Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-23 to 2009-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 225: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (October 2007)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The concentrations of C20/22 ATQ trocken were analytically verified at day 0 (insertion of test organisms), day 14 as well as at day 28 via LC-MS/MS. The analytical verification was performed at the test item concentrations of 250, 500 and 750 mg/kg sediment dry weight. The overlying water and the sediment were analysed.

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking: The respective test item concentration was weighed out and dissolved in 2.5 mL methanol per replicate. Subsequently, the solution was thoroughly mixed with 10 g quartz sand per replicate in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment. Subsequently, the sediment was mixed thoroughly and 45 g sediment dry weight was filled into each test vessel.
- Equilibration time: 2 Days prior to insertion of test organisms
- Equilibration conditions: Same as test
- Controls: Controls with test sediment and quartz sand. Vehicle controls with test sediment and quartz sand treated with acetone.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent methanol
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes, complete evaporation

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- Common name: Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta
- Source: Bundesanstalt für Gewässerkunde (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany
- Age of parental stock (mean and range, SD): Synchronized from cultured organisms 11 days before test insertion
- Breeding conditions: 20 +/- 2 °C, diffuse light (100 - 1000 lx, 16 h photoperiod) dissolved oxygen concentration > 60 % of the air saturation value
- Handling of egg masses and larvae: 11 days before insertion into the test, the worms were synchronized. Therefore a sufficient amount of worms was taken from the breeding and cutted into half. After regeneration of heads, the worms were inserted into the test.
- Age of animals at beginning of exposure: 11 days after synchronization, completely regenerated head region. Synchronization was done to minimize uncontrolled regeneration and reproduction during the test.
- Feeding during test
- Food type: Powder of Urtica dioica (Heinrich Klein GmbH & Co KG, 97525 Schwebheim, Germany)
- Amount:0.5 % Urtica powder
- Frequency: Urtica powder was mixed into the sediment once at test start.


ACCLIMATION
- Acclimation period: 11 days (after synchronization)
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Same as test
- Feeding frequency: Same as test (Once)
- Health during acclimation (any mortality observed): No mortality observed

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

Day Parameter Control Solvent Control 62.5 125 250 500 750
mg test item/kg sediment dry weight
0
(before
insertion of worms)

Total hardness [mg/L CaCO3] 269 272 246 246 249 244 244

28

Total hardness [mg/L CaCO3] 436 393 260 319 392 299 427

Test temperature:

Day Parameter Control Solvent Control 62.5 125 250 500 750
mg test item/kg sediment dry weight
0
(before
insertion of worms)
Temperature [°C] 20.9 20.8 20.7 20.6 20.4 20.2 20.6



7
Temperature [°C] 20.4 20.2 20.1 20.1 20.2 20.1 20.3


14
Temperature [°C] 20.3 20.1 20.4 20.2 20.5 20.5 20.7


21
Temperature [°C] 19.9 19.5 19.7 19.9 19.9 20.0 20.0


28
Temperature [°C] 20.6 20.5 20.6 20.8 20.7 20.7 20.7

pH:

Day Control Solvent Control 62.5 125 250 500 750
mg test item/kg sediment dry weight
0
(before
insertion of worms)
pH 7.57 7.62 7.57 7.57 7.54 7.54 7.52


7
pH 8.18 8.16 7.99 8.00 8.01 8.00 8.00

14
pH 8.06 8.08 8.07 8.09 8.08 8.10 8.12

21
pH 8.19 8.34 8.18 8.18 8.17 8.22 8.28

28
pH 8.18 8.25 8.13 8.14 8.14 8.20 8.29

Dissolved oxygen:

Day Control Solvent Control 62.5 125 250 500 750
mg test item/kg sediment dry weight
0
(before
insertion of worms)

O2 [mg/L]* 8.53 8.31 8.44 8.52 8.49 8.52 8.43

7
O2 [mg/L]* 8.42 8.25 8.25 8.36 8.50 8.56 8.31

14
O2 [mg/L]* 8.67 8.54 8.37 8.54 8.56 8.60 8.40

21
O2 [mg/L]* 8.54 8.65 8.34 8.41 8.51 8.39 8.39

28
O2 [mg/L]* 8.41 8.43 8.27 8.32 8.37 8.27 8.30

Salinity:

not measured, freshwater conditions

Ammonia:

Ammonium-N [mg/L]
Day after worm insertion Control Solvent Control 62.5 125 250 500 750
mg test item/kg sediment dry weight
0 0.488 0.396 0.370 0.203 0.190 0.541 0.226
3 1.98 0.039 1.52 1.45 0.958 0.423 0.911
5 1.39 0.153 1.36 1.42 1.33 0.946 1.01
7 1.38 0.331 0.948 1.19 1.33 0.482 0.600
10 0.565 0.066 0.543 0.997 1.10 0.454 0.639
12 1.16 0.586 0.907 1.16 1.44 0.994 0.918
14 1.41 1.20 1.45 1.63 1.81 1.45 1.38
17 2.24 2.40 2.35 2.05 2.22 2.24 2.20
19 2.56 2.77 2.88 2.47 2.10 2.13 2.22
21 3.36 2.98 4.03 3.28 2.83 2.87 3.00
24 6.17 6.95 8.29 6.50 5.75 7.18 5.88
26 5.06 4.33 5.96 5.02 3.97 4.43 4.13
28 7.79 9.08 9.93 8.60 7.83 6.94 8.51

Nominal and measured concentrations:

For details please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test container (material, size):250 mL glass beakers, 6 cm in diameter
- Sediment volume: 45 g DW per vessel, ca. 2 cm from the ground
- Overlying water volume: 150 mL
- Depth of sediment and overlying water: sediment depth ca. 2 cm, water depth ca. 10 cm
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration (2 bubbles per second)
- Replacement of evaporated test water, if any: Yes, once weekly


EXPOSURE REGIME
- No. of organisms per container (treatment): 10 per container (60 per treatment)
- No. of replicates per treatment group: 6 replicates per treatment group
- No. of replicates per control / vehicle control: 6 replicates per control/vehicle control
- Feeding regime: Once at test start
- Type and preparation of food: Powder of Urtica dioica mixed into the sediment before insertion of test organisms
- Amount of food: 0.5 %


OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Reconstituted water acc. to OECD 203




CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate): 5 % peat air dried and finely ground, 20 % kaolin, 75 % quartz sand, 0.33 % calcium carbonate
Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 2.2 6.64 1.78



OTHER TEST CONDITIONS
Environmental Conditions
Constant Water / Room temperature [°C] Photoperiod [h] Light intensity [lx]
Nominal 20 +/- 2 16 100 - 1000
Actual 18 - 22 16 415 ± 44.6



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Visual observations for behavioural effects were made 3 times per week for each test vessel.
Behavioural differences to the controls were recorded. The endpoints of the study were the total number of living and dead worms per replicate and the total dry biomass per replicate.


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
Range Finding Test (28 d): Mortality, Reproduction and Biomass
Test Item Concentration
Mortality
of adult worms Mean number of worms Mean dry weight
per replicate
[mg test item/kg soil dry weight] [%] [mg]
Control 0 28.6 19.5
Solvent Control
1 0 32.0 16.2
10 0 30.5 23.2
100 0 36.5 20.4
1000 0 10.0 4.40

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

169 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (143-208)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

125 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: n.a.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg sediment dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: n.a.

Details on results:

- Mortality of test animals at end of exposure period: No mortality
- Total mass of test animals at beginning of test: 10 worms were equal to ca. 18.7 mg dry mass
- Changes in body weigthH58 of live adults (% of initial weight) at end of exposure period:
No mortality of complete worms was observed in the controls as well as at the test item concentrations of 62.5 and 125 mg/kg sediment dry weight after an exposure to the test item over 28 days. At 250, 500 and 750 mg/kg sediment dry weight at mortality of 15, 22.5 and 50 % occurred, respectively

- No. of offspring produced:
The number of incomplete worms and thus the reproduction did not statistically significantly differ to the pooled control at the test item concentration of 62.5 mg/kg sediment dry weight after 28 days of exposure. At all other test item concentrations the number of incomplete worms was statistically significantly reduced compared to the control . The EC50 for reproduction was determined to be at 169 mg/kg sediment dry weight

