Hexadecyltrimethoxysilane

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Data waiving:

other justification

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

biodegradation in water: ready biodegradability

Remarks:

prolonged to 60 d

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jul - 24 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Municipal sewage treatment plant, Hildesheim, Germany
- Pretreatment: Sludge was washed twice with chlorine free tap water. After the second washing settled sludge was resuspended in mineral salts medium and was maintained in an aerobic condition by aeration with CO2 free air until test start (2 d). The amount of inoculum used to initiate inoculation was 3.83 mg/L (25 mg/L dw)
- Initial cell/biomass concentration: Colony Forming Units (CFU) of the inoculum were determined prior to test start by standard dilution plate count. The CFU concentration of the inoculum corresponds to approx. 0.52 x 10^7 CFU/L in the final test solution.
- Water filtered: no

Duration of test (contact time):

60 d

Initial conc.:

20 mg/L

Based on:

test mat.

Initial conc.:

51.2 other: mg O2/L

Based on:

ThOD

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: according to guideline
- Solubilising agent: silicone oil (500 µL in 250 mL test solution, 100 mg test item / 10 mL silicone oil)
- Test temperature: 21 - 21.7 °C
- pH: inoculum control: 7.51 - 7.65, functional control: 7.64 - 7.68, inoculum control with silicone oil: 7.51 - 7.73, test item: 7.5 - 7.71, toxicity control: 7.64 - 7.73
- pH adjusted: no
- Continuous darkness: yes
- Other: continuous stirring

TEST SYSTEM
- Culturing apparatus: 500 mL brown glass bottles filled with 250 mL
- Number of culture flasks/concentration: 2
- Measuring equipment: OxiTop measuring heads
- Test performed in open system: no
- Details of trap for CO2 and volatile organics if used: A rubber sleeve with soda lime was hung into the opening of the bottles to absorb evolved CO2.

SAMPLING
- Sampling frequency and method: The oxygen consumption was determined in the incubation vessels by the OxiTop measuring system at 360 measuring points (every 240 min) during the 60 d incubation period.

CONTROL AND BLANK SYSTEM
- Inoculum control: 2 replicates (test medium without test and / or reference item)
- Inoculum control with silicone oil: 2 replicates (test medium without test and / or reference item and 500 µL silicone oil)
- Toxicity control: 1 replica (test item (incl. 500 µL silicone oil) and reference item)
- Functional control: 1 replica
- Functional control with silicone oil: 1 replica

Reference substance:

benzoic acid, sodium salt

Remarks:

30 mg/L

Reference substance:

benzoic acid, sodium salt

Remarks:

30 mg/L sodium benzoate and 500 µL silicone oil / 250 mL test solution

Parameter:

% degradation (O2 consumption)

Value:

65

Sampling time:

28 d

Parameter:

% degradation (O2 consumption)

Value:

81

Sampling time:

60 d

Details on results:

- The biodegradation of the substance failed the 10 - d window criterion.
- O2 depletion in inoculum control with silicon oil: 28.6 mg O2/L on Day 28 and 33.5 mg/L on Day 60.
- Toxicity control: 47% degradation of test item after 14 d and 86% after 60 d.

Results with reference substance:

- Functional control: 86% biodegradation of sodium benzoate on Day 14.
- Functional control with silicone oil: 98% biodegradation on Day 60.

Table 1: % Biodegradation of test item, functional control and toxicity control during 60 d.

[d]	% Biodegradation
	Functional Control	Functional control with silicon oil	Test Item 1	Test Item 2	Tox. Control
2	30	30	3	1	14
4	64	66	3	3	33
6	75	78	3	3	39
8	83	86	4	4	43
10	84	90	9	9	45
12	85	90	23	20	46
14	86	90	36	34	47
16	81	90	40	41	48
18	78	93	46	45	55
20	77	94	51	52	62
22	78	94	56	57	67
24	78	93	59	60	70
26	78	94	62	64	72
28	79	94	64	66	74
30	80	95	65	68	76
32	80	94	67	70	78
34	80	96	67	72	79
36	79	96	70	74	80
38	77	97	70	74	80
40	80	95	71	76	80
42	81	94	72	78	81
44	80	96	73	79	82
46	82	97	75	79	83
48	82	96	75	80	83
50	81	96	75	80	83
52	80	97	76	80	84
54	80	97	76	81	84
56	78	97	76	81	84
58	79	97	78	81	85
60	77	98	79	83	86

Table 2: Validity criteria for OECD 301 F.

Criterion from the guideline	Outcome	Validity criterion fulfilled
Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.	4%	yes
Percentage degradation of the reference compound reached the pass level by day 14 (≥ 60%).	Functional control: 86% Functional control with silicone oil: 90%	yes
The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.	47%	yes
The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg/L in 28 days.	Functional control: 32% Functional control with silicone oil: 28.6%	yes
the pH value should be in the range 6-8.5.	7.47 – 7.64	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Interpretation of results:

readily biodegradable, but failing 10-day window

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

adsorption / desorption, other

Remarks:

adsorption

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Principles of method if other than guideline:

QSARs for soil and sediment sorption for different chemical classes (Sabljic and Güsten, 1995)

GLP compliance:

no

Type of method:

other: calculation

Media:

soil

Type:

Koc

Value:

170 608.2 L/kg

Remarks on result:

other: predicted using the TGD (Technical Guidance Document) non-hydrophobics log Kow prediction method

Type:

log Koc

Value:

5.232 dimensionless

Remarks on result:

other: predicted using the TGD (Technical Guidance Document) non-hydrophobics log Kow prediction method

Conclusions:

A log Koc of 5.232 was predicted using the TGD (Technical Guidance Document) non-hydrophobics log Kow prediction method.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb - 08 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

yes

Remarks:

LC-MS/MS and TOC

Details on sampling:

