Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

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:

2015-06-24 to 2015-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All concentration levels of Antimussol ZJ and the control were analytically verified by LC-MS/MS. The concentrations of the test item were analysed on the day of worm insertion (day 0) as well as on day 7 and at test end (day 28) in the water layer, the sediment layer and in the pore water. Three additional replicates per treatment were set up for the analytical verification on day 0, 7 and 28.

Vehicle:

no

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 acetone per replicate. Subsequently, the solution was mixed thoroughly with 10 g artificial sediment per replicate in a glass vessel. After complete evaporation of the solvent, the treated portion of artificial sediment was given to the remaining sediment. Demineralised water was added to adjust the humidity of the sediment to 30 %. Subsequently, the sediment will be mixed thoroughly and an amount corresponding to 45 g sediment dry weight were filled into each test vessel.
- Equilibration time:
2 days prior to insertion of test organisms
- Equilibration conditions:
Same as test conditions
- Controls:
Six replicates with untreated artificial sediment
-Solvent controls:
6 replicates with artificial sediment, treated with acetone but not with test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Acetone
- Concentration of vehicle in test medium (stock solution and final test solution):
2.5 mL Acetoneper replicate
- Evaporation of vehicle before use:
Complete

PREPARATION OF SPIKED WATER
- Details of spiking:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in final test solution:

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 14 days before insertion
- Breeding conditions:
20 +/- °C, diffuse light (100 - 1000 lx, 16 h photoperiod), dissolved oxygen concentration > 60 % of the air saturation value
- Handling of egg masses and larvae:
14 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 regenerationof heads, the worms were inserted into the test.
- Age of animals at beginning of exposure:
14 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:
0.4 % Urtica powder
- Frequency:
Once at test start

ACCLIMATION
- Acclimation period:
14 days (after synchrinization)
- Acclimation conditions (same as test or not):
Same sa 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

Post exposure observation period:

No

Hardness:

T
Day Parameter Control Solvent Control 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms)
Total hardness [mg/L CaCO3] 265 262 320 328 271 324 333
28 Total hardness [mg/L CaCO3] 247 285 221 278 255 230 233

Test temperature:

Day Parameter Control Solvent Control 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) Temperature [°C] 19.7 19.5 19.6 19.6 19.5 19.4 19.6
7 Temperature [°C] 20.2 19.4 19.6 19.4 19.3 19.4 19.6
14 Temperature [°C] 20.0 20.2 20.2 20.2 20.2 20.1 20.3
21 Temperature [°C] 21.2 21.3 21.3 21.2 21.3 21.3 21.2
28 Temperature [°C] 20.6 20.7 20.7 20.6 20.7 20.7 20.8

pH:

Day Parameter Control Solvent Control 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms)
pH 8.24 8.27 8.27 8.09 8.13 8.07 8.01
7 pH 8.36 8.43 8.41 8.24 8.42 8.45 8.43
14 pH 8.46 8.42 8.24 8.23 8.17 8.25 8.34
21 pH 8.27 8.38 8.55 8.42 8.45 8.47 8.52
28 pH 8.47 8.38 8.57 8.57 8.51 8.45 8.49

Dissolved oxygen:

Table 14: Water Parameters (pH-value, Temperature, Oxygen-Concentration, Hardness)
Day Parameter Control Solvent Control 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms)
O2 [mg/L]* 9.16 9.17 9.27 9.06 9.10 8.96 9.17
7 O2 [mg/L]* 8.44 8.84 9.14 9.25 9.25 9.15 8.91
14 O2 [mg/L]* 8.90 9.00 9.41 8.84 9.24 8.96 8.18
21 O2 [mg/L]* 7.70 8.53 9.36 8.83 8.99 9.12 9.19
28 O2 [mg/L]* 8.71 7.82 8.76 8.64 8.58 8.48 8.55
*) 70 % of the air saturation value corresponds to 6.2 mg O2/L.