- Morphological abnormalities: None
- Behavioural abnormalities: No changes in the worm behaviour or pathological symptoms were observed in the controls as well as at 62.5 and 125 mg/kg sediment dry weight throughout 28 days of exposure. Beginning from day 19 changes in the worm behaviour and dead worms were observed at 250, 500 and 750 mg/kg sediment dry weight.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
Summary of all Effects and resulting LOEC, NOEC of the Reference Item Study


Effects Potassium Chloride
[g/kg sediment dry weight]
Mortality 4.0
Biomass 4.0
Reproduction 4.0
LOEC
(Mortality, Biomass, Reproduction) 4.0
NOEC
(Mortality, Biomass, Reproduction) 2.0
EC50
(Mortality) 4.06
EC50
(Biomass) 2.52
EC50
(Reproduction) 2.91

Reported statistics and error estimates:

All data were checked for outliers using Grubb’s test for outlier (Barnett & Lewis, 1994), no outliers were found.
One Way Analysis of Variance (ANOVA) was used for LOEC and NOEC calculations. Prior to running a One Way Analysis of Variance a Normality Test and an Equal Variance Test were performed. P-values for both Normality and Equal Variance Test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was a=0.05.
The control and solvent control were statistically analysed for significant differences with t-tests. Since no significant differences occurred the controls were pooled for further statistical analyses.
EC50-value and confidence intervals were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals was carried out using standard procedures.

Mortality after 28 Days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Number of Complete						MV	SD ±	Recovery [%]	Mortality [%]
	1	2	3	4	5	6
Control	10	10	10	10	10	10	10	0	100	0
Solvent Control	10	10	10	10	10	10
62.5	10	10	10	10	-		10	0	100	0
125	10	10	10	10			10	0	100	0
250	9	9	10	6			8.50	1.73	85.0	15.0
500	5	9	9	8			7.75	1.89	77.5	22.5
750	5	5	4	6			5.00	0.82	50.0	50.0

MV = Mean          = Standard Deviation

Total Worm Number after 28 Days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Total Number of
	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Control	34	33	39	29	31	35	35.1	2.94	-	-
Solvent Control	37	38	36	37	37	35
62.5	33	36	33	31	-		33.3	2.06	5.23	No
125	28	29	33	26			29.0	2.94	17.3	Yes
250	11	11	21	15			14.5	4.73	58.7	Yes
500	9	10	12	10			10.3	1.26	70.8	Yes
750	11	9	10	10			10.0	0.82	71.5	Yes

Reproduction after 28 days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Number of Incomplete
	1	2	3	4	5	6	MV	SD	% Inhibition	Significance*
Control	24	23	29	19	21	25	25.1	2.94	-
Solvent Control	27	28	26	27	27	25
62.5	23	26	23	21	-		23.3	2.06	7.31	No
125	18	19	23	16			19.0	2.94	24.3	Yes
250	2	2	11	9			6.00	4.69	76.1	Yes
500	4	1	3	2			2.50	1.29	90.0	Yes
750	6	4	6	4			5.00	1.15	80.1	Yes

Worm Dry Biomass after 28 Days of Exposure

Nominal concentration [mg/kg sediment dry weight]	Dry weight [mg] per replicate
	Replicate						MV ± SD	Significance*
	1	2	3	4	5	6
Control	22.5	26.3	29.7	24.4	22.5	26.3	26.2 ± 2.32	-
Solvent Control	27.4	27.9	26.3	25.5	26.2	29.6
62.5	25.4	24.6	23.2	18.0	-		22.8 ± 3.33	No
125	15.3	18.6	19.6	15.2			17.2 ± 2.26	Yes
250	11.0	9.90	14.5	15.1			12.6 ± 2.56	Yes
500	5.30	6.70	10.3	7.70			7.50 ± 2.11	Yes
750	8.60	4.50	5.40	5.40			5.97 ± 1.80	Yes

Validity criteria fulfilled:

yes

Conclusions:

In this study C20/22 ATQ trocken induced a mortality of 15, 22.5 and 50 % at the test item concentrations of 250, 500 and 750 mg/kg sediment dry weight, respectively. The reproduction as well as the worm dry biomass was statistically significant reduced at 125, 250, 500 and 750 mg/kg sediment dry weight.
Thus, the lowest observed effect concentration (LOEC) was set to be at 125 mg/kg sediment dry weight. The overall no observed effect level (NOEC) was determined to be at 62.5 mg/kg sediment dry weight. The EC50 for reproduction was determined to be at 169 mg/kg sediment dry weight and the EC50 for biomass was set at 221 mg/kg sediment dry weight. All effect levels given are based on the nominal concentrations of C20/22 ATQ trocken.