- Concentrations: Control, 10 mg/L (nominal)
- Sampling for determination of the test item: at least once within 7 d in fresh media at the start of an exposure interval and in old media at the end of an exposure interval (48 or 72 h). Furthermore once during the test, samples of the saturated solution and the control taken during the preparation phase of the saturated solution and during the exposure phase (i.e. at 24, 20, 0 and 48 h) were analyzed via LC-MS/MS and also via TOC-analysis to demonstrate the establishment of an equilibrium of the test item concentration.
- Sampling for the analytical monitoring: during the preparation of the saturated solution and at the start of an exposure interval, samples of the fresh media were taken at 24 h (after addition of the test item and a few minutes of stirring), at 20 h (approx. 4 h after the start of the stirring period during the preparation of the saturated solution) and at 0 h (after preparation of the saturated solution). At the end of an exposure interval (48 or 72 h), samples for analyses were taken from the test vessels. For analysis of the TOC, separate replicates of the saturated solution and the control were prepared without daphnids and food algae and analyzed. For the longest exposure interval of 72 h, samples were taken at the start (0 h) and at the end of the exposure interval (72 h) once within the test period.
- Sample storage conditions before analysis: all samples were stored at room temperature until sample preparation and in an autosampler until start of analysis, if necessary

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the solution was prepared with a nominal concentration of 10 mg/L in Elendt M4 medium one day prior to the start of each exposure interval. 22.48 µL test item were slowly added by pipette to 2L Elendt M4 medium. The mixture was stirred slowly for 24 h with a magnetic stirrer at room temperature. Subsequently the solution was allowed to stand for 1 h for separation of the phases and then the saturated solution was removed from the center of the aqueous phase. Once during the test the establishment of an equilibrium of the test item concentration was shown by analysis during the preparation phase (0 and 4 h after stirring) and during the exposure phase (0 and 48 h).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: clone 5
- Source: daphnids were bred at the test facility (origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany)
- Culturing conditions: in glass vessels (2 - 3 L) with approx. 1.8 L medium (Elendt M4), at approx. 20 °C, in an incubator, 16 h illumination; light intensity of max. 1500 Lux. Daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae with an algae cell density of > 10^6 cells/mL
- Age of daphnids at test start: < 24 h. Juvenile daphnids were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within this period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
- Feeding during test: yes
- Food type and amount: Pseudokirchneriella subcapitata (1.06 - 1.40 mL) and Desmodesmus subspicatus (0.623 -0.786 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. There was variation according to the density of the algae suspension, but it was the same for all test groups on each feeding day.
- Frequency: daily

ACCLIMATION
- Acclimation period: non necessary since culture medium same as test medium

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

158 - 172 mg CaCO3/L

Test temperature:

19.8 - 20.4 °C (water temperature)

pH:

7.06 - 8.45

Dissolved oxygen:

7.63 - 9.64 mg/L

Nominal and measured concentrations:

Nominal loading rate: 10 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): beakers were loosely covered with watch glasses
- Fill volume: 50 mL
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): three times per week
- No. of organisms per vessel: 10 (1 daphnid per replicate was used in the control)
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4, according to OECD 211
- Culture medium different from test medium: no
- Intervals of water quality measurement: once within 7 d, in fresh media at the start of an exposure interval (0 h) and in old media at the end of an exposure interval (48 or 72 h), in one replicate of the control and the saturated solution. The water quality parameters in fresh media were measured in an additional replicate without daphnids of the saturated solution and the control. At the end of an exposure interval, the water quality parameters of the old media were measured in a test vessel of the saturated solution and the control, which contained daphnids and food algae. The temperature in the incubator was recorded throughout the test.

OTHER TEST CONDITIONS
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: 1500 Lux

EFFECT PARAMETERS MEASURED: immobile daphnids were observed and recorded once per day and offspring was counted. Dead specimens were removed. The neonates were removed after counting and before addition of algae. The number of aborted eggs or dead offspring was recorded. Abnormalities (e.g. swimming behavior, number of males and winter eggs) were observed and recorded daily. At the end of the test, the total length excluding the anal spine of each survived parental daphnid and the mean dry weight of the survived parental daphnids of the saturated solution and the control were determined (these information were not used for determination of a NOEC). The time to first brood, the intrinsic rate of population increase and the number and size of first brood per animal were reported, but not used for endpoint calculations.

TEST CONCENTRATIONS
- Range finding study: yes (static conditions, 48 h, 2 replicates)
- Test concentrations: control, 2 mg/L
- Results used to determine the conditions for the definitive study: no mortalities were observed in the range finding test

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Observations on body length and weight: the mean values of the body length (excluding the anal spine) of the parental daphnids in the saturated solution were 4.20 mm per daphnid and 4.50 mm per daphnid in the control group. The mean values of the dry weight of the parental daphnids were 0.80 mg per daphnid in the saturated solution and 0.64 mg per daphnid in the control.
- Other biological observations: stillborn juveniles and aborted eggs produced by the parental daphnids after 21 days was 0. No males were observed in the control or in the test groups during the test. No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 2 mg/L (95% CI: 1.8 - 2.28 mg/L)

Reported statistics and error estimates:

Since no mortality appeared in this study, statistical evaluation for the adult mortality was not necessary. Further information on statistics can be found in the field any other information on results incl. tables.

ANALYTICAL RESULTS

The measured concentrations of the test item determined via LC-MS/MS during the preparation phase of the saturated solution and during the exposure were all below the LOQ (152 µg/L) of the analytical method. The measured TOC concentrations were around the LOQ of 2 mg C/L in all analyzed samples. This indicates that the test item is hardly soluble as well as completely hydrolyzed during the exposure. Since the test item concentrations could not be quantified, the nominal concentration of the test item was used for the evaluation of the NOEC and LOEC.

Table 1: Measured TOC Concentrations during the Definitive Test

Sampling date	Day 0 Fresh media (0 h)	Day 2 Old media (48 h)	Day 9 Fresh media (0 h)	Day 12 Old media (72 h)	Day 13 Preparation of the saturated solution (0 h)	Day 13 Preparation of the saturated solution (after 4 h stirring)	Day 14 Fresh media (0 h)	Day 16 Old media (48 h)
Nominal test item concentration [mg/L]	Total Organic Carbon (TOC)
	Measured concentration [mgC/L]
10.0 (saturated solution)	2.70	< 2.00	< 2.00	< 2.00	< 2.00	3.65	< 2.00	< 2.00
Control	2.66	< 2.00	3.02	< 2.00	< 2.00	< 2.00	< 2.00	< 2.00

BIOLOGICAL RESULTS

Table 2: Effects on Reproduction for all parental Daphnids

Nominal test item concentration	Mean number of offspring per survived / introduced parental daphnid
[mg/L]	Mean	SD	CV
10.0	82.7	9.89	12.0
Control	93.7	13.6	14.5

Table 3: First Appearance of Living Juveniles and Mean Number of Broods in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles at the parental daphnid in replicate no.										First
											appearance
	1	2	3	4	5	6	7	8	9	10	mean day
10.0 (saturated solution)	8	8	8	9	8	9	9	9	8	8	8.4
Control	9	9	8	9	9	9	9	9	8	8	8.7

STATISTICAL ANALYSIS

No statistically significant difference of the reproductive output in comparison to the reproductive output in the control was determined at the saturated solution.

