Reaction mass of decamethyltetrasiloxane; dodecamethylpentasiloxane; hexamethyldisiloxane; octamethyltrisiloxane

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:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with some non-standard elements. Results at the 70-h time point are valid, results at the 95-hour time point are considered not valid, because the pH deviated by more than 1.5 units.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

other: EU C.3

Qualifier:

according to guideline

Guideline:

other: EPA Guideline EG8

Principles of method if other than guideline:

The test was extended to 4 days according to the EPA Guideline EG8 requirements.  Modifications of the test design were implemented to allow for a closed-system test.  A reduced initial algal density (1x10E3 cells/mL), an increased NaHCO3 concentration (300 mg/L), the addition of Fe-citrate and a reduced medium pH (7.0) were utilized in order to avoid substantial pH changes or carbon limited growth in the closed test vials.  pH was adjusted to 7 using HCl.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The concentrations in the test medium were measured at the start and at the end of the test (t=95 hours). Duplicate samples were taken at the start of the test from test vials containing the test substance concentrations of 0, 0.33, 1.0, 1.8 and 3.3 mg/L (nominal), without algae. At the end of the test single samples were taken from two vials per test substance concentration of the same concentration series, containing algae.
Samples of 16 mL were taken by syringe from the test vials. Each sample was injected through the septum of a 40 mL sample vial containing 7 mL hexane, 16 mL of a 120 g/L salt (NaCl) solution and a magnetic stirrer. One sample series was transferred to the Residue Analysis Department (Zeist) to be analysed; the other series was stored in a refrigerator as a spare.

The sampling method is assumed to have avoided collection of any undissolved material that may have been present.

Vehicle:

yes

Details on test solutions:

- Controls: Algal growth medium

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Tertiary butyl alcohol (TBA) was used as the carrier solvent. 

- An amount of 329.1 mg of the test substance was dissolved in 10 ml tertiary butanol (TBA). Dilutions were then prepared in TBA so as to yield a test substance concentration series of (0.10, 0.33, 1.0, 1.8 and 3.3) x 10E4 mg/L.

- Four µl of each test substance dilution in TBA was injected through the septum of the appropriate vials using a syringe into 40 ml of algal growth medium. The resulting test substance concentration series was 0, 0.11, 0.34, 1.1, 3.6 and 11.2 mg/L.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM

- Source (laboratory, culture collection): The fresh-water green alga Selenastrum capricornutum (CCAP 278/4)1), which belongs to the order of Chlorococcales (class Chlorophyceae), was used as the test organism. The culture was supplied by the CCAP, The Freshwater Biological Association, the Ferry House, Far Sawrey, Ambleside, Cumbria LA22 OLP, England.

A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, and in accordance with EPA requirements it was sub-cultured two times during 4 days using the medium

- Method of cultivation: A preculture of algae in the exponential growth phase was prepared as detailed in OECD Guideline no. 201, and in accordance with EPA requirements it was sub-cultured two times during 4 days using the mediumprepared from concentrated stock solutions in ultra pure water. It was sterilized by micropore filtration and contained 300 mg/L NaHCO3 (not 50 mg/L as specified in the OECD Guideline). This in order to improve the buffer capacity of the medium especially when is culturing algae in a closed system. Furthermore, the medium contained Fe-citrate, because the growth of the algae would be erratic in the absence of complexed iron. The pH was adjusted to pH 7 using a HCl-solution.

Test type:

other: sealed system

Water media type:

freshwater

Limit test:

no

Total exposure duration:

95 h

Hardness:

No data

Test temperature:

24ºC+/-2ºC

pH:

After 70 hours of incubation the pH in the test media cultures ranged from pH 7.4 – 7.9 (pH 7.7-7.9 in the control and solvent control), whereas the pH of the test solutions without algae were found to be pH 6.3.
After 95 hour of incubation the pH in the test media cultures ranged from pH 8.4 - 10.0 (pH 9.5 - 10.0 in the control and solvent control), and in the corresponding solutions without algae, pH 6.9-7.0. In the opinion of the present reviewers, the elevated pH in test and control cultures at the 95-h time point makes it particularly difficult to interpret the dose-response and significance.