Salinity:

Not measured, fresh water conditions

Ammonia:

Ammonium-N [mg/L]
Day after worm insertion Control Solvent Control 62.5 125 250 500 1000
mg test item/kg sediment dry weight
Ammonium-N [mg/L]
0 0.73 0.7 0.72 0.64 0.68 0.65 0.68
2 2.1 1.98 1.67 1.37 1.45 1.17 1.44
5 0.65 0.38 0.17 0.21 0.35 0.05 0.04
7 0.08 0.1 0.18 0.2 0.18 0.1 0.08
9 0.09 0.11 0.33 0.32 0.25 0.14 0.08
12 0.11 0.14 0.36 0.4 0.5 0.28 0.12
14 0.14 0.17 0.37 0.28 0.45 0.29 0.16
16 0.16 0.18 0.37 0.24 0.47 0.29 0.19
19 0.18 0.17 0.35 0.24 0.49 0.34 0.21
21 0.2 0.18 0.4 0.26 0.44 0.34 0.27
23 0.27 0.27 0.56 0.33 0.54 0.41 0.29
26 0.31 0.26 0.5 0.38 1.01 0.44 0.33
28 0.28 0.23 0.52 0.39 0.55 0.44 0.29

Nominal and measured concentrations:

Measured Concentrations and Recovery Rates of Antimussol ZJ
(Aqueous Layer, Pore Water and Sediment) for Day 0
Sampling date: 2015-06-24, Start of analysis: 2015-06-253)
Concentration test item [mg/kg DW] 1000 500 250 125 62.5 Control Solvent Control
Amount per test vessel [mg]1) 45.0 22.5 11.3 5.63 2.81
Aqueous Layer Meas. amount of Antimussol ZJ
[mg] 0.208 0.212 0.110 0.0697 < LOQ < LOQ2) < LOQ
% 0 1 1 1 < LOQ < LOQ < LOQ
Pore
Water Meas. amount of Antimussol ZJ
[mg] 0.00518 0.00397 0.00270 0.002582) < LOQ < LOQ < LOQ
% 0 0 0 0 < LOQ < LOQ < LOQ
Sediment Meas. amount of
Antimussol ZJ
[mg] 45.1 21.8 11.3 5.34 3.02 < LOQ < LOQ
% 100 97 100 95 107 < LOQ < LOQ
Total
Amount [mg] 45.3 22.0 11.4 5.41 3.02 < LOQ < LOQ
% 101 98 101 96 107 - -

Measured Concentrations and Recovery Rates of Antimussol ZJ
(Aqueous Layer, Pore Water and Sediment) for Day 28
Sampling date: 2015-07-22, Start of analysis: 2015-07-22 (Aqueous Layer, Pore water)
Start of analysis: 2015-07-232) (Sediment)

Concentration test item [mg/kg DW] 1000 500 250 125 62.5 Control Solvent Control
Amount per test vessel [mg]1) 45.0 22.5 11.3 5.63 2.82
Aqueous Layer Meas. amount of Antimussol ZJ
[mg] 0.0888 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ
% 0 < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ
Pore Water Meas. amount of Antimussol ZJ
[mg] 0.0122 0.00547 0.00247 0.000856 < LOQ < LOQ < LOQ
% 0 0 0 0 < LOQ < LOQ < LOQ
Sediment Meas. amount of Antimussol ZJ
[mg] 47.6 22.3 10.2 4.89 2.33 < LOQ < LOQ
% 106 99 90 87 83 < LOQ < LOQ
Total
Amount [mg] 47.7 22.3 10.2 4.89 2.33 < LOQ < LOQ
% 106 99 90 87 83 - -
Meas. amount = Measured amount , mean value of two injections (dilution factor taken into account)
% = Percent of the nominal concentration of the test item Antimussol ZJ

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass beakers (6 cm diameter)
- Sediment volume: 45 g dry weight per vessel / ca. 1.0 - 3.0 cm from the ground
- Overlying water volume: 150 mL
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration via Pasteur pipettes. Aeration was checked each working day.
- Replacement of evaporated test water, if any: Water levels were topped up with demineralised water each working day during the study to compensate losses due to evaporation.

