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Diss Factsheets

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP, All validity criteria fulfilled, complete identification of test substance, including chemical analyses

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl, chlorides
EC Number:
274-846-6
EC Name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl, chlorides
Cas Number:
70750-47-9
IUPAC Name:
Quaternary ammonium compounds, coco alkylbis(hydroxyethy​l)​methyl, chlorides
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cocobis(2-hydroxyethyl) methylammonium chloride (CAS No. 70750-47-9)
- Physical state: Paste-like, yellow-brownish
- Active ingredient content: 99.4%
97.9 % Cocobis(2-hydroxyethyl) methylammonium chloride
1.5 % Cocobis(2-hydroxyethyl) amine
- Purity test date: 2009-06-02
- Lot/batch No.: S001700
- Expiration date of the lot/batch: 2019-04-28
- Storage condition of test material: Room temperature, protected from moisture and light, under nitrogen gas.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Two concentration levels 30.0 and 270 µg/L and the control were analysed at least once within 7 days 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 item 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.

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 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

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
Concentration
[µg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
Feb. 05th Feb. 08th Feb. 15th Feb. 17th Feb. 22nd Feb. 24th
810 106 127 n.a. n.a. n.a. n.a.
270 -- -- 95 101 110 110
Control 116 119 105 102 100 108
Test temperature:
Temperature [°C]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
Feb. 05th Feb. 08th Feb. 15th Feb. 17th Feb. 22nd Feb. 24th
810 20.4 21.8 n.a. n.a. n.a. n.a.
270 -- -- 19.8 20.1 20.0 21.1
Control 19.9 22.0 19.5 20.7 19.1 21.2
pH:
pH-Values
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
Feb. 05th Feb. 08th Feb. 15th Feb. 17th Feb. 22nd Feb. 24th
810 7.55 7.42 n.a. n.a. n.a. n.a.
270 -- -- 7.47 7.39 7.83 7.53
Control 7.88 7.62 7.53 7.39 8.07 7.54
Dissolved oxygen:
Dissolved Oxygen Concentration [mg/L]
Nominal
Concentration
[µg/L] I F I F I F
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
Feb. 05th Feb. 08th Feb. 15th Feb. 17th Feb. 22nd Feb. 24th
810 8.15 3.82 n.a. n.a. n.a. n.a.
270 -- -- 9.23 5.96 9.12 7.01
Control 9.43 7.90 9.48 6.07 9.32 6.81
Salinity:
Not measured, freshwater
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.

Location D-31171 Nordstemmen
Sampling Date 2009-12-15

Weather conditions
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 80 90 85 100 100 100
0.1 0 0 0 0 0 0
Control 0 0 0 0 0 0
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
268 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
465 µg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: (268 - 805 ug/L)
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:
EC50
Effect conc.:
468 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (270 - 810 ug/L)
Details on results:
- Mortality of parent animals: The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality
(≤ 20 %) of parent animals was observed. The EC50 for adult mortality after 21 days was calculated to be 468 µg/L (95 % confidence limits: 270 - 810 µg/L).
- No. of offspring produced per day per female:
Four to five broods were released by all surviving animals of the control and concentration levels 10.0, 30.0 and 270 µg/L. In the concentration levels 90.0 to 270 µg/L the coefficients of variation were ≥ 25 %, which is regarded to be biologically significant.
The reasons for the noticeable increased variation of the reproductive output in the concentration levels 90 and 270 µg/L are as follows: In concentration level 90.0 µg/L a reduced number of broods (lower than 4 broods) or comparatively small broods were observed at 2 of 10 parental daphnids. In concentration level the number of broods was observed to be in the expected range of 4 to 5 broods at all parental daphnids, but it is noticeable, that either very small or very big broods were released by the parental daphnids.
The coefficient of variation of the mean number of living offspring produced per parent alive at the control group was calculated to be 13 %. At the concentration levels 10.0 and 30.0 µg/L the calculated coefficients of variation of 20 and 14 % were comparable to the control. At the concentration levels 90.0 and 270 µg/L the coefficients of variation were > 30 %.
The average number of juveniles per parent in the control group was 122 after 21 days. The reproductive output was not statistically significant reduced or increased at any of the tested concentration levels. The number of juveniles of the treatment groups were compared to the control by KRUSKAL-WALLIS One Way Analysis on ranks, DUNNETT’S method (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 270 µg/L were determined to be in the range of 0.72 to 0.91 mg per daphnid and 0.87 mg per daphnid at the control group.
The mean values of the total body length at the concentration levels 10.0 to 270 µg/L were in the range of 5.30 to 5.80 mm per daphnid and comparable to the mean value of 5.28 mm per daphnid at the control group (One Way Analysis of Variance, p = 0.05).