Dissolved oxygen:

No data

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations:  0 (Control), 0.10, 0.33, 1.0, 1.8 and 3.3 mg/L. It is possible that the limit of solubility may have been reached or exceeded in the upper test concentrations in this range.

Corresponding geometric mean measured concentrations: Not determined, Not determined, 0.045, 0.15, 0.28 and 0.55 mg/L

Chemical analysis: Measured concentration at the start of the test were found to be an average of 20.3+/- 1.2% of the nominal concentrations at the three highest concentrations. At the end of the test, the test concentrations were found to have decreased. The study report states that the concentrations are geometric mean measured concentrations based on analysis at the 0-h and 95-h time points. However, at the 70-h NOEC (0.1 mg/l, nominal) loading level, no analytical results are reported. The geometric mean concentrations are based on the overall average recovery for the three highest loading rates. The recovery at the 0.33 mg/l nominal concentration was excluded from the averaging.

Measured concentrations at the 70-h time point are not available, though at the shorter duration it is perhaps likely that levels of HMDS would have been higher than those measured at 95-h.

The results were interpreted with reference to mean measured concentrations.

Details on test conditions:

TEST SYSTEM

- Modifications to standard test: The test was extended to 4 days according to the EPA Guideline EG8 requirements.  Modifications of the test design were implemented to allow for a closed-system test. 

- Test vessel: Due to the high volatility of the test substance, the test was conducted in closed, silanized, 40 mL EPA vials with Teflon lined septum caps.  

- Test medium: The test medium was prepared from concentrated stock solutions in ultra pure water, sterilized by micropore filtration, containing 300 mg/l NaHCO3, in order to improve the buffering capacity of the medium since the test was run in a closed system.

- Initial cells density: A reduced initial algal density (1x10E3 cells/mL), an increased NaHCO3 concentration (300 mg/L), the addition of Fe-citrate and a reduced medium pH (7.0) were utilized in order to avoid substantial pH changes or carbon limited growth in the closed test vials.  

- The test was carried out in triplicate with three controls containing algae only, three controls containing algae and carrier solvent (TBA) and a single background concentration series containing test substance without algae


OTHER TEST CONDITIONS

- pH adjustment: pH was adjusted to 7 using HCl.

- Light source: fluorescent lamps

- Photoperiod: Continuous

- Light intensity: 78 to 86 µmol/s/m2


OBSERVATIONS

- Cell density measurements: The initial density was calculated from the measured density in the algal pre-culture used for the inoculation, divided by the appropriate dilution factor. After 23, 43.5, 70 and 95 hours of incubation, algal densities (cells/mL) and algal biovolume (µm3/mL) were determined with the electronic particle counter (Coulter Multisizer IIe). For each measurement a separate vial was sacrificed. Measured values were corrected for the background values in the appropriate blanks.

- Post-exposure observations: In order to test whether or not the effects seen were algistatic or algicidal, cultures containing 0 and 3.3 mg/L (nominal) test substance were diluted with fresh medium and incubated for 7 and 10 days.

Reference substance (positive control):

yes

Remarks:

 K2Cr2O7 and/or 3,5 dichlorophenol.

Duration:

95 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

95 h

Dose descriptor:

LOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

95 h

Dose descriptor:

EC10

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

95 h

Dose descriptor:

EC50

Effect conc.:

> 0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

95 h

Dose descriptor:

EC10

Effect conc.:

0.05 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

95 h

Dose descriptor:

EC50

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

95 h

Dose descriptor:

EC90

Effect conc.:

> 0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

70 h

Dose descriptor:

EC50

Effect conc.:

> 0.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

70 h

Dose descriptor:

EC50

Effect conc.:

0.18 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

70 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

70 h

Dose descriptor:

EC10

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

70 h

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: This NOEC is reported in the study report, however there is significant uncertainty associated with this value.

