Hexamethylcyclotrisiloxane

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-12-11 to 2006-12-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Although test substance concentrations in the solvent stock solutions were measured prior to the start of the study, concentrations in the test vessels were not measured. Methods to maintain test concentrations (e.g. flow-through) in algae studies are unavailable, hence this study is representative of the potential effect on algae initially exposed to the test substance but subsequently and progressively exposed to its hydrolysis products.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At test initiation, a single sample was removed from each treatment level stock solution prepared (i.e. 1.0, 2.0, 4.0, 8.0 and 16 mg/mL. The samples were analysed for the test substance.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 16 mg/mL stock solution was prepared prior to test initiation by placing 0.4001 g of the test substance in a 25-mL volumetric flask and bringing it to volume with acetone. Test solutions were prepared from dilutions of the 16 mg/mL stock solution using AAP medium.

- Controls: Algal growth medium and algal growth medium plus acetone (0.1 mL/L)

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM

Test organism: Pseudokirchneriella subcapitata

Source: Springborn Smithers culture

Age of inoculum: 3 days since previous transfer

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24 ºC

pH:

pH at test initiation: 7.0 to 7.2

pH at test termination: 9.2 to 9.3 (the increase in pH during exposure is common in static algal cultures and is due to photosynthesis by the algae)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentrations: 0 (Control), 0 (Solvent control), 0.10, 0.20, 0.40, 0.80 and 1.6 mg/L.  The concentration of 1.6 mg/L, the highest concentration tested, is considered representative of the functional limit of solubility.  All test solutions were observed to be clear and colourless with no visible undissolved test substance.

Details on test conditions:

TEST SYSTEM

- Test conditions: Closed system. Due to the volatile nature of the test substance volatile organic analysis (VOA) vials with Teflon ®-lined screw caps were used rather than traditional caps which allow air exchange.  This procedure was used to minimize the loss of test substance.  

- Twelve replicates per treatment level and the control and 24 replicates for the solvent control were prepared.  The additional two replicates per treatment level and controls were used for analytical and water quality measurements.  

- Initial cell density: 1.5x10E4 cells/mL.

- Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 96-hour exposure period.

- Dilution water: Algal Assay Procedure (AAP) medium

- Continuous illumination at 420 to 540 footcandles (4500 to 5800 lux), shaking rate of 100 rpm.  

- Effect criteria: 0- to 96-hour cell density and 0- to 72-hour average growth rate (μ) and biomass expressed as yield relative to the performance of the solvent control data.

- 0 to 96-hour cell density and 0 to 72-hour average growth rate (μ ave) and biomass expressed as yield were determined relative to the performance of the solvent control. Yield was calculated as biomass (cell density) at each interval of the test minus the initial biomass at the start of the test. 

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

>= 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass expressed as yield

Remarks on result:

other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass expressed as yield

Remarks on result:

other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

Details on results:

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

Reported statistics and error estimates:

No treatment resulted in > 50 % inhibition of growth rate or biomass. It was therefore not possible to determine definitive EC50 values for these end points.

No observed effect concentrations (NOECs) for growth rate and biomass (yield) were determined using Bonferroni's t-Test. The calculations were performed using TOXSTAT® version 3.5 software.

Table 1. Test results

Nominal concentration (mg/L)	Initial cell concentration (cells/mL)	Cell concentration after 24 hours (cells/mL)	Cell concentration after 48 hours (cells/mL)	Cell concentration after 72 hours (cells/mL)	Inhibition of growth rate after 72 hours (%)	Inhibition of biomass (yield) after 72 hours (%)
0 (Control)	15000	51700	194200	399200	-	-
0 (Solvent control)	15000	56700	172900	406700	-	-
0.10	15000	62500	181700	368300	3	10
0.20	15000	51700	177500	348300	5	15
0.40	15000	55000	192500	394200	1	3
0.80	15000	54200	192500	323300	7	21
1.6	15000	54200	176700	375800	3	8

The following acceptance criteria are required by OECD Guideline 201: the cell growth in the solvent control must increase
from the initial density (1.5 x 10E4 cells/mL) by more than 16 times after 72 hours of growth.  Additionally, the mean
coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2, and 2 to 3) in the solvent control replicates should not exceed 35 %.  The CV for the average growth rate of the solvent control for
the entire test period (0- to 72-hour growth rate) should not exceed 7 %.  During this study, the 72-hour cell density
in the solvent control was 39.92 x 10E4 cells/mL, which meets the criterion of 24 x 10E4 cells/mL at 72 hours.  The
mean daily CV for growth rates observed in the test was 28 % and the CV for 0- to 72-hour growth rate was 2.6 %. These values are within the acceptability criteria.