Adult mortality was not observed in this study. Therefore, no statistical evaluation was carried out for this parameter.

Number of living juveniles per survived parental Daphnid:

- Shapiro-Wilk's test on normal distribution: normality check was passed (p > 0.01).

- Levene's Test on variance homogeneity (with Residuals): the Levene test indicates variance homogeneity (p <= 0.010). Variance homogeneity check was passed (p > 0.01). Normal-distribution and variance-homogeneity requirements are fulfilled.

- Two-sample Welch-t-test Procedure (two sided test) with cumulative offspring per survived parent at 21 d: two-sample comparison of treatments with "Control". Significance was Alpha = 0.050, two-sided; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha (Control(c) and treatment(t) variance was applied: s^2(c)/nc + s^2(t)/nt, each).

Table 4: Two-sample Welch-t-test Procedure

Treatm. [mg/L]	mean	s	df	%MDD	t	t*	sign.
control	93.7	13.58
10	82.7	9.89	16	12	-2.07	0.55	non-sign.

VALIDITY CRITERIA

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test.	% mortality of the Adult Daphnids after 7, 14 and 21 days of exposure was 0.	yes
The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60.	The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93.7 in the control group	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables".

Conclusions:

The study conducted according to the OECD guideline 211 and GLP did not show any long term effects to the test organism Daphnia magna resulting in a NOELR (21 d) >= 10 mg/L (nominal).

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

water solubility

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction

Principles of method if other than guideline:

The result was obtained using an appropriate QSAR method (see QMRF and QPRF for details).

Water solubility:

0.002 mg/L

Temp.:

20 °C

pH:

7

Estimated value, no data available for pH.

Conclusions:

Interpretation of results: insoluble (< 0.1 mg/L)
A solubility of 2.0E-3 mg/l at 20°C was predicted for the substance based on an appropriate calculation method. The result is considered to be reliable.

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-03 to 1991-06-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study followed a protocol based on the OECD 201 (1984) guideline. It should be noted, that only two parallels were investigated for both control and test series. Thus no thorough statistical assessment of effect values is possible. The study duration was 96 hours which is not in accordance with OECD standards. Thus a recalculation of effect values for 72 h has been performed in order to meet CLP and OECD standards.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

yes

Remarks:

Methanol and tertiary butanol (TBA) were used as solvents to aid test substance dispersion, test duration was 96 hours, only two parallels for both control and test series, respectively.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Two stock solutions (nominally 300 g/L) were prepared in methanol and tertiary butanol (TBA). The stock solutions were added to the algal culture medium to produce the desired test concentrations.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): It is likely that all exposure concentrations exceeded the solubility of the test substance in the test medium.
- Controls: Algal culture medium

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM

- Strain: SAG 86.81

- Source: Culture supplied by the Collection of Algal Cultures, Institute of Plant Physiology, University of Göttingen, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

no data

Test temperature:

23+/-1ºC

pH:

Medium prepared using methanol stock solution: 8.3-8.9 over 3 days, 8.8-10.5 over 4 days.

Medium prepared using TBA stock solution: 8.4-8.7 over 3 days, 8.4-9.9 over 4 days.

Dissolved oxygen:

no data

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations in media prepared with methanol stock solution: 0(Control), 0.01, 0.032, 0.10, 0.32, 1.0, 3.2, 10.2 and 31.8 mg/L.
Nominal concentrations in media prepared with TBA stock solution: 0(Control), 0.009, 0.029, 0.094, 0.29, 0.94, 2.9, 9.4 and 29.5 mg/L.
It is likely that all exposure concentrations exceeded the solubility of the test substance in the test medium.

Details on test conditions:

TEST SYSTEM

- Test vessel: Conical flasks

- Type: closed with sponge cap

- Material, size, fill volume: glass, 200 mL containing 100 mL of test medium

- Aeration: no

- Initial cells density: 12000 cells/mL (methanol), 10000 cells/mL (TBA)

- Control end cells density: 2200000 cells/mL (methanol), 3000000 cells/mL (TBA)

- No. of vessels per concentration (replicates): 2

- No. of vessels per control (replicates): 2


GROWTH MEDIUM

- Standard medium used: yes except that the medium contained 150 mg/l of NaHCO3 instead of the 50 mg/l specified in the OECD guideline.

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Milli-Q filtered water

- Culture medium different from test medium: no

- Intervals of water quality measurement: start and end of test


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: continuous

- Light intensity and quality: 60-120 μm/s/m2


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell density at 0, 24, 48, 72 and 96 hours (approx)

- Determination of cell concentrations: Coulter Counter Model TAII


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3.2 approx

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 31.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: methanol

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 29.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: TBA

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

>= 31.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: methanol

Remarks:

values calculated in retrospect from original cell numbers

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

20.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solvent: TBA

Remarks:

values calculated in retrospect from original cell numbers

Details on results:

- Exponential growth in the control (for algal test): yes

- Any stimulation of growth found in any treatment: some evidence of stimulation at concentrations >/=0.3 mg/L but it is not certain if it is significant.

- Effect concentrations exceeding solubility of substance in test medium: It is likely that all exposure concentrations exceeded the water solubility of the test substance i(0.0029 mg/L).

Reported statistics and error estimates:

The area under the growth curve (biomass) and growth rate parameters were calculated from the cell concentration data in accordance with the description of the test method.