Executive summary:

The effects of the test item C20/22 ATQ trocken (batch no. DEG4047322) on the oligochaete Lumbriculus variegatus in a water-sediment system were determined at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany , from September 23^rdto October 26^th, 2009. The study was carried out according to OECD Guideline 225 (2007). Test duration was 28 days after insertion of the test organisms. The study was performed by spiking the test item into the sediment with five concentrations of 62.5 – 125 – 250 – 500 and 750 mg/kg sediment dry weight. Six replicates per control as well as solvent control and four replicates per test item concentration were set up for the biological investigation. Water quality parameters as temperature, pH-value, O₂-concentration, ammonium and total hardness were determined throughout the study.

The concentrations of C20/22 ATQ trocken were analytically investigated at the test item concentrations of 250, 500 and 750 mg/kg sediment dry weight at day 0 (worm insertion), day 14 and day 28 in the sediment and in the overlying water via LC-MS/MS.

The measured concentrations of C20/22 ATQ trocken (C20 fraction) in the sediment at the beginning of the test were in the range of 95 - 98 % of the applied concentrations. After 14 days, the recoveries of C20/22 ATQ trocken (C20 fraction) in the sediment were at all levels between 91 – 93 % of the applied concentrations. At the end of the study the recoveries were in the range of 83 – 86 %.

The measured concentrations of C20/22 ATQ trocken (C22 fraction) in the sediment at the beginning of the test were in the range of 79 - 86 % of the applied concentrations. After 14 days, the recoveries of C20/22 ATQ trocken (C22 fraction) in the sediment were at all levels between 86 – 88 % of the applied concentrations. At the end of the study the recoveries were in the range of 71 – 79 %.

These recoveries demonstrate that the nominal range of the test item concentrations was met.

After 28 days of exposure C20/22 ATQ trocken induced a mortality of 15, 22.5 and 50 % at the test item concentrations of 250, 500 and 750 mg/kg sediment dry weight. The total number of worms and also the reproduction was statistically significantly reduced at 125, 250, 500 and 750 mg/kg sediment dry weight compared to the pooled control (Table 1). The EC₅₀ for reproduction was determined to be at 169 mg/kg sediment dry weight. Also, the biomass was statistically significantly reduced compared to the pooled control after 28 days of exposure to the test item at 125, 250, 500 and 750 mg/kg sediment dry weight (Table 1). The EC₅₀for worm biomass was set at 221 mg/kg sediment dry weight. 

Concluded from the results, the Lowest Observed Effect Concentration (LOEC) was set at 125 mg/kg sediment dry weight. Thus, the No Observed Effect Concentration (NOEC) was determined to be at 62.5 mg/kg sediment dry weight

Reference 2

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-09 to 2009-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Draft ISO/DIS 10872 (2008)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates:
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.5 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 50 %.
- Details of spiking: The respective test item amount for each concentration was weighed out and dissolved in 10 mL methanol. Subsequently, the solution was thoroughly mixed with quartz sand (20 % of the entire sediment) in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment and thoroughly mixed with the sediment. Additional M9 medium* was added to the sediment to obtain a water content of 44 %. Subsequently, 0.5 g sediment wet weight was filled into each test well.
- Equilibration time: 1 day
- Equilibration conditions: 8 +/- 2 °C, dark
- Controls: Moistened test sediment with the addition of quartz sand was tested under the same conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent, methanol
- Concentration of vehicle in test medium (stock solution and final test solution): 5 mL per 100 g sediment
- Evaporation of vehicle before use: Yes

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wilde type strain N2 var Bristol
- Source: Caenorhabditis Genetics Center, Minneapolis
- Breeding conditions:Breeding is performed at the test facility at 20 +/- 2 °C in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP 50.
- Age of animals at beginning of exposure: First stage juveniles were obtained by filtering nematode solutions from culture plates through nylon nets (10 µm and subsequently 5 µm mesh size). The size of 30 representative juvenile nematodes, which were not used in the test, was determined prior to nematode insertion.
- Feeding during test
LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 17 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.