EXPOSURE REGIME
- No. of organisms per container (treatment): 10 synchronized worms per replicate (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 (stinging nettle) was mixed into the sediment.
- Amount of food: 0.4 % of thge sediment dry weight

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

Day Parameter Control
0 (before insertion of worms)
Temperature [°C] 19.7
pH 8.24 8.27 8.27 8.09 8.13 8.07 8.01
O2 [mg/L]* 9.16 9.17 9.27 9.06 9.10 8.96 9.17
Total hardness [mg/L CaCO3] 265 262 320 328 271 324 333

CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- 5 % peat, air-dried and finely ground
- 20 % kaolin, with a kaolinite content > 30 %
- 75 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.47 % calcium carbonate (CaCO3) to achieve a pH of 6.55

Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 2.5* 6.55 30
*) external measurement, non-GLP

OTHER TEST CONDITIONS
Environmental Conditions
Room temperature [°C] Photoperiod [h light] Light intensity [lx]
Nominal 20 ± 2 16 100 - 500
Actual 18 - 22 16 247 ± 67

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Visual observations of behavioural differences to the controls were recorded three times per week. The endpoints of the study as determined after day 28 were the number of surviving worms per replicate and the total dry biomass of worms per replicate. Worms were considered dead if showing no response to a mechanical stimulus, signs of decomposition or by absence. Living worms were assigned as adult (large complete worms) or offspring (small complete worms with regenerated body regions or incomplete worms), if possible.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1000 - 100 - 10 - 1 mg test item/kf sediment dry weight
- Results used to determine the conditions for the definitive study:
Preliminary Range Finding Test (28 d): Mortality, Reproduction and Biomass (non-GLP)
(Application with acetone, 3 replicates, each with 10 synchronized worms)
Nominal Test Item Concentration Mortality
of adult worms Mean number of worms per replicate Mean dry weight
per replicate
[mg test item/kg sediment dry weight] [%] [n] [mg]
Control 0 31.7 23.8
Solvent Control
1 0 30.7 24.1
10 0 33.3 27.8
100 0 28.7 29.0
1000 13.3 8.67 8.50

Reference substance (positive control):

yes

Remarks:

Potassium chloride

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

125 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

250 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

432 mg/kg sediment dw

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 338 - 549

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

712 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Remarks on result:

other: 287 - 1702

Details on results:

- Changes in body weight of live adults (% of initial weight) at end of exposure period:
At test start the worms had a body weight of 6.5 mg dry weight/10 worms (mean weight of two determinations). At test end, the biomass dry weight of the worms was not statistically significantly different at the test item concentrations 62.5 and 125 mg/kg sediment dry weight compared to the pooled control. However, at 250, 500 and 1000 mg/kg sediment dry weight the biomass was statistically significantly reduced compared to the pooled control. The EC50-value of total biomass per replicate was determined to be 712 mg/kg sediment DW.
- No. of offspring produced:
At the test item concentrations 125 mg/kg sediment dry weight and above, the number of total worms was statistically significantly reduced. Accordingly, the reproduction did statistically significantly differ from the pooled control at test item concentrations > 125 mg/kg sediment dry weight after 28 days of exposure. The EC50–value for total number of worms/reproduction was determined to be 432 mg/kg sediment dry weight.
- Morphological abnormalities: None
- Behavioural abnormalities:
No evident changes in the worm behaviour or pathological symptoms were observed in the controls as well as in the test item concentrations 62.5 and 125 mg/kg sediment dry weight during the 28 days of exposure. However, worms stayed on the sediment surface more frequently in the replicates of the test item concentrations 250, 500 and 1000 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 NOEC, LOEC of the Reference Item Study
Effects Potassium Chloride
[g/kg sediment dry weight]
NOEC
Total number of worms/reproduction < 1.0
NOEC
Total Biomass per replicate 1.0
LOEC
Total number of worms/reproduction ≤ 1.0
LOEC
Total Biomass per replicate 2.0
EC50
Total number of worms/reproduction 2.6 (mean: 2.51)*
EC50
Total Biomass per replicate 2.4 (mean: 2.49)*

*) Due to a lack of literature for Potassium Chloride and L. variegatus, internal reference data of the last 6 years were collected and the resulting means are presented.

Reported statistics and error estimates:

Controls
The control and solvent control were statistically analysed for significant differences with t-test (Student). Since no statistical difference concerning total number of worms/reproduction and total biomass per replicate were determined, both controls were pooled for further statistical analyses.
NOEC/LOEC-values and statistical analyses
Williams Multiple Sequential t-Test was used for LOEC and NOEC calculations. When running the test, a Normality Test and an Equal Variance Test were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.
EC-values and statistical analyses
ECx-values for reproduction and biomass and confidence intervals were calculated by 3-parameter normal CDF regression analysis.

