1,1,5,5,5-hexamethyl-3,3-bis[(trimethylsilyl)oxy]trisiloxane

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 February 2010 to 29 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were collected from alternating replicate test chambers of each treatment and control group eleven and seven days prior test initiation to confirm the operation of the diluter.
Water samples also were collected from alternating replicate test chambers at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to determine concentrations of the test substance in the test system. Additionally, water samples were collected on Day 2 of the test after the Day 0 analytical results showed differing results from the pre-test.
- Sampling method: During the two pre-tests, on Day 0 and Day 2 the following procedure was used to collect samples for analytical verification: A 100 mL glass was submerged to mid depth in the appropriate test chamber then solution poured into a labelled 100 ml glass volumetric flask. On Day 7, 14 and 21, the following procedure was used to collect samples for analytical verification - One 100 ml glass volumetric flask was used for each concentration to collect samples. The flasks were externally rinsed with reverse-osmosis water into a waste bucket, then rinsed externally with the appropriate test solution into a waste bucket. Next, flasks were submerged completely into the test solution with stoppers on. The stoppers were removed and the flasks filled completely, then stoppered again. A Portion of the sample was removed with a glass pipette to achieve a final volume of 100 ml. Flasks were labelled after sampling. All samples were collected mid-depth, and processed immediately for analysis.
- Sample storage conditions before analysis: All samples were processed immediately for analysis.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solutions were prepared two times during the test. For each preparation, a primary stock solution was prepared in DMF at a nominal concentration of 10,000 ng/ml (Primary stock 'A'). A second primary stock was prepared in DMF at a nominal concentration of 1510 ng/ml (Primary stock 'B'). Both primary stock solutions were mixed by inversion and both appeared clear and colourless. Proportional dilutions of primary stock 'B' were made to prepare four additional secondary stock solutions at nominal concentrations of 94, 190, 380 and 760 ng/ml. The secondary stock solutions were inverted to mix, and appeared clear and colourless.
Primary stock 'B' and the four secondary stock solutions were delivered to the diluter mixing chambers (at a rate of 15.5 µl/minute) where they were mixed with dilution water (at a rate of 155 ml/minute) to achieve the desired test concentrations of 9.4, 19, 38, 76 and 151 ng/l. The solvent control was prepared by injecting DMF into the mixing chamber assigned to the solvent control. The concentration of DMF in the solvent control and all treatment groups was 0.1 ml/l.
- Controls: Solvent control: DMF in dilution water at a concentration of 0.1 ml/l with no test substance. Control: dilution water without test substance or solvent.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMF
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1 ml/l
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No: Test solutions in the test chambers for all treatment groups and control groups appeared clear and colourless at test initiation and termination.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna - water flea
- Strain/clone: not reported
- Justification for species other than prescribed by test guideline: n/a
- Source: Laboratory cultures
- Age of parental stock (mean and range, SD): Neonates <24 hours old.
- Feeding during test
- Food type: A mixture of yeast, cereal grass media, and trout chow (YCT) as well as a suspension of freshwater green algae.
- Amount: Each test chamber was fed 0.75 ml of YCT and 1.5 ml of algae.
- Frequency: 3 times per day up to day 6, 4 times per day after.

ACCLIMATION
- Acclimation period: Adults daphnids were cultured in water from the same source at the same temperature as used during the test.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: As testing
- Feeding frequency: Not reported for culturing conditions
- Health during acclimation (any mortality observed): No

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The three adult daphnids used to supply neonates for the test were held for at least 12 days prior to collection of the juveniles for testing, and had produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers (e.g. glass beakers) until each chamber contained five daphnids. Each group of daphnids then was transferred to an indiscriminately assigned test compartment. All transfers were made below the water surface using wide-bore pipettes.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Negative control: 138 - 144 mg/l as CaCO3
191 ng/l treatment group: 140 - 144 as CaCO3

Test temperature:

19.7 - 20.0 °C

pH:

8.1 - 8.3

Dissolved oxygen:

5.8 - 9.4 mg/l

Conductivity:

Negative control: 349 - 372 µS/cm
191 ng/l treatment group: 368 - 401 µS/cm

Nominal and measured concentrations:

Nominal: control, solvent control, 9.4, 19, 38, 76 and 151 ng/l.
Mean Measured: control, solvent control, 9.1, 13, 28, 54 and 191 ng/l (97, 70, 73, 71 and 126% of nominal concentrations, respectively).