Table 1. Cell concentrations [cells/mL] when methanol was used as a solvent

Conc. [mg/L]		10^4 cells/mL corrected for background
	Replicates	0 d	1 d	2 d	3 d	4 d
Control	1	1.5	2.0	11.9	79.2	231.2
	2	1	1.9	11.0	74.1	233.5
Solv. Control	1	1.2	2.1	10.5	76.7	205.9
	2	1	1.9	11.5	76.4	232.6
0.01	1	1.6	2.2	10.9	81.2	240.2
	2	1	2.0	12.7	81.2	233.5
0.032	1	1.4	2.1	11.1	76.8	244.1
	2	1	2.0	11.4	87.5	224.0
0.1	1	0.9	1.9	11.1	73.8	222.3
	2	0.8	2.2	12.3	95.4	286.6
0.32	1	1.1	1.9	11.5	77.1	218.3
	2	0.9	2.1	10.3	86.1	233.2
1	1	0.8	1.8	10.3	67.4	214.4
	2	0.9	2.1	9.4	69.1	219.9
3.2	1	0.9	2.3	9.8	57.8	199.4
	2	0.9	2.2	9.1	56.6	168.7
10.2	1	0.8	1.7	7.8	46.8	155.8
	2	1.1	1.7	9.1	44.5	131.8
31.8	1	0.8	9.1	6.4	45.3	170.1
	2	0.9	-0.9	2.3	49.5	173.5

 red value: was excluded from the calculation due to negative cell number

 

Table 2.Cell concentrations [cells/mL] when TBA was used as a solvent

Conc. [mg/L]		10^4 cells/mL corrected for background
	Replicates	0 d	1 d	2 d	3 d	4 d
Control	1	1	3.1	14.5	109.1	297.3
	2	1	3.1	13.2	97.5	291.6
Solv. control	1	1.1	3.1	14.4	108.5	319.4
	2	1	2.7	14.1	91.2	308.6
0.009	1	1	3.2	13.3	102.3	305.5
	2	1	2.6	14.9	103.1	277.1
0.029	1	1	3.1	12.6	101.2	317.8
	2	1	3.3	16.8	105.2	324.6
0.094	1	0.8	2.9	14.7	105.5	294.5
	2	0.9	3.3	16.3	115.1	316.1
0.29	1	1	2.7	15.2	104.7	259.7
	2	1	3.3	15.1	115.4	287.2
0.94	1	1	2.5	13.9	79.9	262.3
	2	1	3.1	14.4	107.9	329.6
2.9	1	1	2.8	12.9	83.8	287.1
	2	1	2.6	10.3	68.9	262.0
9.4	1	0.9	3.3	10.0	70.2	211.9
	2	0.9	3.2	10.1	60.5	192.5
29.5	1	1.1	2.6	8.7	62.1	176.3
	2	1.2	6.4	9.6	57.9	167.2

 

96 h -EC10 values mentioned in the report were 11 mg/L when TBA was used as a solvent and 7 mg/L when methanol was used as a solvent. No effects causing 50% inhibition of the growth rate were observed. The EC values were calculated by means of a parametric model developed by Kooijman et al. 1983.

Re-evaluation of data:

The 72 -h effect concentrations were calculated in retrospect according to the current OECD 201 (2011) guideline and the original cell numbers given in the report.

No differences were observed between the control and the solvent control. However since only duplicates were used, no statistical analysis could be performed. The solvent control was used as a worst case for the calculation of the growth rate inhibition rates.

1) Results on growth rate inhibition when methanol was used as a solvent:

Table 3: % Inhibition of growth rate at 72 h when methanol was used as a solvent, as calculated in retrospect according to the current OECD 201 standards using the orginal cell densities from the study report.

Nominal concentrations [mg/L]	% Growth Rate Inhibition at 72 h
0.01	1.94
0.032	0.18
0.1	-8.23
0.32	-3.78
1	-3.36
3.2	2.23
10.2	8.51
31.8	5.26

No growth rate inhibition up to 10% was observed after 72 h when methanol was used as a solvent resulting in an ErC10 (72 h) >= 31.8 mg/L.

2) Results on growth rate inhibition when TBA was used as a solvent:

Table 4: % Inhibition of growth rate at 72 h when TBA was used as a solvent, as calculated in retrospect according to the current OECD 201 standards using the original cell densities from the study report.

Nominal Concentrations [mg/L]	% Grwoth Rate Inhibition at 72 h
0.009	-1.75
0.029	-1.88
0.094	-6.89
0.29	-3.26
0.94	0.49
2.9	4.84
9.4	5.9
29.5	13.08

The EC10 value was calculated based on the dose-response curve. The ErC10 (72 h) was 20.914 mg/L (nominal) when TBA was used as a solvent.

No growth rate inhibition up to 50% was observed after 72 h of exposure when TBA was used as a solvent resulting in an ErC50 (72 h) > 29.5 mg/L (nominal).

Validity criteria fulfilled:

yes

Remarks:

The validity criteria outlined in the OECD guideline 201 version of 1984 are fulfilled

Conclusions:

The study resulted in an ErL50 (72 h) and ErL10 (72 h) of > 30.8 mg/L when methanol was used as a solvent. The ErL50 (72 h) and ErL10 (72 h) when TBA was used as a solvent were > 29.5 mg/L and 20.91 mg/L (nominal), respectively. The 72 h effect values were not presented in the report and therefore the calculations were made in retrospect based on the current OECD guideline 201 calculation criteria and the original cell numbers given in the study report.

Cross-reference 6

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sediment toxicity: long-term

Data waiving:

other justification

Justification for data waiving:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

Test organisms (species):

Chironomus sp.

Study type:

laboratory study

Water media type:

freshwater

Cross-reference 7

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jul - 31 Aug 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2016

Deviations:

yes

Remarks:

Food was provided on day 0 instead of day 1 due to good experience with this procedure. The room temperature increased to maximum 23 °C for about 8 hours on day 55. These deviations are considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil: respective test item amount was weighed out for each test item concentration, solved in Acetone and mixed with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil.
- Controls: artificial soil moistened with demineralised water without test or reference item
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium: 2.5 mL per replicate
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: breeding stock culture maintained at the test facility
- Age at test initiation: 2-12 (age did not deviate by more than 1 month)
- Weight at test initiation: 0.46 - 0.47 g (wet mass)

ACCLIMATION
- Acclimation period: 2 days prior to test start

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 - 23°C

pH:

6.11 - 6.44

Moisture:

19.4 - 24.4%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round 2 L plastic boxes (diameter: 15 cm, bottom surface: 177 cm2, height: 14 cm) with transparent and perforated lids
- Amount of soil or substrate: 600 g dw (5 cm height)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 5 % peat (air-dried and finely ground), 20 % kaolin (kaolinite content > 30 %), 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm), 0.20 % calcium carbonate (CaCO3)

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h light:dark
- Light intensity: 612 ± 77.2 lux

EFFECT PARAMETERS MEASURED: body weight of the adult earthworms (day 0, 28), adult earthworm mortality (day 28), the number of unhatched cocoons and number of offspring (juveniles) hatched from the cocoons (day 56)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0, 1, 10, 100, 1000 mg/kg dw
- Results used to determine the conditions for the definitive study: No mortality observed at any test concentration after 14 days.