Total Worm Number after 28 Days of Exposure

Test item concentration [mg/kg sediment dry weight]	Number of Surviving						MV	SD ±	Inhibition to pooled control[%]	Sign.*
	1	2	3	4	5	6
Control	43	25	33	30	34	28	33.3	6.40	-	-
Solvent Control	38	34	34	22	35	43
62.5	33	29	36	40	-		34.5	4.65	-3.76	No
125	28	27	30	28			28.3	1.26	15.0	Yes
250	27	26	29	28			27.5	1.29	17.3	Yes
500	8	11	10	12			10.3	1.71	69.2	Yes
1000	10	9	9	10			9.5	0.58	71.4	Yes

*) = Statistically significantly different compared to pooled control (Williams Sequental t-test Procedure)

Reproduction after 28 days of Exposure

Test item concentration [mg/kg sediment dry weight]	Number of Reproduced						MV	SD ±	Inhibition to pooled control[%]
	1	2	3	4	5	6
Control	33	15	23	20	24	18	23.3	6.40	-
Solvent Control	28	24	24	12	25	33
62.5	23	19	26	30	-		16.3	13.2	29.8
125	18	17	20	18			12.2	9.47	47.7
250	17	16	19	18			11.7	9.09	49.8
500	0	1	0	2			0.50	0.84	97.9
1000	0	0	0	0			0	0	100

MV = Mean Value SD = Standard Deviation

Worm Dry Biomass after 28 Days of Exposure

Test item concentration [mg/kg sediment dry weight]	Dry weight[mg] per replicate						MV	SD ±	Inhibition to pooled control[%]	Sign.*
	1	2	3	4	5	6
Control	26.7	22.9	28.2	21.7	26.2	22.8	26.5	3.3	-	-
Solvent Control	29.2	29.1	27.9	22.0	30.6	30.3
62.5	31.3	22.6	30.7	27.4	-		28.0	4.0	-5.78	No
125	26.5	30.3	29.1	28.3			28.6	1.6	-7.86	No
250	21.3	19.7	20.0	23.0			21.0	1.5	20.7	Yes
500	14.1	16.4	16.2	16.9			15.9	1.2	39.9	Yes
1000	13.5	12.6	14.2	11.4			12.9	1.2	51.2	Yes

MV = Mean value    SD = Standard deviation                                   
*) Statistically significantly different compared to pooled control (Williams Sequential t-test Procedure)

Validity criteria fulfilled:

yes

Conclusions:

After 28 days of exposure, Antimussol ZJ induced no evident mortality (< 10 %) in all test item concentrations. Since no statistical or biological differences concerning total number of worms/reproduction and total biomass per replicate were determined between the control and the solvent control, both controls were pooled for statistical analyses.
The total number of worms and thus the reproduction were statistically significantly reduced at the treatment rates 125 mg/kg sediment dry weight and above compared to the pooled control.
Furthermore, compared to the pooled control, 250, 500 and 1000 mg/kg sediment dry weight the total biomass per replicate was statistically significantly reduced.
The EC50-values for total biomass per replicate and total number of worms/reproduction were determined to be 712 and 432 mg/kg sediment dry weight, respectively.

Executive summary:

The effects of the test item Antimussol ZJ (batch no.ESD0017844) on the oligochaeteLumbriculus variegatusin a water-sediment system were determined at Dr.U.Noack Laboratorien, Sarstedt, Germany, from June 22nd to July 23rd, 2015 with the definitive exposure phase from June 24th to July 22nd, 2015. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms.The study was performed by spiking artificial sediment with five test item concentrations: 62.5 – 125 – 250 – 500 – 1000 mg/kg sediment dry weight. Six replicates per control and solvent control and four replicates per test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O₂-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for each control and test item concentration.
The concentrations of Antimussol ZJ in the sediment and the overlying water were analytically verified in all test item concentrations on day 0 (worm insertion), day 7 and day 28 via LC-MS/MS. The measured concentrations of Antimussol ZJ at the beginning of the test (day 0, after equilibration for two days) were in the range of 95 - 106 % of the nominal values (total amount in sediment, aqueous layer and pore water). On day 7, the measured concentrations of Antimussol ZJ remained at 80 - 93 %. At the end of the exposure period (day 28), the measured concentrations were 82 - 106  % of the nominal values. During all sampling dates only low amounts ofAntimussol ZJwere measured in the aqueous layer and in the pore water (≤ 1.22 %).
After 28 days of exposure, Antimussol ZJ induced no evident mortality (< 10 %) in all test item concentrations. Since no statistical or biological differences concerning total number of worms, reproduction and biomass were determined between the control and the solvent control, both controls were pooled for further statistical analyses.