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of > 1.6 mg/L and NOEC of ≥ 1.6 mg/L have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. A concentration of 1.6 mg/L corresponds to the approximate water solubility of the substance. It is likely that the test organisms were predominantly exposed to the hydrolysis products of the substance.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008-08-05 to 2008-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples of each test medium were taken at the start and end of the test. They received no further treatment prior to analysis.

Vehicle:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM

- Source (laboratory, culture collection): Test organisms were obtained from in-house cultures. The source of the original brood stock was strain UTEX 1648, from the University of Texas at Austin, The Culture Collection of Algae, Botany Department, Austin, Texas, in March 1998. The identity of the species was verified by the supplier.

- Method of cultivation: Algal cultures were maintained at 24 ± 2 ºC on a rotary shaker set at approximately 100 rpm, under continuous cool white fluorescent lighting at a range of 400 ± 100 foot candles (4300 ± 1075 lux). The cultures were periodically transferred axenically to new AAM.

- Culture medium: Algal Assay Medium was prepared based on ASTM guideline E 1218-04. The final concentration of each nutrient in the medium is presented in Appendix A. All stocks used to prepare the medium were prepared with sterile distilled water and were filter sterilized (0.22 μm). Medium was prepared with commercially available distilled water that is analyzed annually for contaminants. No contaminants have been shown to be present which might adversely impact the viability of the cultures or study

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

23.8 to 24.0 ºC

pH:

pH at test initiation ranged from 7.4 to 7.5. At test termination, pH in the flasks that contained algae ranged from 8.0 to 9.0 and pH of media blanks ranged from 7.6 to 7.8

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentrations: 0 (Control), 7.5, 15, 30, 60 and 120 mg/L

Mean measured test concentrations in treated media: 8, 18, 28, 57 and 118 mg/L; which is 106, 122, 93, 96 and 99 % of nominal, respectively.

The results are expressed relative to mean measured concentrations.

Details on test conditions:

TEST SYSTEM

- Test Apparatus: Test vessels were sterile 250-mL Erlenmeyer flasks covered with a sponge closure containing approximately 100 mL of test solution. Test vessels were labelled with the study number, concentration, and replicate.

- Initial cell density: 10000 cells/ml

- Test Conditions: Test vessels were placed in a temperature-controlled incubator and positioned on rotary shakers that were set at approximately 100 rpm. In addition, the incubator was set at a temperature of 24 ± 2 ºC and under continuous cool white fluorescent lighting at a range of 412 – 825 foot-candles (4440 - 8880 lux).

Temperature was measured in a beaker of water adjacent to the shakers in the incubator and light readings were recorded at five indiscriminate areas on the shaker tables daily during the test. Readings of pH were completed on bulk preparations of each treatment group at test initiation. To maintain axenic conditions, pH readings for each test vessel were measured only at 72 hours.

- Observations: Samples for cell counts were collected on Day 0 for the original culture and test inoculums. Samples for cell counts were also collected from each test vessel at 24, 48 and 72 hours (within 1 hour). Samples were diluted with Isoton® II diluent solution and counted immediately using a Coulter® Counter. Dilution volumes were recorded in the raw data. Dilution samples of the medium and diluent blanks were counted at 24, 48, and 72 hours to account for any interference of particles. At test termination, samples were pooled by treatment and visually inspected for atypical cell morphology. Visual examination showed no concentration-dependent changes in shape, colour or size.

- Light intensity: 446 to 620 foot-candles

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 118 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 118 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield and growth rate

Details on results:

- Exponential growth in the control (for algal test): yes, Control cell density for this study increased 122 fold by 72 hours.

Reported statistics and error estimates:

ECx values for yield and growth rate at 72 hours were calculated using linear interpolation or estimated by visual examination of the data.

Yield (biomass) and growth rate data were analyzed for normality and homogeneity of variance using the Shapiro-Wilk’s test and Bartlett’s test, respectively. The no observed effect concentration (NOEC) values were determined for growth rate and yield at 72 hours using Dunnett’s test. All statistical analyses were performed using TOXSTAT Version 3.5 software

Table 1. Measurement of Test Concentrations

Nominal Concentration (mg/L)	Day 0 Measured Concentration  (mg/L)	Day 3 Measured Concentration (mg/L)	Mean Measured Concentration (mg/L)	Percent of Nominal
Control	ND	ND	--	--
7.5	5.9	10	8	106
15	18	19	18	122
30	25	31	28	93
60	55	60	57	96
120	114	122	118	99
ND = Not Detected

 

Table 2. Mean Cell Densities (cells/mL)

Mean Measured Concentration (mg/L)	0-hour	24-hour	48-hour	72-hour
Negative Control	~10000	43433	240424	1222333
8	~10000	41551	246551	1276867
18	~10000	37384	232924	1216000
28	~10000	38551	217216	1129333
57	~10000	42022	228560	1090778
118	~10000	35773	201429	990800

Algal cell growth in the control was exponential during the 72-h exposure period. Cell densities were used to calculate the average specific growth rates and yield for each test concentration. 