Details on results:

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

- At the end of the incubation, growth in the 3.3 mg/l flasks was higher than in the control, confirming the algistatic nature of the test substance.

- 70 h results should be used, as 95 h pH deviates by > 1.5 units.

- ErC50 result of >0.55 mg/l has been extrapolated to 1.3 mg/l (0.98 -1.82 mg/l). The limit of solubility for the test substance is 0.93 mg/L, therefore ErC50 is greater than the water solubility.

Reported statistics and error estimates:

EbC and ErC were calculated by means of a parametric model developped by Kooijman. EbC were calculated as recommended in the OECD guideline.

NOEC (Calculated no-effect-concentration) at 95 hours (model calculations using the DEBtox software)

Table 1. Results of analysis of test media without algae at start of test and with algae at end of test

Nominal concentration (mg/L)	Measured concentration at start of test (mg/L)	Percentage of nominal	Measured concentration at end of test (mg/L)	Percentage of nominal	Geometric mean measured concentration (mg/L)
0 (Control)	0		0
0.1	Not analysed	-	Not analysed	-	-
0.33	0.23	69.2	0.01	4.1	0.045
1.0	0.21	21.3	0.06	5.5	0.15
1.8	0.34	18.9	0.19	10.4	0.28
3.3	0.68	20.7	0.72	21.9	0.55
Average (excluding 0.33 mg/L nominal)		20.3		12.6

Table 2. Areas under the growth curves (A) and percentage reductions (IA) in each treatment

Parameter	Nominal concentration of hexamethyldisiloxane (mg/L)
	Control	TBA Control	0.10	0.33	1.0	1.8	3.3
A t=0-70h	489	437	426	323	257	184	106
IA	0	0	8	30	44	60	77
A t=0-95h	2151	2076	2002	1904	1373	860	530
IA	0	0	5	10	35	59	75

Table 3.Average-specific growth rates and percentage inhibition in each treatment

Nominal concentration of hexamethyldisiloxane (mg/L)	Growth rate  (per day) (3d)	% inhibition (3d)	Growth rate  (per day) (4d)	% inhibition (4d)	Mean inhibition %
					3d	4d
Control	1.98	-2.3	1.70	3.0
Control	1.98	-2.3	1.77	-1.1
Control	1.94	-0.1	1.77	-1.2	0.0	0.0
TBA Control	1.97	-1.6	1.73	1.5
TBA Control	1.91	1.4	1.79	-1.9
TBA Control	1.85	4.8	1.76	-0.2
0.10	1.95	-0.4	1.75	0.2
0.10	1.83	5.5	1.72	1.6	2.0	0.3
0.10	1.92	0.9	1.77	-0.8
0.33	1.69	12.7	1.74	0.8
0.33	1.78	8.4	1.76	-0.7	8.4	-0.8
0.33	1.86	4.1	1.79	-2.3
1.0	1.70	12.4	1.65	5.7
1.0	1.59	17.8	1.70	3.0	12.5	4.6
1.0	1.80	7.3	1.66	5.0
1.8	1.65	14.9	1.47	16.2
1.8	1.49	23.2	1.55	11.5	18.3	12.5
1.8	1.61	16.9	1.58	9.7
3.3	1.25	35.7	1.29	26.2
3.3	1.20	38.0	1.21	30.7	31.6	21.8
3.3	1.53	21.1	1.61	8.3

Table 4. Test results (based on the geometric mean measured concentrations)

Parameter	70 hours	95 hours
NOEC	0.01 mg/l	0.15 mg/l
NEC	n.d.	0.05 mg/l
EbC10	0.01 mg/l	0.05 mg/l
EbC50	0.18 mg/l	0.22 mg/l
EbC90	>0.55 mg/l	>0.55 mg/l
ErC10	0.09 mg/l	0.14 mg/l
ErC50	>0.55 mg/l	>0.55 mg/l
ErC90	>>0.55 mg/l	>>0.55 mg/l