The total number of worms and thus the reproduction were statistically significantly reduced at the treatment rates 125 mg/kg sediment dry weight and above compared to the pooled control.

Compared to the control, the total biomass per replicate was not statistically significantly different at the test item concentrations up to 125 mg/kg sediment dry weight. However, at 250 mg/kg sediment dry weight and above the biomass was statistically significantly reduced.

Summary of all Effects

Effects	Antimussol ZJ
	[mg/kg sediment dry weight]
NOEC (Total number of worms/Reproduction)	62.5
NOEC (Total Biomass per replicate)	125
LOEC (Total worms/Reproduction)	125
LOEC (Total Biomass per replicate)	250
EC50 (Total number of worms/Reproduction)	432 (338 - 549)
EC50 (Total Biomass per replicate)	712 (287 - 1702)

 

Reference 2

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-24 to 2015-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 10872 (2010)

Deviations:

yes

Remarks:

To kill the nematodes the multi dishes were heated for 20 minutes at 80°C instead of 10 minutes. This deviation is considered to have no impact on the quality and integrity of the study.

Principles of method if other than guideline:

To kill the nematodes the multi dishes were heated for 20 minutes at 80°C instead of 10 minutes.
This deviation is considered to have no impact on the quality and integrity of the study.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Analytical evaluation of the application solutions and the control of the test item Antimussol ZJ was carried out via LC-MS/MS using an external standard calibration. Detection was performed with a mass selective detector (MS).

Vehicle:

yes

Details on sediment and application:

Test medium
A loamy sand (LUFA 2.2 soil) was used as sediment in the study. The soil was received from the LANDWIRTSCHAFTLICHE UNTERSUCHUNGS-UND FORSCHUNGSANSTALT SPEYER, Obere Langgasse 40, 67346 Speyer, Germany. The soil was sampled at June 8th, 2015 and stored at greenhouse conditions at the test facility until use.

PREPARATION OF SPIKED SEDIMENT
A loamy sand (LUFA 2.2 soil) was used as sediment in the study. The soil was received from the LANDWIRTSCHAFTLICHE UNTERSUCHUNGS-UND FORSCHUNGSANSTALT SPEYER, Obere Langgasse 40, 67346 Speyer, Germany. The soil was sampled at June 8th, 2015 and stored at greenhouse conditions at the test facility until use.
- Details of spiking:
Ca. 24 hours before insertion of the test organisms, the respective test item amount for a stock solution was weighed out and dissolved in acetone (50 mL). Afterwards, the stock solution was further diluted with acetone to get the application solutions. 1.25 mL of the application solution was added to 5 g sediment and thoroughly mixed, each. After complete evaporation of the solvent, the treated soil was given to an untreated portion of the sediment (5 g sediment dry weight). 5.57 mL M9 medium* was added to obtain a water content of approx. 40 %.
Subsequently, the sediment was thoroughly mixed to ensure a homogenous distribution of the test item. Finally, 0.5 g wet weight sediment was filled into each test well.

*) M9 medium: 7.52 g Na2HPO4 + 2 H2O, 3 g KH2PO4, 5 g NaCl and 0.25 g MgSO4 x 7H2O dissolved in 1000 mL demineralised water and autoclaved.

Control
Moistened test sediment was tested under the same conditions as the test replicates.

Solvent control
Test sediment treated with acetone as described above, but without the test item.

Liquid control
Demineralised water (0.5 mL) without sediment was tested under the same test conditions as the test replicates.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in final test solution: 1.25 mL Acetone per replicate

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wild type strain N2 var Bristol
- Source: CAENORHABDITIS GENETICS CENTER, Minneapolis
- Breeding conditions: Breeding is performed at 20  2 °C at the test facility in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP50.
- 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 nematodes was determined, these nematodes were not used in the test.
- Feeding during test: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. After overnight growth 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.
*) 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

Type of sediment:

natural sediment

Limit test:

no

Duration:

96 h

Exposure phase:

total exposure duration

Test temperature:

The room temperature ranged from 18.5 to 22 °C.

pH:

The initial pH of the sediment at test start was 5.93.

