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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-30 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
liquid
Analytical monitoring:
yes
Remarks:
measured the parent substance
Details on sampling:
- Concentrations: (nominal) control, solvent control, 0.0313, 0.0625, 0.125, 0.250, 0.500 mg/L
- Sampling method: LC-MS/MS once within every 7 days in fresh media at the start of an exposure interval (0 hours) and in old media at the end of an exposure interval (24 hours).
- Sample storage conditions before analysis: Samples were analysed immediately (as far as technically possible) following sampling, All original samples were stored at room temperature. Prepared samples were stored at room temperature in an autosampler until analysis.
Vehicle:
yes
Remarks:
Solvent used: dimethylformamide, a solvent control was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were freshly prepared for each exposure interval and each concentration level. The stock solutions were prepared by direct addition of the appropriate amount of test item by pipette (the relative density of the test item of 0.877 was taken into account) into DMF. The concentrations of the stock solutions were as follows: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 g/L
- Eluate: not reported
- Differential loading: not reported
- Controls: control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide (DMF) was prepared and tested under the same conditions as the test item concentrations and the control.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.05 mL/L in every test concentration and solvent control, 0 ml/L in control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test item solutions were mixed thoroughly by manual agitation, no reported evidence of undissolved material
- Other relevant information:
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: STRAUS Clone 5
- Justification for species other than prescribed by test guideline: N/a
- Age at study initiation (mean and range, SD): Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
- Weight at study initiation (mean and range, SD): not reported
- Length at study initiation (length definition, mean, range and SD): not reported
- Stage and instar at study initiation: not reported
- Valve height at study initiation, for shell deposition study (mean and range, SD): N/a
- Peripheral shell growth removed prior to test initiation: N/a
- Method of breeding: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx
- Source: Institut Dr. Nowak, Mayenbrook 1, 28870 Ottersberg, Germany
- Age of parental stock (mean and range, SD): not reported
- Feeding during test - yes
- Food type: Pseudokirchneriella subcapitata and Desmodesmus subspicatus suspension was provided as food corresponding to 0.2 mg C per daphnid and day.
- Amount: 0.2 mg C per daphnid and day.
- Frequency: Daily feeding per test vessel

ACCLIMATION
- Acclimation period: Acclimatization was not necessary, because the composition of the dilution water used for test medium preparation was equivalent to the culture medium.
- Acclimation conditions (same as test or not): N/a
- Type and amount of food: N/a
- Feeding frequency: N/a
- Health during acclimation (any mortality observed): N/a

QUARANTINE (wild caught)
- Duration: N/a
- Health/mortality: N/a

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: N/a
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
as CaCO3.
In control group: 216-257.
In solvent control: 224-280
In test concentrations 0.199 and 0.562: 225-260
Test temperature:
In control group: 20.1-20.7
In solvent control: 19.7-20.7
In test concentrations 0.199 and 0.562: 19.7-20.6
pH:
In control group: 7.05-7.47
In solvent control: 7.30-7.44
In test concentrations 0.199 and 0.562: 7.31-7.45
Dissolved oxygen:
In control group: 6.66-9.19
In solvent control: 7.49-9.56
In test concentrations 0.199 and 0.562: 7.95-9.18
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal: 0.0313, 0.0625, 0.125, 0.250, 0.500 mg/L
Measured: 0.025, 0.043, 0.103, 0.199, 0.562 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: (4.5 (ID) x 9.5 (H) cm) with screw caps were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.
- Aeration: Test vessels were not aerated during the test.
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static test so N/a
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed daily. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 10 replicates for all concentrations
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- Biomass loading rate:N/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Same composition as the culture medium (culture medium Elendt M4)
- Total organic carbon: Not reported
- Particulate matter: not reported
- Metals: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Pesticides: not reported
- Chlorine: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Alkalinity: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Ca/mg ratio: as standard in Elendt M4 culture medium (OECD 202 Annex 3)
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: once every seven days in fresh media and at the end of an exposure interval (24h)