Nominal and measured concentrations:

Nominal concentration: 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg dw

Reference substance (positive control):

yes

Remarks:

Carbendazim (0.5, 1, 2, 4 mg/kg dw)

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

biomass

Details on results:

- Mortality at end of exposure period: No significant effects observed in the controls and all test item concentrations on day 28
- Total mass of adults at beginning of test: 0.46 - 0.47 g (wet mass)
- Changes in body weight of live adults at end of exposure period: at the test item concentration 1000 mg/k dw the earthworm biomass was statistically significantly reduced
- No. of offspring produced: at the test item concentration 1000 mg/k dw earthworm reproduction was statistically significantly reduced
- Behavioural abnormalities: No significant effects observed in the controls and all test item concentrations on day 28

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: NOEC: 0.5 mg/kg dw, LOEC 1 mg/kg dw

Reported statistics and error estimates:

- Adult Mortality: Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response), Chi2 2x2 Table Test with Bonferroni Correction
- Reproduction and Biomass: Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity (with Residuals), Trend Analysis by Contrasts (Monotonicity of Concentration/Response), William’s Multiple Sequential t-test Procedure
- The α-value (acceptable probability of incorrectly concluding that there is a difference) was α = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and controls were calculated.

Table 1: Validity criteria for OECD 222.

Criterion from the guideline to be satisfied in the controls	Outcome	Validity criterion fulfilled
Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.	≥ 30	yes
The coefficient of variation of reproduction is ≤ 30%.	< 30 %	yes
Adult mortality over the initial 4 weeks of the test is ≤ 10%.	≤10 %	yes

Table 2: Body weight changes

Application rate [mgtest item/kg SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.46	0.51	0.05	10.9	0.06 ± 0.02	11.8 ± 4.13	–
	2	0.47	0.55	0.08	17.0
	3	0.46	0.49	0.03	6.5
	4	0.47	0.53	0.06	12.8
	5	0.46	0.51	0.05	10.9
	6	0.47	0.53	0.06	12.8
	7	0.46	0.54	0.08	17.4
	8	0.47	0.50	0.03	6.4
Solvent Control	1	0.46	0.50	0.04	8.7	0.05 ± 0.02	10.8 ± 3.26	–
	2	0.47	0.52	0.05	10.6
	3	0.46	0.50	0.04	8.7
	4	0.47	0.50	0.03	6.4
	5	0.46	0.52	0.06	13.0
	6	0.47	0.54	0.07	14.9
	7	0.46	0.53	0.07	15.2
	8	0.47	0.51	0.04	8.5
Pooled Control						0.05 ± 0.02	11.3 ± 3.64	-
62.5	1	0.46	0.51	0.05	10.9	0.06 ± 0.01	11.8 ± 2.65	No
	2	0.47	0.54	0.07	14.9
	3	0.46	0.50	0.04	8.7
	4	0.47	0.53	0.06	12.8
125	1	0.46	0.54	0.08	17.4	0.07 ± 0.01	15.1 ± 3.21	No
	2	0.47	0.54	0.07	14.9
	3	0.46	0.54	0.08	17.4
	4	0.47	0.52	0.05	10.6
250	1	0.46	0.52	0.06	13.0	0.07 ± 0.03	15.5 ± 5.75	No
	2	0.47	0.56	0.09	19.1
	3	0.46	0.50	0.04	8.7
	4	0.47	0.57	0.10	21.3
500	1	0.46	0.48	0.02	4.3	0.06 ± 0.03	12.3 ± 5.35	No
	2	0.47	0.54	0.07	14.9
	3	0.46	0.53	0.07	15.2
	4	0.47	0.54	0.07	14.9
1000	1	0.46	0.48	0.02	4.3	0.03 ± 0.02	6.93 ± 3.15	Yes
	2	0.47	0.52	0.05	10.6
	3	0.46	0.48	0.02	4.3
	4	0.47	0.51	0.04	8.5

Table 3: Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil dry weight]	Replicate	Number of juveniles	Mean±SD	CV              [%]	[%] of pooled control	Sig.
Control	1	94	117 ± 16.9	14.5	94.4	-
	2	137
	3	115
	4	127
	5	102
	6	141
	7	106
	8	110
Solvent Control	1	188	131 ± 28.5	21.8	106	-
	2	111
	3	158
	4	102
	5	135
	6	122
	7	118
	8	117
Pooled Control			124 ± 23.9	19.3	-	-
62.5	1	127	108 ± 34.3	31.8	87.1	No
	2	70
	3	145
	4	89
125	1	100	119 ± 25.4	21.4	96.0	No
	2	115
	3	105
	4	156
250	1	126	135 ± 22.8	16.9	109	No
	2	114
	3	167
	4	132
500	1	110	121 ± 25.2	20.8	97.6	No
	2	122
	3	155
	4	96
1000	1	73	80 ± 19.3	24.1	64.5	Yes
	2	68
	3	71
	4	109

Table 4: NOEC, LOEC and EC-values

Endpoint	[mg test item/kg SDW]
LOECmortality	> 1000
LOECbiomass	1000
LOECreproduction	1000
NOECmortality	1000
NOECbiomass	500
NOECreproduction	500
EC-valuesmortality, biomass (95 % Confidence Interval)	EC10, 20, 50: > 1000 (n.d.)
EC-valuesreproduction (95 % Confidence Interval)	EC10: 820 (753– 964) EC20: 907 (831 to n.d.) EC50: > 1000 (973 to n.d.)

n.d. = not determined

Validity criteria fulfilled:

yes

Remarks:

For details please refer to “Any other information on results incl. tables”.