Details on test conditions:

TEST SYSTEM
- Test vessel: Continuous flow diluter: 25 litre Teflon lined stainless steel aquaria. Daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 ml glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative test compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. The test chambers were indiscriminately placed in a temperature-controlled water bath to maintain the temperature throughout the test period. All test chambers were labelled with the project number, test concentration and replicate.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Teflon lined stainless steel aquaria, 25 litre capacity filled with 22 litres test solution.
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow diluter
- Renewal rate of test solution (frequency/flow rate): Five volume additions of test water in each test chamber per day.
- No. of organisms per vessel: 20: Two replicate test chambers were tested for each treatment and control. Each replicate contained two compartments with five daphnids, resulting in a total of 20 Daphnia magna in each treatment and control group.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: Not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the laboratory site. The well water is characterized as moderately-hard water. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered (0.45 µm) to remove fine particles and was passed through an ultraviolet (UV) sterilizer to remove microorganisms.
- Total organic carbon: Not reported
- Alkalinity: Negative control: 170 - 184 mg/l as CaCO3
191 ng/l treatment group: 182 - 186 as CaCO3
- Conductivity: Negative control: 349 - 372 µS/cm
191 ng/l treatment group: 368 - 401 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature and pH were measured on days 0, 7, 14 and 21. Dissolved oxygen was measured on day 0 and 21 and approximately 3 times per week.
Hardness, alkalinity and specific conductance were measured on day 0 and then at weekly intervals.
TOC was measured at day 0, 7 and 21.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light, 8 hours dark, 30-minute transition period.
- Light intensity: Fluorescent light bulbs that emit wavelengths similar to sunlight.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of dead and immobile daphnids were recorded along with any clinical signs of toxicity (e.g. inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Immobility was defined as a lack of movement, except for minor spontaneous random movement of the appendages. The presence of eggs in the brood pouch, aborted eggs, males or ephippia also were recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Concentrations were based on the results of exploratory range finding toxicity data and the reported water solubility of the test substance (150.7 ng/l).

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 191 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: highest concentration tested

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

> 191 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: highest concentration tested

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

191 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: highest concentration tested

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

191 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: highest concentration tested

Details on results:

The first day of brood production in the negative and solvent control and in all M4Q treatment groups was between Days 7 and 9 of the test, indicating that there was no apparent delay in the onset of production at any concentration tested. No males or ephippia were produced during the test.

Adult daphnids in the negative and solvent control groups produced an average of 8.8 and 9.0 young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the treatment groups were compared to the pooled control. Daphnids in 9.1, 13, 28, 54 and 191 ng/l treatment groups produced an average of 9.1, 7.9, 8.5, 8.7 and 9.6 young per reproductive day, respectively. Dunnett's test indicated that there were no statistically significant differences (p > 0.05) in reproduction in any of the treatment groups when compared to the pooled control. Consequently, the NOEC for reproduction was 191 ng/l and the LOEC was >191 ng/l.

Daphnids in the negative control group averaged 4.4 mm in length and 1.1 mg in dry weight, while daphnids in the solvent control group averaged 4.4 mm in length and 1.1 mg in dry weight. There were no statistically significant differences (p >0.05) in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the 9.1, 13, 28, 54 and 191 ng/l treatment groups had mean lengths of 4.3, 4.3, 4.3, 4.2 and 4.3 mm, respectively, and mean dry weights of 1.2, 1.1, 1.1, 1.1 and 1.1 mg, respectively.
Dunnett's test indicated that there were no statistically significant (p >0.05) decreases in mean dry weight in any of the M4Q treatment groups in comparison to the pooled control. There was a statistically significant (p < 0.05) decrease in mean length in the 54 ng/l treatment group in comparison with the pooled control using Dunnett's test, however, the decrease was not considered to be treatment related since it was not concentration-responsive. Consequently, the NOEC for growth (length and dry weight) was 191 ng/l, and the LOEC was > 191 ng/l.