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to pH 7.0-7.5
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Max. 1500 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality of parental daphnids (once a day at least), neonates (checked daily), condition of parental daphnids (daily), total body length/mean dry weight of parental daphnids (at the end of the test), first time to brood, intrinsic rate of population increase, number and size of first brood per animal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.0313 - 0.0625 - 0.125 - 0.250 - 0.500 mg/L
- Justification for using less concentrations than requested by guideline: N/a
- Range finding study
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: n/a
Reference substance (positive control):
yes
Remarks:
Test is undertaken once a month at the test facility
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.189 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
(C.I 0.119-0.300)
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.199 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.261 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
(C.I. 0.0177-3.16)
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.562 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and adult mortality
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.234 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult mortality
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.264 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
adult mortality
Details on results:
- Behavioural abnormalities: None observed
- Observations on body length and weight: mean length of survived parents for control, solvent control, 0.025, 0.043, 0.103, 0.199, 0.562 mg/L : 4.63, 4.63, 4.70, 4.63, 4.58, 4.50mm and N/a as mortality was 100% in the 0.562 treatment.
- Other biological observations: In the geometric mean measured concentration level of 0.199 mg/L, one parental daphnid was identified as a male during the exposure period. This parental daphnid was treated as accidental mortality and was not taken into account for any further evaluation. No stillborn juveniles or aborted eggs were observed in the control or solvent control during the exposure period of 21 days. In the geometric mean measured concentrations levels of 0.025 mg/L and 0.043 mg/L, a total of 1 and 47 stillborn juveniles were observed, respectively. First appearance of living juveniles mean day for all treatments and controls = day 9 (except for 0.562 mg/L as 100% mortality of parents occurred). No ephippia were observed in the control or in the test groups during the test.
- Mortality of control: 0% mortality in the solvent control and the control
- Other adverse effects control: no adverse effects observed in the control or solvent control
- Immobilisation of control: 0%
- Abnormal responses: described in 'Other biological observations'
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The measured test item concentration in fresh media at the start of the respective exposure intervals (0 hours) were in the range of 101 to 296% of the nominal concentrations and 16 to 100% in old media at the end of the respective exposure intervals (48 hours). The exact reasons for the determined outliers > 126% are unknown. Possible factors of influence may be the tested concentrations near the functional water solubility of the test item and recoveries near the LOQ of the analytical method. Further, the test item is not stable under test conditions and degradation products may influence recovery rates.
- Effect concentrations exceeding solubility of substance in test medium: no - solvent used
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 1.66 mg/L of potassium dichromate (C.I. <1.00-4.00)
- Other: The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018-03-13 to 2018-03-14.
Reported statistics and error estimates:
Reproduction NOEC/LOEC based on geometric mean concentrations:
Shapiro Wilk's Test on normal distribution: Number of residuals = 55; Shapiro-Wilk´s W = 0.782; p(W) = <0.001; p(W) is smaller than or equal to the selected significance level of 0.010; therefore, treatment data significantly differ from normal distribution.
Levene's Test on Variance Homogeneity: The Levene test indicates variance heterogeneity (p <= 0.010). Variance homogeneity check failed. Also the degree of normal distribution is poor. Therefore, the mediantest (2-by-2 table) with Bonferroni correction is performed.
Multiple Sequentially-rejective Median (2x2-Table) Test After Bonferroni-Holm: A NOEC of 0.1990 mg/L is suggested by the program.
Validity criteria fulfilled:
yes
Conclusions:
A 21-day reproduction NOEC of 0.199 mg/L, EC10 value of 0.189 mg/l and a LOEC of 0.562 mg/L (geometric mean measured concentrations) have been determined for the effects of the test substance on reproduction of Daphnia magna. A 21-day adult mortality NOEC of 0.199 mg/L and a LOEC of 0.562 mg/L (geometric mean measured concentrations) have been determined for the effects of the test substance on mortality of Daphnia magna. It is likely that the test organisms were primarily exposed to the parent substance.

Description of key information

Long-term toxicity to invertebrates: 21-day reproduction EC10 0.189 mg/l and NOEC 0.199 mg/l (OECD TG 211), for the test substance triethoxy(octyl)silane (CAS 2943-75-1).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.189 mg/L

Additional information

A 21-day EC10 value of 0.25 mg/l and NOEC value of 0.25 mg/l have been determined for the effects of triethoxy(octyl)silane (CAS 2943-75-1) on reproduction of Daphnia magna based on nominal concentrations. The EC10 and NOEC values are 0.189 mg/l and 0.199 mg/l, respectively, based on geometric mean measured concentrations.

The focus of the chemical safety assessment for this substance is the parent substance, due to its hydrolysis half-life of 30 hours at pH 7. Measures were taken during the Daphnia reproduction test to maximise exposure to the parent, including use of a solvent for media preparation, keeping the pH neutral and using a semi-static daily renewal system. Although these steps were taken to maximise exposure to the parent substance, measured analytical concentrations of the parent substance were inconsistent and degradation was observed in between test solution renewals. The variability of analytical concentrations is thought to be due to the very low solubility of the substance and its potential to degrade in the test system. The results have been reported in terms of nominal test substance concentrations and geometric mean measured concentrations of the parent substance.

In view of the exposure regime it is likely that the test organisms were exposed predominantly to the parent substance at the beginning of the media renewal period, with increasing amounts of hydrolysis products prior to renewal at 24 hours.

To support the data with the registration substance, data have been read-across from two structurally analogous substances; triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3) and trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4).

A 21-day NOELR of 32 mg/l (nominal) and a NOEC 0.058 mg/l (arithmetic mean measured concentration) has been determined for the effects of triethoxy(2,4,4-trimethylpentyl)silane (CAS 35435-21-3) on reproduction of Daphnia magna under semi-static test conditions. However the result of the study should be treated with caution as the effects were recorded at a nominal concentration 25 times higher than the limit of solubility of the parent substance and it is not possible to discern between effects of the parent and effects of the hydrolysis product. The hydrolysis half-life of this substance is 42 hours at pH 7. The test media preparation included 15 minutes of ultrasonication followed by 96 hours of stirring before introduction of test organisms. It is therefore likely that the test organisms were exposed to high concentrations of the hydrolysis products. The analysis of the parent substance support this theory, with poor measured recoveries of parent test substance. Hence this study is used only as supporting evidence on toxicity of the silanol hydrolysis product.

A 21-day NOEC 32 mg/l (initial mean measured equivalent test substance concentration based on measured TOC) has been determined for the effects of trichloro(2,4,4-trimethylpentyl)silane (CAS 18379-25-4) on reproduction of Daphnia magna. The parent substance hydrolyses very rapidly (<1 minute) at pH 7, therefore it is likely that the test organisms were exposed to high concentrations of the hydrolysis products.

It is likely that the test organisms in the above studies were predominantly exposed to the hydrolysis products of the substances. These results indicate that the silanol hydrolysis product of the registered substance has low long-term toxicity to aquatic invertebrates and would not cause toxic effects at concentrations that correspond with the solubility limit of the registered substance.

The read-across to the registered substance is considered scientifically justified. Refer to the Section 6 endpoint summary (Section 7.0 of CSR) for further discussion of the approach to chemical safety assessment for this registration substance, and justification for read-across used.