Validity criteria fulfilled:

yes

Conclusions:

A 70-hour ErC50 value of >0.55 mg/L, ErC10 value of 0.09 mg/L and NOEC of 0.1 mg/L have been determined for the effects of the test substance on growth rate of Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata). However, given the uncertainty in the calculation of the NOEC, the present reviewers consider it is not a reliable result of the test, therefore the ErC10 value is considered to be the most useful long term test result from this study.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-08-07 to 2009-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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: Water samples were daily from each test chamber. Water samples (10 mL) were collected using a 10 mL volumetric pipette and placed in a 20-mL glass scintillation vial.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The nominal test concentrations were selected on the basis of the reported water solubility of the test substance 34 μg/L. A preliminary stock solution was prepared by adding 0.00333 g of the substance to a 10 mL volumetric flask (stock concentration 333 mg/L). The flask was then brought to volume with acetone. The specific activity of the primary stock solution was determined to be 173.4 μCi/mg. Four additional stock solutions were prepared at concentrations of 167, 83, 42 and 21 mg/L by 50% serial dilution of the primary stock with acetone. Acetone only (0.1 mL/L) was used for the solvent control.

To prepare the test solutions, 0.5 mL of the appropriate stock was added to 5 L algal assay medium in a 5 L volumetric flask. The flask was then inverted to mix. The resulting solutions were clear and colourless. Approximately 300 mL of test solution was added to each of thirteen replicates and the media blank for each treatment group. Remaining test solution in the volumetric flasks was used for initial water quality measurements.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM

- Strain: UTEX 1648, obtained in March 1998 from the University of Texas at Austin

- Source (laboratory, culture collection): In-house cultures

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24.0 to 24.1 ºC

pH:

7.0 - 7.3 at start of test

7.1 - 7.5 on Day 1

7.2 to 7.5 on Day 2

8.9 to 9.7 on Day 3

pH of test media blanks ranged between 7.2 and 7.3

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Solvent control), 2.1, 4.3, 8.5, 17 and 34 μg/L.

Geometric mean measured concentrations: -, -, 0.5, 1.2, 2.3, 4.6 and 9.4 μg/L

The geometric mean measured concentrations in the treated media were 24, 28, 27, 27 and 28% of the nominal values respectively

The test results are reported and interpreted with reference to the geometric mean measured concentrations.

Details on test conditions:

TEST SYSTEM

- Test vessel: 300 mL BOD bottles

- Type (delete if not applicable): sealed with a glass stopper

- Material, size, headspace, fill volume: glass, 300 mL, completely full and containing two glass marbles to aid continuous suspension of the algae throughout the test.

- Aeration: None

- Initial cells density: 5000 cells/mL

- Control end cell density: 755844 (Control), 699756 (solvent control)

- No. of vessels per concentration (replicates): 13

- No. of vessels per control (replicates): 13

- No. of vessels per vehicle control (replicates): 13


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: Algal assay Medium (AAM) based on ASTM Guideline E 1218-04 except that the concentration of NaHCO3 was increased from 15 mg/L to 300 mg/L to supply the CO2 needed in the closed bottle test system.

- Culture medium different from test medium: No

- Intervals of water quality measurement: Daily


OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: yes

- Photoperiod: Continuous

- Light intensity and quality: Continuous cool fluorescent white lighting at a range of 412 - 825 foot-candles (4440 to 8880 Lux).


EFFECT PARAMETERS MEASURED: Cell concentrations measured daily

- Determination of cell concentrations: Coulter Counter


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield (equivalent to biomass)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.4 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield (equivalent to biomass)

Details on results:

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

Reported statistics and error estimates:

The NOEC concentrations were determined using Dunnett's test. The analysis was performed using TOXSTAT Version 3.5 software.