Executive summary:

Effects of test substance concentrations between 62.5 and 1000 mg/ kg dw on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016). All validity criteria recommended by the test guidelines were fulfilled. The NOEC for mortality was determined to be 1000 mg/kg dw. The derived NOEC for biomass and reproduction was 500 mg/kg dw.

Cross-reference 8

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 - 21 Aug 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): respective test item amounts were weighed out for each test item concentration, dissolved in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After evaporation of the solvent, spiked quartz sand was added to the artificial soil (2485 g). Humidity of the soil to a moisture of 54 % of the WHC max was adjusted by demineralised water (321 g). Then test medium was mixed thoroughly to ensure a homogenous distribution, and about 555 g SDW were filled into each test vessel.
- Other: soil moisture content and the max water holding capacity were determined prior to experimental starting

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: own breeding, kept in covered plastic vessels containing potting compost
- Age at test initiation (mean and range, SD): adult earthworms
- Weight at test initiation (mean and range, SD): 0. 46 ± 0.06 to 0.47 ± 0.07 g/worm
- Feeding during breeding: litter of dried stinging nettle leaves and porridge oats. The amount of food is portioned due to the feeding rate and due to the density of the earthworm population in the vessels.
- Pretreatment: earthworms were washed in demineralised water, dabbed dry on a paper towel, weighed individually and placed onto the surface of the test medium

ACCLIMATION
- Acclimation period: 2 d
- Acclimation conditions (same as test or not): in artificial soil

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

18.5 - 22 °C

pH:

5.39 - 5.94

Moisture:

24.1 - 27.4%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass dishes, 1.5 L volume (during the test they were covered with perforated plastic film to prevent the test medium from drying)
- Amount of soil or substrate: 555 g SDW/vessel
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per solvent control: 4 (artifical soil with addition of quartz sand spiked with acetone and moistened with demineralised water without test item)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
-Peat, air-dried and finely ground: 10 %
- Kaolin, kaolinite content > 30 %: 20 %
- Air-dried quartz sand (sand with > 50 % particles size of 0.05 to 0.2 mm): 69%
- Calcium carbonate (CaCO3) to achieve a pH of 6.0 ± 0.5: 0.40 %
- Maximum water holding capacity (in % dry weigth): 52.3 g/100 g SDW

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: 445 ± 30 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): pH-value and moisture content were analysed in all vessels, the control and solvent control using mixed samples of all replicates, at the start and end of the test. Live weight of earthworms was determined individually on the day of application (day 0) and on day 14. The mean weight change was assessed at day 14 after application. Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 d after application.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: control, solvent control, 1, 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: no mortalities were observed up to the highest test concentration. 4 concentrations were chosen based on these results for the definitive test.

Nominal and measured concentrations:

0 (control), 62.5, 125, 250, 500 and 1000 mg product/kg SDW (nominal)

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: LC50 28.2 mg/kg SDW

Reported statistics and error estimates:

The NOEC/LOEC was determined by calculation of statistical significance of mortality and biomass. Dunnett's Multiple t-Test. Procedure was carried out for the determination of statistically significant differences compared to the control. When running the test, a Normality Test and an Equal Variance Test were done first. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

No LCx-values were determined, since there were no effects after 14 days of exposure.

No significant pathological symptoms and no changes in the earthworm behaviour were observed in the control as well as in the test item concentrations after 7 and 14 d of exposure.

During the test the biomass loss (inhibition) of the earthworms was below 20% in the control.

Validity criteria fulfilled:

yes

Conclusions:

A 14-d NOEC value of >= 1000 mg/kg SDW and a NOEL of >1000 mg/kg SDW have been determined for the effects of the test substance on mortality of Eisenia fetida, based on nominal concentrations of the test substance.

Cross-reference 9

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to terrestrial plants: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Aug - 17 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 208 (Terrestrial Plants, Growth Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatliches Gewerbeaufsichtsamt Hildesheim, Germany

Analytical monitoring:

no

Vehicle:

no

Species:

Allium cepa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Onion
- Plant family: Amaryllideaceae
- Variety: Exhibition
- Lot: 1300968
- Source of seed: Hild Samen GmbH, Marbach, Germany
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

- Common name: Oats
- Plant family: Poaceae
- Variety: KWS Contender
- Lot: F17-BRA-CON-3
- Source of seed: KWS Lochow GmbH, Bergen, Germany
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Sugar beet
- Plant family: Amaranthaceae
- Variety: Fiorella KWS
- Source of seed: KWS Saat SE, Einbeck, Germany
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Species:

Brassica napus

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Rape
- Plant family: Brassicaceae
- Variety: Sherlock
- Source of seed: KWS Saat SE, Einbeck, Germany
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Species:

Lactuca sativa

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Lettuce
- Plant family: Asteraceae
- Variety: Larissa
- Lot: 16193.011
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Species:

Glycine max (G. soja)

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Soybean
- Plant family: Fabaceae
- Variety: Obelix
- Lot: DSP-96-8-1-18295-5
- Prior seed treatment/sterilization: untreated seed
- Seed storage: stored dark at the test facility at room temperature (22 ± 10 °C) and protected from moisture until use

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

21 d

Remarks:

The test for onion was prolonged until day 28.