Table: Immobility and observations of first generation daphnids

		Day 7		Day 14		Day 21
Mean measured test concentration (ng/l)	Number of Daphnids exposed	% Immobile	Observations	% Immobile	Observations	% Immobile	Observations	Percent survival
Negative control	20	0	20 AN	5	19 AN	15	17 AN	85
Solvent control	20	0	20 AN	0	1 D, 19 AN	5	19 AN	95
9.1	20	0	20 AN	0	20 AN	5	19 AN	95
13	20	0	20 AN	5	19 AN	10	18 AN	90
28	20	0	20 AN	0	20 AN	0	20 AN	100
54	20	0	20 AN	0	20 AN	0	20 AN	100
191	20	0	20 AN	0	20 AN	5	19 AN	95

Observations: AN = Appear Normal; D = discolouration (pale in colour)

Table: Summary of reproduction of first generation daphnids

Mean measured test concentration (ng/l)	Compartment	First day of reproduction	Number of young produced per reproductive day	Treatment mean number of young produced per reproductive day (±SD)^1
Negative control	A	7	9.4	8.8 ± 0.51
	B	7	8.5
	C	7	9.1
	D	9	8.3
Solvenht control	A	7	7.9	9.0 ± 0.79
	B	7	9.6
	C	7	9.5
	D	9	8.8
Pooled control		-	-	8.9 ± 0.62
9.1	A	7	8.5	9.1 ± 0.94
	B	8	8.2
	C	7	10.2
	D	7	9.6
13	A	9	7.6	7.9 ± 0.83
	B	8	9.1
	C	7	7.5
	D	7	7.3
28	A	7	9.0	8.5 ± 0.79
	B	7	8.3
	C	7	7.4
	D	7	9.1
54	A	8	8.9	8.7 ± 0.87
	B	8	9.6
	C	9	7.5
	D	8	8.6
191	A	8	9.1	9.6 ± 1.49
	B	7	8.2
	C	8	9.4
	D	7	11.7

Table: Summary of length and dry weight of surviving first generation daphnids

Mean measured concentration	Compartment	Number surviving	Replicate mean length (mm)	Treatment mean length (mm) (±SD)	Replicate mean dry weight (mg)	Treatment mean dry weight (mg) (±SD)
Negative control	A	3	4.6	4.4 ± 0.13	1.26	1.08 ± 0.15
	B	5	4.6		1.08
	C	5	4.3		0.89
	D	4	4.4		1.08
Solvent control	A	4	4.5	4.4 ± 0.10	1.22	1.11 ± 0.094
	B	5	4.3		1.06
	C	5	4.3		1.15
	D	5	4.3		1.01
Pooled control	-	36	-	4.4 ± 0.11	-	1.09 ± 0.12
9.1	A	4	4.2	4.3 ± 0.0058	1.05	1.17 ± 0.096
	B	5	4.2		1.21
	C	5	4.3		1.27
	D	5	4.3		1.13
13	A	3	4.3	4.3 ± 0.10	1.23	1.13 ± 0.074
	B	5	4.3		1.08
	C	5	4.1		1.15
	D	5	4.3		1.07
28	A	5	4.3	4.3 ± 0.10	1.03	1.07 ± 0.11
	B	5	4.3		1.22
	C	5	4.1		1.07
	D	5	4.3		0.97
54	A	5	4.3	4.2 ± 0.12*	1.16	1.08 ± 0.12
	B	5	4.3		1.16
	C	5	4.1		1.07
	D	5	4.1		0.91
191	A	5	4.4	4.3 ± 0.050	1.11	1.10 ± 0.13
	B	5	4.3		1.15
	C	5	4.3		1.21
	D	4	4.3		0.91

*There was a statistically significant difference in mength compared to the pooled controls (Dunnett's test)

Validity criteria fulfilled:

yes

Conclusions:

A 21-day EC50 value of >191 ng/l, a NOEC value of 191 ng/l and a LOEC value of >191 ng/l (highest concentrations tested), based on mean measured concentrations, have been determined for the effects of the test substance on survival, growth and reproduction of Daphnia magna, in accordance with OECD Guideline 211.

Description of key information

EC₅₀: >191 ng/l; NOEC ≥191 ng/l; LOEC >191 ng/l (highest concentrations tested) (mean measured concentrations): effects of the test substance on survival, growth and reproduction of Daphnia magna.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

>= 191 ng/L

Additional information

A 21-day EC₅₀ value of >191 ng/l, a NOEC value of ≥191 ng/l and a LOEC value of >191 ng/l (highest concentrations tested), based on mean measured concentrations, have been determined for the effects of the test substance on survival, growth and reproduction of Daphnia magna, in accordance with OECD Guideline 211.

The test solution preparation method and test system design indicate that the test organisms were predominantly exposed to the parent substance.