reproductive outputdetermined at the tested concentration level 0.320 mg/L when compared to the control by One Way Analysis of Variance, Holm-Sidak method, p = 0.05. The NOEC was assessed to be 0.128 mg/L (the highest test concentration with no significant reduction of the reproductive output). The EC₅₀- for the reduction of the reproductive output was estimated directly from the observation data to be > 0.320 mg/L, because effects > 50 % did not occur within the tested concentration range. An EC₁₀- value for the reduction of the reproductive output could not estimated, because the effects were not concentration related. Due to mortality of all parental daphnids at the concentration levels 0.800 and 2.00 mg/L no reproduction was observed. Details are presented in Table 1.

·   The coefficient of variation of the number of living offspring produced per parent was 4 % at the control. At the concentration levels 0.0152 and 0.128 mg/L the coefficient of variation was 8 and 11 %, respectively (Table 1). At the concentration level 0.320 mg/L the calculation of the CV was not possible, because only one parent daphnid survived.

·   The Lowest Observed Effect Concentration (LOEC_Reproduction) and the No Observed Effect Concentration (NOEC_Reproduction) after 21 days based on the reduction of the reproductive, output as the most sensitive effect in this study are summarized in Table 1.

·   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. The mean IR of the surviving daphnids was 0.51 at the control. At the concentration levels 0.0512 to 0.320 mg/L the mean IR was in a range of 0.40 to 0.54. Due to mortality of all parental daphnids at the concentration levels 0.800 and 2.00 mg/L no IR was calculated. For details see Table 1.

·   The first brood was released till day 8 by all parent animals of the control and the concentration levels 0.0512 and 0.128 mg/L. Four to five broods were observed at the concentration levels 0.0512 to 0.320 mg/L and control during the course of the study.
At the concentration level 0.320 mg/L the only surviving parental daphnid released its first brood biologically significantly delayed on day 11. Release of the first brood after day 9 is regarded to be too late and biologically significant.

·   No stillborn juvenilesand aborted eggs were produced by the control group. Related to the total number of produced juveniles (dead + alive) the percentage of dead juveniles was biologically not significant ( 1 %) at the tested concentration levels.

Table 1:        Test Item Related Effects on Reproduction (NOEC, LOEC, EC₅₀)

                      (based on the nominal test item concentrations)

Effects	C20/22 ATQ trocken Nominal Test Item Concentration [mg/L]
	Control	0.0512	0.128	0.320	0.800	2.00
Mean Number of Juveniles per Producing Parent (Reproduction Rate ± SD)	124 ± 6	119 ± 9	125 ± 14	94 ± 0^1)	--	--
Coefficient of Variation of the Mean Number of Juveniles per Producing Parent [%]	4	8	11	0	--	--
Mean Intrinsic Rates of Natural Increase	0.51	0.52	0.54	0.40	--	--
NOECReproduction	0.128 mg/L
LOECReproduction	0.320 mg/L
EC50, Reproduction	> 0.320 mg/L

SD = Standard deviation

¹⁾    = Statistical significance (One Way Analysis of Variance, Holm-Sidak method, p = 0.05)

--    = Not applicable due to the mortality of all parental animals

A summary of test item related effects concerning the adult mortality and growth (dry weight, body length) is presented in Table 2.

·   The test item induced biologically significant adult mortality at the concentration levels 0.320 mg/L (90 %), 0.800 mg/L (100 %) and 2.00 mg/L (100 %). At the tested concentration levels 0.0512 and 0.128 mg/L and the control no adult mortality was observed within the exposure phase of 21 days. At the control all parental daphnids survived till the end of the test after 21 days. The EC₅₀for adult mortality after 21 days was calculated respectively to be 0.295 mg/L (Cl: 0.128- 0.800 mg/L). Details are presented in Table 2.

·          The mean values of the body length of the surviving parental daphnids at the test groups in the range of 5.00 to 5.45 mm per daphnid were comparable to the control group with 5.33 mm per daphnid.
The mean dry body weight was determined to be in the range of 0.30 to 0.80 mg per daphnid at the test groups and 0.92 mg per daphnid at the control, respectively