Nominal and measured concentrations:

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

Concentrations of Antimussol ZJ in the application solutions at the start of the test measured via LC-MS/MS analysis were in the range of 105 to 119 % of the nominal test item concentration, indicating the correct preparation of the application solutions.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): NUNC Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g treated sediment wet weight
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: No
-Replacement of evaparated 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. After overnight growth 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.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.

SOURCE OF NATURAL SEDIMENT
Sampling Site:
Germany / Rheinland-Pfalz / Hanhofen

Pesticides:
Sampling year and 4 former years: None

Plants:
2015: Meadow
2014: Meadow
2013: Meadow
2012: Meadow
2011: Meadow

Fertilization:
No organic fertilization
2015: None
2014: 2000 kg/ha CaO;
833 kg/ha MgO
2013: None
2012: None
2011: None

At experimental starting (Day -1) the soil was moistened with M9 medium to a water content of approx. 40 %. The initial sediment moisture was 9.7 %.

Parameters of Natural LUFA soil
Parameter

LUFA-soil 2.2
Batch-No. sp2.22415
pH-value 5.93
Soil moisture before application [%] 9.7
Maximum water capacity* [g/100 g soil dry weight] 43.5 ± 2.8
Particle size distribution acc. to DIN*
Sand:
0.63 – 2.0 mm % 0.5 ± 0.1
0.2 – 0.63 mm % 40.9 ± 1.5
0.063 – 0.2 mm % 33.3 ± 2.7
Silt:
0.02 – 0.063 mm % 8.0 ± 1.3
0.006 – 0.02 mm % 5.2 ± 0.9
0.002 – 0.006 mm % 3.7 ± 0.9
Clay:
< 0.002 mm % 8.3 ± 1.3
Organic carbon content %* 1.59 ± 0.13
Cation exchange capacity [meq/100 g]*
at pH 9.7 ± 0.4
Soil type# loamy sand (IS)

*) data provided by LUFA Speyer
#) acc. to German DIN classification

HANDLING OF NATURAL SEDIMENT
- Storage conditions: The soil was sampled at June 8th, 2015 and stored at greenhouse conditions at the test facility until use.

OTHER TEST CONDITIONS
- Light quality: Continous dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality / fertility of the inserted nematodes were 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 counting the juveniles in subsamples under a microscope in petri dishes with an optical raster. The total number of offspring per replicate was calculated by using the entire volume of extract compounded of 0.5 mL food solution, 0.5 mL Bengal rose solution and 9.5 mL LUDOX solution.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
A preliminary non GLP range finding test was performed with 4 test item concentrations
of 1 – 10 – 100 – 1000 mg/kg sediment dry weight and three replicates each.

Range Finding Test (96 h): Recovery, Mortality and Reproduction
Test Item Concentration
Recovery of adult nematodes Fertility of adult females Reproduction
[mg test item/kg
sediment dry weight] [%] [%] [Offspring
per replicate]
Solvent Control 75 100 170
1 85 100 190
10 85 97.5 185
100 95 97.7 184
1000 77.5 100 78

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

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride (98.3 %)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, fertility, reproduction, growth

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, fertility, reproduction, growth

Details on results:

- Mortality of test animals at end of exposure period:
After four days of exposure to the test item, Antimussol ZJ did not induce biologically significant mortalities.
- No. of offspring produced:
All female nematodes in the controls as well as at the test item concentrations were observed to be fertile. This also applies to the surviving nematodes in the reference item concentrations.
The reproduction per inserted female test organism in sediment was not statistically significant reduced compared to the solvent control at all test item concentrations.
-Growth:
The initial mean size of the introduced juvenile nematodes was 264 µm.
After four days of exposure to the test item, Antimussol ZJ did not affect the nematodes at all test item concentrations. A statistically significant but not dose – related promotion of growth was found at the test item concentration 250 mg test item/kg sediment dry weight.
- Morphological abnormalities:
Not observed
- Behavioural abnormalities:
Not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Effects Reference Item Benzyldimethylhexadecylammonium chloride
[mg/L]
Mortality 10
Fertility > 2
Reproduction 10
Growth 2
LOEC
(Mortality, reproduction) 10
NOEC
(Mortality, reproduction) 2
LOEC
(Growth) 2
NOEC
(Growth) < 2
LC50 (CI)
(Mortality) 3.95 (3.29 – 4.69)
EC50
(Reproduction) 5.37 (4.01 – 7.18)
EC50 (CI)
(Growth) 3.28 (0.15 – 70.8)*
*) The EC50 for growth was below the range of 8 to 22 mg as given in the guideline, which supports the
finding of a comparatively high sensitivity of the nematodes used in the present study.