Table 1. Results of analysis of test media

Nominal concentration (μg/L)	Geometric mean (μg/L)	Percent of nominal
0 (Control)	<LoQ*	-
0 (Solvent control)	<LoQ*	-
2.1	0.50	24
4.3	1.2	28
8.5	2.3	27
17	4.6	27
34	9.4	28

*Limit of Quantitation

Table 2. Test results

Geometric mean measured concentration (μg/L)	Mean cell density at start of test (cell/mL)	Mean cell density after 24 hours (cell/mL)	Mean cell density after 48 hours (cell/mL)	Mean cell density after 72 hours (cell/mL)	Inhibition of growth rate (%)*	Inhibition of yield (biomass) (%)*
0 (Control)	5000	12109	102562	755844	-	-
0 (Solvent control)	5000	16647	92947	699756	-	-
0.50	5000	16527	106527	662889	1.1	5.3
1.2	5000	17698	108558	662978	1.1	5.3
2.3	5000	16489	111276	648533	1.6	7.4
4.6	5000	15196	105311	654933	1.3	6.5
9.4	5000	14084	98676	774267	-2.0	-11

*Compared with solvent control

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of >9.4 μg/L and NOEC of ≥9.4 μg/L have been determined for the effects of the test substance on growth rate and biomass (yield) of Pseudokirchnerella subcapitata based on mean measured concentrations.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-29 to 2008-06-05

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:

Radioactivity in samples was measured daily using liquid scintillation counting (LSC). Samples were collected from each test vessel at mid-depth of the test solutions. Samples (10 mL) were collected using a 10-mL volumetric pipette and transferred to a 20-mL glass scintillation vial.

Vehicle:

yes

Details on test solutions:

The nominal test concentrations were selected based on the reported solubility of the test article (6.74 μg/L). The nominal test concentrations were: 0.42, 0.84, 1.7, 3.4 and 6.7 μg/L. A primary stock was prepared by isotopic dilution of the 14C-L4 with nonradiolabeled L4. Approximately 2 mL of DMF (dimethylformamide) was transferred to a 10-mL conical vial with a septum cap. A 7.0 μL aliquot of 14C-L4 (0.00660 g) and a 37.0 μL aliquot of L4 (0.03167 g) was added to the vial through the septum cap. The total weight of 14C-L4 and L4 was 0.03827 g. The contents of the vial were transferred to a 500-mL volumetric flask, brought to volume with DMF, and mixed well giving a final concentration of 77 mg/L. Three 0.05 mL aliquots of the primary stock were added to 10 mL of scintillation cocktail and counted by LSC. The average of the 3 aliquots was 413, 154 dpm. The specific activity of the primary stock was calculated as 48.6 μCi/mg.

Four additional stock solutions were prepared at concentrations of 39, 20, 10 and 5 mg/L by serial dilution of the primary stock with DMF. DMF (0.1 mL/L only was used for the solvent control. To prepare the test solutions, 0.5 mL of the appropriate stock was added to 5 L of algal assay medium (AAM) in a 5-L volumetric flask. The flask was then inverted to mix. Approximately 300 mL of test solution was added to each of the thirteen
replicates and the media blank for each treatment group. Remaining test solution in the volumetric flask was used for initial water quality measurements.

Test organisms (species):

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

Details on test organisms:

Source
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.

Organism Holding Conditions
Algal cultures were maintained at 24 ± 2 °C 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.

Test organisms were impartially added to the test vessels by transferring a specific density of algal cells (nominal concentration equal to 5,000 cells/mL) from a test inoculum into each vessel. In addition, the test vessels were indiscriminately positioned daily on 4 shaker tables in an incubator.