- Number of males and females (parental):
No males were observed in either the control or the test groups during the test.
- Time to first brood release or time to hatch:
The first brood of all daphnids in all test item and control groups producing living juveniles was observed between day 7 and day 10.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.60 (CI 1.44 - 1.84)
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 intrinsic rates of natural increase and the number of juveniles was carried out using KRUSKAL-WALLIS, One Way Analysis of Variance, DUNNETT’S method (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 was calculated by
EC-VALUES 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 EC50 for adult mortality was calculated as the geometric mean of the lowest concentration causing 100 % adult mortality (810 µg/L) and the highest concentration causing 0 % adult mortality (270 µg/L), because only 0 % and 100 % adult mortality was observed in this study. These concentrations were also used as confidence limits.
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.

Any other information on results incl. tables

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

177

177

161

 25

192

199

191

163

 68

125

1478

10

 148   ±

58

39

-21.3

no

 90.0

119

 59

103

 61

103

 90

116

131

 21

121

 924

10

  92.4±

35

38

 24.3

no

 30.0

126

139

105

153

150

121

115

124

115

159

1307

10

 131   ±

18

14

 -7.38

no

 10.0

124

115

130

141

 97

121

102

 64

137

132

1163

10

 116   ±

23

20

 4.92

no

Control

124

139

122

116

137

118

114

132

 84

137

1223

10

 122±

16

13

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

 9

10

 8

 7

 8

 8

 8

8.2

 90.0

 8

 8

 8

 8

 8

 8

 8

 8

 8

 8

8.0

 30.0

 8

 8

 8

 8

 8

 8

 8

 8

 8

 8

8.0

 10.0

 8

 8

 8

 8

 8

 8

 8

 8

 8

 8

8.0

Control

 8

 8

 8

 8

 8

 8

 8

 8

 8

 7

7.9

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

  0

 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

5.75

5.25

5.25

5.50

5.50

6.00

6.00

6.00

6.00

6.25

5.75

10

7.6

0.76

 90.0

5.75

5.75

5.75

6.00

5.75

6.25

5.75

5.50

5.50

6.00

5.80

10

9.1

0.91

 30.0

6.00

6.00

5.50

5.75

5.75

5.75

5.25

5.75

5.75

5.75

5.73

10

7.8

0.78

 10.0

5.50

5.50

5.75

5.75

4.50

5.25

4.50

5.00

5.50

5.75

5.30

10

7.2

0.72

Control

5.50

5.75

5.00

5.00

5.25

4.75

4.75

6.00

4.75

6.00

5.28

10

8.7

0.87

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The adult mortality at the concentration level 810 µg/L was the most sensitive effect in this study.
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 statistically significant effects on the reproductive. Effect values are as following:

NOECAdverse : 270 µg/L

EC10, Reproduction : Not applicable
EC50, Reproduction : Not applicable

EC50, Adult Mortality : 468 µg/L (95 % confidence limits: 270 - 810 µg/L)
NOEC, Adult Mortality : 270 µg/L

The recoveries in the fresh media were in the range of 84 to 103 % of the nominal values. In the old media (after 48 h or 72 h) the recoveries decreased to values in the range of 54 to 93 %. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame between the refreshments of the test solutions. No adsorption (< LOQ; day 12) of the test item to the glassware was observed. The adsorbed fraction of the test item concentration was determined to be < 10 % of the nominal concentration at the concentration level 30.0 µg/L. The limited concentration decrease between fresh and old media is therefore most likely caused by thermodynamically more favourable redistribution of the sorbed fraction resulting in an additional sorption to suspended matter and DOC. The results of the chemical analyses show that the test organisms were fully exposed to the test substance during the test. Therefore, all effect values given are based on the nominal test item concentrations.
Executive summary:

The long-term toxicity to aquatic invertebrates of the test substance, Quaternary ammonium compounds, coco alkylbis(hydroxyethyl)methyl chlorides, CAS Number 70750 -47 -9, EC Number 274-846-6, [Quaternary ammonium compounds, C12-18-alkylbis(hydroxyethyl)methyl, chlorides, CAS Number 71808 -53 -2, EC Number 276 -038 -9], was investigated in a 21 d-reproduction study conducted according to OECD guideline 211 (Daphnia Magna Reproduction Test).

The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Under the conditions of the study effects concerning the reproduction were not observed in this study. The adult mortality at the concentration level 810 µg/L was the most sensitive effect in this study. A summary of all test item related effects on reproduction is presented inTable 1.

Table1:       Test Item Related Effects on Reproduction, EC10and EC50

Effects

Nominal Concentration [µg/L]

Control

10.0

30.0

90.0

270

810

Mean Number of Juveniles per Producing Parent (Reproduction Rate±SD)

122±16

116±23

131±18

92.4±35

148±58

--

Coefficient of Variation of theMean Number of Juveniles per Producing Parent

13

20

14

38

39

--

Number of Dead Juveniles and Aborted Eggs

0

 7

58

58

38

--

Mean Intrinsic Rates of Natural Increase

0.51

 0.50

 0.51

 0.471)

  0.50

--

Appearance of First Brood [Mean Day]

7.9

 8.0

 8.0

 8.0

 8.2

--

Mean Number of broods

5.0

 4.9

 4.9

 4.5

 4.8

--

NOECAdverse

270 µg/L

EC50, Reproduction

with Confidence Interval (CI) p = 95 %

Not determinable

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

·  The test item induced significant adult mortality at the concentration level 810 µg/L (100 %) after 21 days. At the other concentration levels and in the control no significant mortality (≤ 20 %) of parent animals was observed. The EC50for adult mortality after 21 dayswas calculated respectivelyto be 468 µg/L (95 % confidence limits: 270 - 810 µg/L).

·  The mean dry body weight and total body lengths of the surviving parental daphnids, determined at the end of the study, at the concentration levels 10.0 to 270 µg/L were comparable to the control group.
The mean dry body weight at the concentration levels 10.0 to 270 µg/L was determined to be in the range of 0.72 to 0.91 mg per daphnid and 0.87 mg per daphnid at the control group.
The mean values of the total body length at the concentration levels 10.0 to 270 µg/L were in the range of 5.30 to 5.80 mm per daphnid and comparable to the mean value of 5.28 mm per daphnid at the control group

Table2:   Test Item Related Effects on Adult Mortality (EC50) and Growth (Weight, Length)

Effects

Nominal Concentration [µg/L]

Control

10.0

30.0

90.0

270

810

Adult Mortality after 21 Days [%]

0

0

0

 0

 0

1001)

Parent Animals: Mean Dry Weight [mg]

0.87

0.72

0.78

 0.91

 0.76

--

Parent Animals: Mean Body Length [mm]

5.28

5.30

5.73

 5.80

 5.75

--

EC50, Adult Mortality

with Confidence Interval (CI) p = 95 %

468 µg/L

(CI: 270 - 810 µg/L)

1)      =Mortality> 20 % biologically significant

--      = Not applicable due to 100 % mortality of all parental animals

·      No males and ephippia (winter eggs) were observed at the control or test groups.

·  Water quality parametersas pH-value, dissolved oxygen, water hardness and temperature were determined to be within the acceptable limits.

·         In order to prove the validity of the test system and test conditions at the test facility, an acute immobilization test according to DIN 38412 L 11 was carried out with potassium dichromate as reference item once per month.
The EC50of the reference item at 1.60 mg/L after 24 h was within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according toAQS P 9/2 (05/1996) 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.