Test temperature:

17.5 - 24.5°C

pH:

4.86 ± 0.08

Moisture:

35.8 - 92.5% relative humidity

Details on test conditions:

TEST SYSTEM
- Testing facility: climatic chamber
- Test container: non-porous plastic containers (standard flower pots), diameter: ca. 12 cm
- Amount of soil: 7.6 kg
- Method of seeding: seeding holes made with a seedling pistil, one seed was given into each hole and covered with soil
- No. of seeds per container: 5
- No. of replicates per treatment group: 8
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil): 2:1 mixture of natural soil LUFA 2.2 (batch number: Sp2.22419, loamy sand (DIN classification)) and quartz sand (12a)
- Geographic location: LUFA: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Germany; Dörentrup Quarz GmbH Co. KG,Duingen, Germany
- Soil texture (if natural soil)
- % sand: 93.4% (0.063-2 mm)
- % silt: 3.0% (0.002-0.063 mm)
- % clay: 3.7% (< 0.002 mm)
- Grain size: ≤2 mm
- Maximum water holding capacity: 33.2 g/100 g soil dw


NUTRIENT MEDIUM (if used)
- Description: HAKAPHOS® SOFT SPEZIAL 16+8+22(+3)

GROWTH CONDITIONS
- Photoperiod: 16/8 h light/dark
- Light source: tubes (special light source for plants)
- Light intensity and quality: 4713 ± 453, light at the blue and red ends of the spectrum, Grünes Landhaus, Hildesheim, Germany (0.5 ‰ solution for watering)
- Relative humidity: 35.8 - 92.5%
- Watering regime and schedules: daily bottom watering
- Water source/type: fertilized tap water
- Interval of applications: control and test item treatments were applied once at the start of the exposure
- Method of application: test item amounts were weighed out on quartz sand for each test item concentration, blended thoroughly and added to the soil with an appropriate amount of demineralized water; after mixing, treated soil transfer to test containers

EFFECT PARAMETERS MEASURED: number of emerged seedlings, number of dead plants and visual phytotoxic effects (day 7, 14 and 21, onion additionally on day 28), shoot heights and the fresh weights of the shoots (test end)

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 62.5, 125, 250, 500, 1000 mg/kg dw

Reference substance (positive control):

no

Species:

other: Lactuca sativa, Avena sativa

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot fresh weight

Species:

other: Avena sativa, Beta vulgaris, Brassica napus, Lactuca sativa and Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot height, number of emerged seedlings and survival

Species:

other: Beta vulgaris, Brassica napus and Glycine max (G. soja)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot fresh weight

Species:

Lactuca sativa

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

961 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot fresh weight

Remarks on result:

other: 95% confidence limits: 583 - > 1000 mg/kg soil dw

Species:

Allium cepa

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: shoot height, number of emerged seedlings and survival, shoot fresh weight

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) and Dunnett’s test for the determination of statistically significant differences compared to control replicates. Normality test (Shapiro-Wilk) and an Equal Variance test (Brown-Forsythe) were done first. P-values for both, Normality and Equal Variance test, are 0.05. The α-value for ANOVA (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05. ECx- values were calculated by sigmoidal dose response regression using GraphPadPrism. Four parameters were used for dose-response fitting. Calculation of the confidence intervals for EC-values was carried out using standard procedures provided by GraphPadPrism.

Table 1. Validity criteria defined by OECD guideline 208.

Criterion	Outcome	Criterion fulfilled
Seedling emergence in the controls is at least 70%.	Control emergence (% of seeds) and survival (% of emerged plants): A. cepa: 100% and 100% A. sativa: 83% and 100% B. vulgaris: 93% and 100% B. napus: 98% and 100% L. sativa: 90% and 100% G. max: 96% and 100%	Yes
No visible phytotoxic effects (e.g. chlorosis, necrosis, wilting, deformatons) in control seedlings and the plants of every particular species exhibit normal variation in growth and morphology.	The seedlings exhibited no visible phytotoxic effects and the plants exhibited only normal variation on growth and morphology.	Yes
The environmental conditions for a particular species are identical and the growing media contain the same amount of soil matrix, support media or substrate from the same source.	Environmental conditions and growth media were identical for each plant species.	Yes

Table 2:Oats: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height 1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence 1) [%]	Inhibition     [%]	Survival of  plants 1) [%]	Inhibition     [%]
Control	41.2	-	1293	-	832)	-	100	-
62.5	40.8	1	1206	7	98	-18	100	0
125	39.7	4	1146	11	95	-14	100	0
250	40.9	1	1213	6	93	-12	100	0
500	39.8	3	1125*	13	95	-14	100	0
1000	39.6	4	1023*	21	95	-14	100	0

 

Negative values = Promoted growth                                 

 * = Statistically significant inhibition

1) Normality test failed        

2)= Rate of emergence is inside the natural  margin of deviation, based on experience and the general validity criteria of the guideline

Table 3: Onion: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height 1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence 2) [%]	Inhibition     [%]	Survival of plants1)[%]	Inhibition     [%]
Control	16.7	-	216	-	100	-	100	-
62.5	16.8	-1	222	-3	98	2	100	0
125	18.1	-8	232	-7	85	15	98	3
250	16.4	2	207	4	83**	17	100	0
500	17.0	-2	226	-5	95	5	100	0
1000	17.6	-5	231	-7	100	0	100	0

Table 4: Sugar Beet: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence 2) [%]	Inhibition     [%]	Survival of  plants1) [%]	Inhibition     [%]
Control	7.6	-	465	-	93	-	100	-
62.5	8.1	-7	519	-12	93	0	100	0
125	8.1	-7	520	-12	98	-5	100	0
250	8.1	-7	521	-12	100	-8	100	0
500	8.4	-11	519	-12	100	-8	100	0
1000	7.9	-4	486	-5	100	-8	98	3

 Negative values = Promoted growth

** = statistically significant inhibition, but not test item related (as inhibition does not increase with the concentration); the result is not considered to set endpoints

1) Normality test failed

2) Normality test and equal variance test failed

Table 5: Rape: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height 1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence 1) [%]	Inhibition     [%]	Survival of  plants 1) [%]	Inhibition     [%]
Control	13.0	-	1139	-	98	-	100	-
62.5	14.4#	-11	1140	0	90	8	100	0
125	13.9#	-7	1056	7	98	0	100	0
250	13.8	-6	1033	9	98	0	100	0
500	14.4#	-11	1087	5	93	5	100	0
1000	12.9	1	920	19	90	8	100	0

 

Table 6: Lettuce: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height 1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence 1) [%]	Inhibition     [%]	Survival of  plants 1) [%]	Inhibition     [%]
Control	5.6	-	486	-	90	-	100	-
62.5	4.9**	13	324**	33	90	0	100	0
125	5.9	-5	500	-3	88	2	100	0
250	5.6	0	438	10	88	2	100	0
500	4.9	13	333*	31	93	-3	100	0
1000	4.4*	21	239*	51	78	13	100	0

 

Negative values = Promoted growth 

# = Statistically significant promoted growth

* = Statistically significant inhibition

** = statistically significant inhibition, but not test item related (as inhibition does not increase with the concentration); the result is not considered to set  endpoints