Nutrient Medium
Algal Assay Medium was prepared based on ASTM guideline E 1218-04 with two exceptions. The concentration of NaHC03 was increased from 15 mg/L to 300 mg/L to supply needed C02 in a closed bottle system. The pH of the medium was adjusted to 7.0±0.1 as recommended in ISO 14442. 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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

24.1 to 24.4°C

pH:

Measurements of pH at test initiation ranged from 7.1 to 7.4. On day 1 and 2, pH in the flasks that contained algae ranged from 7.0 to 7.4 and from 7.0
to 7.3, respectively. At test termination, pH in the flasks that contained algae ranged from 8.8 to 9.6 and pH of media blanks ranged from 6.9 to 7.2.

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal test concentrations were 0.42, 0.84, 1.7, 3.4, and 6.7 μg/L with a control and solvent (DMF) control.

Geometric mean measured test concentrations were calculated to be 0.15, 0.33, 0.70, 1.4 and 2.2 μg/L.

Measured test concentrations at test initiation ranged from 64 to 77 percent of nominal. On Day I and 2 measured test concentrations ranged from 38 to 57 percent of nominal and 32 to 45 percent of nominal, respectively. Measured test concentrations at test termination ranged from 14 to 20 percent of nominal.

Details on test conditions:

Test Apparatus
Test vessels were sterile 300-mL biological oxygen demand (BOD) bottles sealed with a glass stopper. Each test vessel contained two glass marbles to enhance mixing of cells. Test vessels were completely filled with test solution without headspace.

Test Conditions
Test vessels were placed in a temperature controlled incubator and positioned on rotary shakers set at approximately 100 rpm. In addition, the incubator was set at a temperature of 24 ± 2 °C 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 termination.

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 three test vessels of each treatment group at 24, 48 and 72 hours (within I hour). Prior to sampling, test solutions were emptied into 500-mL glass beakers. The inside of the test vessels were rinsed two times with the test solution using a 60 cc syringe. They were shaken vigorously to ensure all algal cells had been removed from the sides of the test vessel. Approximately I mL of the test solution was collected and diluted with Isoton® II diluent solution. Dilution volumes were recorded in the raw data. Samples were counted immediately using a Coulter® Counter. 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.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 2.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 2.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): none reported

Reported statistics and error estimates:

There was insufficient inhibition of growth rate to determine an EC50 value. The NOEC was determined using Dunnett's test.

Table1. Measurement of Test Concentrations

 

Nominal Concentration (μg/L)	Day 0	Day 1	Day 2	Day 3	Geometric mean (μg/L)*
Negative Control	<LOQ^1	<LOQ	<LOQ	<LOQ	-
Solvent Control	<LOQ	<LOQ	<LOQ	<LOQ	-
0.42	0.27	0.16	0.17	0.06	0.15
0.84	0.58	0.34	0.38	0.17	0.33
1.7	1.2	0.88	0.77	0.28	0.70
3.4	2.5	1.9	1.2	0.65	1.4
6.7	5.1	2.6	2.1	0.91	2.2

¹The Limit of Quantitation (LOQ) for the analysis was equivalent to 0.042 μg/L.

*The geometric mean was calculated because the deviation from measured initial concentration was not within ±20%.

 

Table2. Test results

 

Nominal Concentration (μg/L)	Mean cell density after 72 hours (cells/mL)	Mean yield after 72 hours (cells/mL)	% inhibition relative to solvent control	Mean growth rate 0-72 hours	% inhibition relative to solvent control
Negative Control	1147978	1142978	-	1.8119	-
Solvent Control	993911	988911	-	1.7631	-
0.42	905156	900156	9.0	1.7327	1.7
0.84	1004778	999778	-1.1	1.7658	-0.2
1.7	875311	870311	12.0	1.7208	2.4
3.4	795022	790022	20.1*	1.6874	4.3*
6.7	886178	881178	10.9	1.7254	2.1

*Indicates a significant difference from the solvent control using Dunnett's test(p ≤0.05).