ACCLIMATION
- Acclimation period: None, test conditions = breeding conditions

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

96 h

Exposure phase:

total exposure duration

Nominal and measured concentrations:

Nominal: 62.5 - 125 - 250 - 500 - 1000 mg test item/kg sediment dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Nunc Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g sediment wet weight per replicate
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: no
- Replacement of evaporated test water, if any: No


EXPOSURE REGIME
- No. of organisms per container (treatment): 40
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: 4
- Type and preparation of food: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 17 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate):
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.5 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 50 %.
- Maturation of artificial substrate (if any): no

OTHER TEST CONDITIONS
- Continuous dark
The initial pH of the artificial sediment at test start was 6.47. The initial sediment moisture was 7.8 %. The room temperature ranged from 18.0 to 21.0 °C.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality of the inserted nematodes was analysed by counting the adult nematodes under a microscope. The body length of each female adult nematode was determined with a microscale.
The reproduction was analysed by inserting the extracted nematodes in a counting chamber. The produced number of offspring was determined in the specified volume of the counting chamber under a microscope. The total number of offspring per replicate was calculated by using the entire volume of extract.


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
Range Finding Test (4 d): Recovery, Mortality and Reproduction
Test Item Concentration
Recovery of adult nematodes Fertility of adult nematodes Reproduction
[mg test item/kg soil dry weight] [%] [%] [Offspring per test organism]
Control 86.7 100 59.0
Solvent Control 93.3 100 56.2
10 96.7 100 63.0
100 93.3 100 56.5
1000 93.3 100 46.5

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortaliy, fertility, reproduction, growth

Remarks on result:

other: n.a.

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

500 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, fertility, reproduction, growth

Remarks on result:

other: n.a.

Details on results:

- Mortality of test animals at end of exposure period:
In the artificial sediment a recovery of 91 % was observed in the pooled control after 4 days of exposure. At the test item concentrations the recovery ranged from 85 to 97.5 %. The resulting mortality of 2.5 to 15 % was not considered to be of biological significance, and was within the range of experimental variation considered acceptable for the control group.
No evident mortality was observed at the reference item concentration of 10 mg/L. However, at 20 mg/L the reference item induced a 100 % mortality of adult nematodes
- No. of offspring produced:
The reproduction in artificial sediment was not statistically significant reduced compared to the pooled control at 62.5, 125 and 250 mg/kg sediment dry weight. However, at 500 and 1000 mg/kg sediment dry weight the number of offspring per inserted test organism was statistically significant reduced compared to the pooled control by about 46 % at 1000 mg/kg sediment dry weight.
- Growth:
The initial mean size of the introduced juvenile nematodes was 229 µm.
In artificial sediment the growth of the adult nematodes did not statistically significantly differ compared to the control at the test item concentrations of 62.5, 125 and 250 mg/kg sediment dry weight. However, at the test item concentrations of 500 and 100 mg/kg sediment dry weight the nematode growth was statistically significantly reduced


- Morphological abnormalities: Not observed
- Behavioural abnormalities: Not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:


Effects Reference Item Benzyldimethylhexadecylammonium chloride
[mg/L]
Mortality 10
Fertility 15
Reproduction 10
Growth 5
LOEC
(Mortality, Fertility, Reproduction, Growth) 5
NOEC
(Mortality, Fertility, Reproduction, Growth) < 5
EC50
(Mortality) 15.0 (14.2 – 15.8)
EC50
(Reproduction) 10.2 (9.75 – 10.9)
EC50
(Size) 15.9 (11.5 – 23.4)

Reported statistics and error estimates:

All data were checked for outliers using Grubb’s test for outlier (Barnett & Lewis, 1994), no outliers were found. One Way Analysis of Variance (ANOVA) was used for LOEC and NOEC calculations. Prior to running a One Way Analysis of Variance a Normality Test and an Equal Variance Test was done. P-values for both Normality and Equal Variance Test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Recovery / Mortality after 4 days of Exposure to C20/22 ATQ trocken