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of > 118 mg/L and a NOEC of ≥ 118 mg/L have been determined for the effects of the test substance on yield and growth rate of Pseudokirchnerella subcapitata.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1979

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

It is not certain that exponential growth was maintained throughout the test period.

Qualifier:

according to guideline

Guideline:

other: Biological  Field and Laboratory Methods (EPA-670/4-73-001)

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Anabaena flos-aquae

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

13 d

Nominal and measured concentrations:

Test concentrations were 0, 1, 10, and 100 mg/L

Details on test conditions:

The test method followed the Algal BioAssay Procedure as described in "Biological Field and Laboratory Methods (EPA-670/4-73-001)".  

Tests were conducted in triplicate in polycarbonate Ehrlenmeyer flasks. 

The test guideline that was followed includes illumination at 200 ft-candles and a temperature range of 24 ± 2 °C.  

Test concentrations were 0, 1, 10, and 100 mg/L.    

Each test vessel was innoculated with a dose sufficient to produce a starting filament concentration of ~7,500 filaments/mL. 

Algae filament numbers were determined by hand counting on days 0, 3, 5, 7, 11, 12 and 13 using a hemocytometer.  Duplicate counts of triplicate samples were conducted on each test vessel.  

The median inhibitory limit (IL50) was calculated for the exponential growth rate and the final yield. 

 A complete description of the calculations can be found in the Handbook of Phycological Methods (1973).

Duration:

13 d

Dose descriptor:

other: IL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Hydrolysis products

Basis for effect:

other: growth rate and final yield

The IL50 was determined to be > 100 mg/l, the maximum concentration tested.

Conclusions:

A 4-day EC50 value of > 100 mg/L has been determined for the effects of the test substance on growth rate and biomass of Anabaena flos-aquae. It is likely that the test organisms were exposed to the hydrolysis products of the substance.

Description of key information

Toxicity to algae: 72-hour EC₅₀ >1.6 mg/l; NOEC ≥1.6 mg/l (nominal) (highest concentration tested), growth rate of Pseudokirchneriella subcapitata.

Additional information

A 72-hour E(L)C₅₀ value of >1.6 mg/l and NOEC value of ≥1.6 mg/l (nominal) (highest concentration tested) has been determined for the effects of hexamethylcyclotrisiloxane (D3, CAS 541-05-9; EC No. 208-765-4) on growth rate and yield of Pseudokirchneriella subcapitata.

The toxicity to algae test with D3 was carried out at the limit of water solubility for D3, 1.6 mg/l, under static conditions. D3 will rapidly hydrolyse to 1,1,3,3,5,5-hexamethyltrisiloxane-1,5-diol (CAS 3663-50-1; EC No. 222-920-3; L3-diol) (half-life 23 mins, pH7 and 25°C), and then on to 1,1,3,3-tetramethyldisiloxane-1,3-diol (CAS 1118-15-6; EC No. 214-258-9; L2-diol) and dimethylsilanediol (DMSD, CAS 1066-42-8; EC No. 213-915-7) (half-life for loss of L3-diol approximately 200 hours at pH 7 and 25°C). The pH was 7.0 at the beginning of the test, and had risen to 9.3 at the end. There were no other pH measurements taken during the test. Given the rapid hydrolysis rate of D3 to L3-diol it can be concluded that the algae was predominantly exposed to the hydrolysis products. It is not possible, however, to determine which hydrolysis product the algae were exposed to. It is likely that the L3-diol was predominant at the beginning of the test, but as the pH rose, the rate of hydrolysis to the L2-diol and DMSD would increase and the algae would predominantly be exposed to the L2-diol and then DMSD.

No effects were seen in the test, therefore it can be said that the L3-diol, the L2-diol and DMSD are not toxic to algae at the limit of solubility of the parent substance (D3, 1.6 mg/l).

 

A 72-hour E(L)C₅₀ value of >118 mg/l and NOEC value of ≥118 mg/l (measured, arithmetic mean) (highest concentration tested) has been determined for the effects of dimethylsilanediol (DMSD, CAS 1066-42-8) on growth rate and yield of Pseudokirchneriella subcapitata. This substance is the ultimate hydrolysis product of D3.