Validity criteria fulfilled:

yes

Conclusions:

A 72-hour EC50 value of >2.2 μg/L and NOEC of ≥2.2 μg/L have been determined for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata. The results are expressed as geometric mean measured concentrations.

Description of key information

HMDS: Toxicity to algae: 70-h ErC₅₀ >0.55 mg/l and ErC₁₀ 0.09 mg/l (geometric mean measured concentration), growth rate of Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata).

L3: Toxicity to algae: 72-h EC₅₀ >9.4 μg/l and NOEC ≥9.4 μg/l (measured, highest concentration tested) growth rate and biomass (yield) of Pseudokirchneriella subcapitata.

L4: Toxicity to algae: 72-h ErC₅₀ >2.2 μg/l and NOEC: ≥2.2 μg/l (geometric mean measured, highest concentration tested), growth rate of Pseudokirchneriella subcapitata.

L5: Toxicity to algae: 72-h EC₅₀: >2.2 μg/l and NOEC: ≥2.2 μg/l (geometric mean measured concentration), growth rate of Pseudokirchneriella subcapitata, read-across from L4.  

 

Additional information

Constituent HMDS

A 70-hour ErC₅₀ value of >0.55 mg/l and ErC₁₀ value of 0.09 mg/l were determined for the effects of HMDS on growth rate of Selenastrum capricornutum (TNO, 2003). The study is considered reliable with restrictions. The restrictions concern the biological variability, pH change in test and control cultures, and issues with analytical recoveries. In view of the duration of the exposure period it is likely that the test organisms were exposed primarily to the parent form of the tested substance, although losses were observed by the end of the study. A NOEC of 0.01 mg/l was also reported but is not considered to be reliable, given the considerable variability in biological responses seen in the study. Results at the 95-hour time point are considered not valid, because the pH deviated by more than 1.5 units.

 

Constituent L3

A 72-hour EC₅₀ value of >9.4 μg/l and NOEC of ≥9.4 μg/l (measured, highest concentration tested) have been determined for the effects of L3 on growth rate and biomass (yield) of Pseudokirchneriella subcapitata based on mean measured concentrations (DCC, 2009). Chemical analysis of the test substance showed a decline in recoveries throughout the test period. This may be due to losses through volatility and adsorption, but may also be due to hydrolysis of the parent substance. The pH of the test media will influence hydrolysis rate. The measured pH was between 8.9 and 9.7 on day three of the test; this is likely to have accelerated the hydrolysis rate. In view of the duration of the exposure period it is likely that the test organisms were exposed primarily to the parent form of the tested substance but it is probable that they were also exposed to the hydrolysis products, particularly towards the end of the test.

 

Constituent L4

A 72-hour ErC₅₀ value of >2.2 μg/l and NOEC of ≥2.2 μg/l (geometric mean measured, highest concentration tested) have been determined for the effects of L4 on growth rate of Pseudokirchneriella subcapitata (Dow Corning, 2008). Chemical analysis of the test substance showed a decline in recoveries throughout the test period. This may be due to losses through volatility and adsorption, but may also be due to hydrolysis of the parent substance. The pH of the test media will influence hydrolysis rate. The measured pH was between 8.8 and 9.6 on day three of the test; this is likely to have accelerated the hydrolysis rate. In view of the duration of the exposure period it is likely that the test organisms were exposed primarily to the parent form of the tested substance but it is probable that they were also exposed to the hydrolysis products, particularly towards the end of the test.

 

Constituent L5

A 72-hour ErC₅₀ value of >2.2 μg/L and NOEC of ≥2.2 μg/L have been determined for the effects of L4 (decamethyltetrasiloxane, CAS 141-62-8) on growth rate of Pseudokirchneriella subcapitata (Dow Corning, 2008) and the results read across to Constituent L5. In view of the duration of the exposure period it is likely that the test organisms were exposed primarily to the parent form of the tested substance but it is probable that they were also exposed to the hydrolysis products, particularly towards the end of the test. The results are expressed as geometric mean measured concentrations.