Nominal test item concentration [mg/kg sediment dry weight]	[%] Recovered test organisms						[%] Mortality
	Replicate				MV	SD
	1	2	3	4
Liquid Control	100	90	100	90	95.0	5.77	5.00
Control	90	100	80	90	91.3	6.41	8.70
Solvent Control	90	90	100	90
62.5	100	90	100	100	97.5	5.00	2.50
125	100	80	100	90	92.5	9.57	7.50
250	90	100	90	90	92.5	5.00	7.50
500	90	90	90	90	90.0	0.00	10.0
1000	90	80	90	80	85.0	5.77	15.0

Nematode Reproduction after 4 Days of Exposure to C20/22 ATQ trocken

Nominal test item concentration [mg/kg sediment dry weight]	Number of offspring per inserted nematode
	Replicate				MV	SD	Inhibition [%]
	1	2	3	4
Control	44	47	44	51	47.4	4.77	-
Solvent Control	41	48	57	47
62.5	48	50	42	52	47.7	4.36	-0.60
125	35	29	37	48	36.9	7.83	22.1
250	43	30	47	38	39.4	6.99	17.0
500	30	41	28	43	35.5	7.35	25.2*
1000	35	22	20	25	25.5	6.82	46.3*

Nematode Growth after 4 Days of Exposure to C20/22 ATQ trocken

Nominal test item concentration [mg/kg sediment dry weight]	Mean Growth [µm]
	Replicate				MV	SD	Inhibition [%]
	1	2	3	4
Control	875	952	893	923	903	30.9	-
Solvent Control	886	857	923	917
62.5	859	881	894	923	889	26.8	1.56
125	938	875	913	907	908	25.6	-0.53
250	887	866	859	929	885	31.3	2.02
500	821	827	838	811	824	11.2	8.75*
1000	695	731	742	704	718	22.1	20.5*

Validity criteria fulfilled:

yes

Conclusions:

In this study C20/22 ATQ trocken did not induce significant mortality of C. elegans after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg sediment dry weight for four days. Also, nematode fertility was not affected by all test item concentrations. However, the reproduction of nematodes was statistically significant reduced at 500 and 1000 mg/kg sediment dry weight. Also, C20/22 ATQ trocken induced a statistically significant reduction of growth at 500 and 1000 mg/kg sediment dry weight.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set to at 500 mg/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 250 mg/kg sediment dry weight. All effect levels given are based on the nominal concentrations of C20/22 ATQ trocken.

Executive summary:

The effects of the test item C20/22 ATQ trocken (batch no.DEG4047322) on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined at Dr.U.Noack-Laboratorien, , , from December 9^thto December 15^th, 2009. The study was carried out according to the Draft guideline ISO/DIS 10872 (2008). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the test item concentrations of 62.5 - 125 - 250 - 500 - 1000 mg test item/kg sediment dry weight (nominal concentration). Four replicates per control, solvent control and each test item concentration were set up.

After four days of exposure C20/22 ATQ trocken did not induce biologically significant mortality of C. elegansat all test item concentrations. Also, adult nematode fertility was not affected at all test item concentrations. However, the reproduction of nematodes was statistically significant reduced at 500 and 1000 mg/kg sediment dry weight. Also, C20/22 ATQ trocken induced a statistically significant reduction of growth at 500 and 1000 mg/kg sediment dry weight.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set to at 500 mg/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 250 mg/kg sediment dry weight. All effect levels given are based on the nominal concentrations of C20/22 ATQ trocken.

Summary of all Effects and resulting LOEC, NOEC based on nominal Concentrations

Effects	C20/22 ATQ trocken
	[mg/kg sediment dry weight]
Mortality	> 1000
Fertility	> 1000
Reproduction	500
Growth	500
LOEC (Mortality, Fertility, Reproduction, Growth)	500
NOEC (Mortality, Fertility, Reproduction, Growth)	250

Description of key information

Two endobenthic organisms. the worm Lumbriculus variegatus and the abundant nematode Caenorhabditis elegans, were used to determine a potential sediment toxicity of C20/22 ATQ. 

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

62.5 mg/kg sediment dw

Additional information

The worm Lumbriculus has a NOEC being slightly lower than the NOEC of the nematode (62.5 mg/kg dw. and 250 mg/kg dw.). The NOEC of the worm Lumbriculus variegatus of 62.5 mg/kg dw. is choosen as a basis for the PNEC freshwater sediment.