1) Normality test failed

Table 6:Soybean: Inhibition [%] of Shoot Height, Shoot Fresh Weight, Rate of Emergence and Survival of Plants

Test concentration [mg/kg DW]	Shoot height 1)   [cm]	Inhibition     [%]	Shoot fresh weight [mg]	Inhibition     [%]	Rate of emer- gence1)[%]	Inhibition     [%]	Survival of  plants 1) [%]	Inhibition     [%]
Control	21.6	-	2673	-	96	-	100	-
62.5	23.0	-6	2577	4	100	-4	100	0
125	22.5	-4	2660	0	100	-4	100	0
250	23.0	-6	2691	-1	92	4	100	0
500	22.0	-2	2555	4	96	0	100	0
1000	21.4	1	2495	7	96	0	100	0

 Negative values = Promoted growth

1) Normality testfailed

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables".

Executive summary:

The phytotoxicity of the test substance to six terrestrial plant species was determined according to OECD 208 (2006) in seedling emergence and seedling growth tests over a period of 21 days. The test for onion was prolonged until day 28 since 50 % emergence of the control seedlings was reached after 14 days.

For shoot height, number of emerged seedlings and survival of plants statistically no significant differences were observed for all tested plant species. For these species the NOEC is 1000 mg/kg dw. For shoot fresh weight statistically significant differences were determined for the plant species oats and lettuce. For these species the NOEC is 250 mg/kg dw. For the plant species onion, sugar beet, rape and soybean the NOEC is 1000 mg/kg dw and the LOEC is ≥ 1000 mg/kg dw.

Cross-reference 10

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

Published method: NMR-Spectroscopic Investigations on the Hydrolysis of Functional Trialkoxysilanes, M. Brand, A. Frings, P. Jenkner, R. Lehnert, H. J. Metternich, J. Monkiewcz, J. Schram, Verlag der Zeitschrift für Naturforschung Tübingen 54b, 155-64, (1999)

Deviations:

yes

Remarks:

Due to the low solubility of the substance, the water/solvent ratio, was adjusted. Furthermore since the hydrolysis speed of the substance is low, the duration of the studies and the measurement intervals were prolonged to 14 d and daily measurement.

Principles of method if other than guideline:

- Principle of test: Three different series of measurements were carried out at pH 7, pH 10 and pH 4 according to the published method “NMR-Spectroscopic Investigations on the Hydrolysis of Functional Trialkoxysilanes, M. Brand, A. Frings, P. Jenkner, R. Lehnert, H. J. Metternich, J. Monkiewcz, J. Schram, Verlag der Zeitschrift für Naturforschung Tübingen 54b, 155-64, (1999)”.

GLP compliance:

no

Remarks:

The laboratory is accredited from the German Akkreditation Body according to DIN EN ISO/IEC 17025

Radiolabelling:

no

Analytical monitoring:

yes

Remarks:

1H-NMR spectra

Details on sampling:

- Sampling intervals for the parent/transformation products: At the start and then on a daily basis over two weeks.
- Sampling method: All tests were done directly within the NMR tubes.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: NMR tubes
- Is there any indication of the test material adsorbing to the walls of the test apparatus: Yes, therefore all NMR tubes were pretreated with Hexamethyldisilazane to prevent interactions with the glass surface, rinsed with water and dried overnight in a drying cabinet.

TEST PREPARATION
pH 7: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. Using a syringe 10 µl of Hexadecyltrimethoxysilane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).
pH 10: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. The solution was adjusted to pH 10 with NaOH. Using a syringe 10 µl of Hexadecyltrimethoxy-silane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).
pH 4: 0.50 mL acetone-d6 and 0.10 mL H2O were transferred to a NMR tube and mixed. The solution was adjusted to pH 4 with HCl. Using a syringe 10 µl of Hexadecyltrimethoxysilane were added (the resulting concentration of silane is 15 g/L or 0.043 mol/L).

OTHER TEST CONDITIONS
- Adjustment of pH: with NaOH or HCl

Duration:

14 d

pH:

4

Initial conc. measured:

94.2 other: mol%

Duration:

14 d

pH:

7

Initial conc. measured:

100 other: mol%

Duration:

14 d

pH:

10

Initial conc. measured:

98.7 other: mol%

Number of replicates:

Not reported

Positive controls:

not specified

Negative controls:

not specified

Preliminary study:

yes (feasibility study), see field "any other information on material and methods incl. tables"

Transformation products:

yes

No.:

#1

Details on hydrolysis and appearance of transformation product(s):

- Pathways for transformation: The three SiOMe-groups of the silane will react with water and deliberate methanol. The SiOH-groups are not stable and generate Si-O-Si bonding and crosslink eventually.

% Recovery:

59.9

pH:

4

Duration:

14 d

% Recovery:

84.4

pH:

7

Duration:

14 d

% Recovery:

82

pH:

10

Duration:

14 d

pH:

7

DT50:

> 14 d

Type:

not specified

pH:

4

DT50:

> 14 d

Type:

not specified

pH:

10

DT50:

> 14 d

Type:

not specified

Hydrolysis at pH 4:

After 14 days 60 mol% of the SiOCH₃groups remained intact and 40 mol% were hydrolyzed:

Hydrolysis at pH 7:

After 14 days 94.4 mol% of the SiOCH₃groups remained intact and only 5.6 mol% were hydrolysed.

Hydrolysis at pH 10:

After 14 days 82 mol% of the SiOCH₃groups remained intact and 18 mol% were hydrolysed.

Table 1: Hydrolysis speed at different pH values over time.

	% Hydrolysis (= mol % methanol)
Time [d]	pH 7	pH 10	pH 4
0	0	1.3	5.8
1	0,6	4.6	7.9
2	1	5.4	10.7
3	1.2	7.8	13.2
4	1.6	-	17.6
5	-	-	21.8
6	-	10.5	23.9
7	2.5	11.8	26.6
8	2.8	13.3	28.9
9	3.2	13.8	31.5
10	3.7	14.5	32.4
11	4.3	-	35
12	-	-	37.4
13	-	17.7	39.1
14	5.6	18	40.1

Validity criteria fulfilled:

not applicable