Reported statistics and error estimates:

LC/EC-values
LC50/EC50-value with confidence interval after 96 hours were not calculated since no reduction was observed at all test item concentrations.
NOEC/LOEC-values and statistical analyses
Differences between the control and the solvent control were
statistically analysed using t-tests. Since the solvent control statistical significantly differed from the control regarding the number of juveniles and reproduction, for these parameters the solvent control was used for further statistics.
Prior to running the test a Normality and an Equal Variance Test were done. P values for both Normality and Equal Variance Test are 0.05. The alpha value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

 Recovery / Mortality after 4 Days of Exposure toAntimussol ZJ

Nominal test item concentration [mg test item/kg sediment dry weight]			[%]Recovered test organisms							Mortality [%]	Significance*
			Replicate					MV	SD
	1	2		3	4	5	6
Control	80	80		90	90	100	90	88.3	7.53	11.7	-
Solvent Control	100	80		80	100	90	80	88.3	9.83	11.7	-
Pooled control	-	-		-	-	-	-	88.3	8.35	11.7	-
62.5	100	100		70	90	60	80	83.3	16.3	16.7	No
125	80	80		60	100	90	80	81.7	13.3	18.3	No
250	80	80		80	100	70	50	76.7	16.3	23.3	No
500	90	80		50	100	90	90	83.3	17.5	16.7	No
1000	100	100		60	70	70	70	78.3	17.2	21.7	No

Male Occurrence after 4 Days of Exposure toAntimussol ZJ

Nominal test item concentration [mg test item/kg sediment dry weight]	[%]Males
	Replicate						MV	SD
	1	2	3	4	5	6
Control	0	0	0	0	10	0	1.67	4.08
Solvent Control	0	0	25.0*	0	11.1	12.5	8.10	10.1
Pooled control	-	-	-	-	-	-	4.89	8.08
62.5	0	20.0	14.3	0	0	0	5.72	9.04
125	0	0	0	0	11.1	0	1.85	4.53
250	12.5	0	12.5	0	0	0	4.17	6.45
500	0	12.5	0	0	0	0	2.08	5.10
1000	0	10.0	0	0	0	0	1.67	4.08

 

Fertility of Female Nematodes after 4 Days of Exposure toAntimussol ZJ

Nominal test item concentration [mg test item/kg sediment dry weight]	[%]Fertility
	Replicate						MV	SD
	1	2	3	4	5	6
Control	100	100	100	100	100	100	100	0
Solvent Control	100	100	100	100	100	100	100	0
Pooled control	-	-	-	-	-	-	100	0
62.5	100	100	100	100	100	100	100	0
125	100	100	100	100	100	100	100	0
250	100	100	100	100	100	100	100	0
500	100	100	100	100	100	100	100	0
1000	100	100	100	100	100	100	100	0

 

* 2 males observed, however, no striking low number of offspring, therefore included in further calculations

Nematode Reproduction (Offspring per Inserted Female Test Organism) after 4 Days of Exposure toAntimussol ZJ

Nominal test item concentration [mg test item/kg sediment dry weight]	Number of offspring per inserted female test organism
	Replicate						MV	SD	Inhibition [%]*
	1	2	3	4	5	6
Control	41.0	37.6	34.0	39.3	49.0	40.5	40.2	4.98	-
Solvent Control	60.2	44.7	78.3	41.6	64.2	50.9	56.7	13.7	-
62.5	55.4	44.6	39.0	75.6	45.8	35.7	49.4	14.5	No
125	51.2	47.0	38.6	69.7	67.4	35.1	51.5	14.4	No
250	41.1	25.0	41.8	56.5	43.5	29.8	39.6	11.1	No
500	53.6	124	28.6	41.2	45.2	34.4	54.5	35.1	No
1000	84.6	133	44.1	48.7	46.4	33.4	65.0	37.6	No

MV = Mean Value        SD = Standard Deviation        *) = Statistically significantly different compared to solvent control

Nematode Growth after 4 Days of Exposure toAntimussol ZJ

Nominal test item concentration [mg test item/kg sediment dry weight]	Mean growth[µm]
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	876	780	792	920	952	1037	893	98.2	-
Solvent Control	1054	1044	968	914	972	943	983	55.6	-
Pooled control	-	-	-	-	-	-	938	89.3	-
62.5	859	888	904	888	888	924	892	21.5	0
125	936	948	936	936	972	972	950	17.7	-6
250	1053	1056	1053	1042	1053	1003	1043	20.3	-17*
500	995	1080	869	917	984	963	968	72.1	-8
1000	936	1069	1064	902	847	984	967	89.1	-8

MV = Mean Value        SD = Standard Deviation        negative value: Increase in growth                -) not determined
*) statistical significance compared to pooled control

Validity criteria fulfilled:

yes

Conclusions:

After four days of exposure to the test item, Antimussol ZJ did not induce biologically significant mortalities. Moreover, nematode fertility, nematode reproduction (offspring per inserted female test organism) and growth were not affected at all test item concentrations. A statistically significant but not dose – related promotion of growth was found at the test item concentration 250 mg test item/kg sediment dry weight.
Overall, the No Observed Effect Concentration (NOEC) for mortality, fertility, reproduction and growth was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of Antimussol ZJ.

Executive summary:

The effects of the test item Antimussol ZJ (batch no. ESD0017844) on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined at Dr.U.Noack-Laboratorien, Sarstedt, Germany, from July 09^th, 2015 to August 03^rd, 2015 with a definitive exposure period from July 24^thto July 28^th, 2015. The study was carried out according to the Guideline ISO 10872 (2010). 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 62.5 – 125 – 250 – 500 – 1000 mg test item/kg sediment dry weight. Six replicates per control, solvent control and test item concentrations were set up.

Concentrations of Antimussol ZJ in the application solutions at the start of the test measured via LC-MS/MS analysis were in the range of 105 to 119 % of the nominal test item concentration, indicating the correct preparation of the application solutions.

After four days of exposure to the test item Antimussol ZJ did not induce biologically significant mortalities. Moreover, nematode fertility, nematode reproduction (offspring per inserted female test organism) and growth were not affected at all test item concentrations. A statistically significant but not dose – related promotion of growth was found at the test item concentration 250 mg test item/kg sediment dry weight.

Overall, the No Observed Effect Concentration (NOEC) for mortality, fertility, reproduction and growth was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of Antimussol ZJ.

Summary of all Effectsand resulting LOEC, NOECbased on Nominal Concentrations

Effects	Antimussol ZJ
	[mg test item/kg sediment dry weight]
Mortality	> 1000
Fertility	> 1000
Reproduction (offspring per inserted female test organism)	> 1000
Growth	> 1000
LOEC (Mortality, Fertility, Reproduction, Growth)	> 1000
NOEC (Mortality, Fertility, Reproduction, Growth)	1000

Description of key information

OECD 225 Lumbriculus

After 28 days of exposure, Antimussol ZJ induced no evident mortality (< 10 %) in all test item concentrations. Since no statistical or biological differences concerning total number of worms/reproduction and total biomass per replicate were determined between the control and the solvent control, both controls were pooled for statistical analyses.

The total number of worms and thus the reproduction were statistically significantly reduced at the  treatment rates 125 mg/kg sediment dry weight and above compared to the pooled control.

Furthermore, compared to the pooled control, 250, 500 and 1000 mg/kg sediment dry weight the total biomass per replicate was statistically significantly reduced.

The EC50-values for total biomass per replicate and total number of worms/reproduction were determined to be 712 and 432 mg/kg sediment dry weight, respectively.

ISO10872 C. elegans

After four days of exposure to the test item, Antimussol ZJ did not induce biologically significant mortalities. Moreover, nematode fertility, nematode reproduction (offspring per inserted female test organism) and growth were not affected at all test item concentrations. A statistically significant but not dose – related promotion of growth was found at the test item concentration 250 mg test item/kg sediment dry weight.

Overall, the No Observed Effect Concentration (NOEC) for mortality, fertility, reproduction and growth was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of Antimussol ZJ.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

62.5 mg/kg sediment dw