Dodecamethylcyclohexasiloxane

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-12-02 to 2008-12-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Prior to test initiation and before division into the replicate exposure chambers, a sample of sediment from each treatment level and control was removed and analysed. In addition, subsamples of the dosing stock solutions used to dose the sediments were also analysed for test substance concentration. Results of the pretest analyses were used to judge whether sufficient quantities of D5 had been applied to the sediment.

During the in-life phase of the definitive study, sediment samples were removed and analysed for D5 concentration on test days 0, 7 and 28. On day 0, samples were removed and analysed from replicate I of all treatment levels and controls. On day 7, samples were removed and analysed from replicate J of all treatment levels and controls, while on day 28, samples were removed and analysed from replicate K of all treatment levels and controls. Overlying water samples were removed from the test vessels by decanting all of the overlying water from each vessel into a graduated cylinder. Following removal of the overlying water, the sediment was then centrifuged at approximately 1200 g for 30 minutes. The pore water generated by centrifugation was then removed by pipet. The sediment samples were then collected from each centrifuge tube with a stainless steel spatula.

In addition, three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure samples throughout the analytical process. These QC samples were prepared in sediment (based on sediment dry weight) at concentrations of D5 similar to the treatment level range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT

- Details of spiking: Six individual dosing stock solutions (49.4, 24.7, 12.4, 6.2, 3.06 and 1.58 mg a.i./mL) were prepared in DMF for application of the test substance to the sediment. All dosing stock solutions were observed to be clear and colourless with no visible undissolved test substance following preparation. For each dose level, a 1.5-kg aliquot of wet sediment (0.8145 kg dry weight based on a percent solids of 54.30%) along with 4.5 L of overlying water (sediment:volume ratio of 3:1) were placed in individual glass jars. Immediately prior to dosing, the contents of each jar were shaken to suspend the sediment. An 8.25-mL volume of each dosing stock solution was then added to each jar. Each jar was shaken again after dosing to distribute the test substance. After a four-hour settling period, the water was decanted from the sediment and the treated sediment was allocated to the individual test vessels.

A solvent control sample was prepared in the same manner as the treated sediment by adding 1.5 kg of wet sediment (0.8145 kg dry weight based on a percent solids of 54.30%) along with 4.5 L of overlying water (sediment:volume ratio of 3:1) and 8.25 mL of DMF in a glass jar and processed in the same manner as the treated sediments. The negative control sediment group was prepared using only untreated sediment and overlying water (no test substance or solvent). The negative control and solvent control vessels were maintained under the same conditions as the treatment vessels.

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM

- Source and culture conditions: The amphipods used during this study were obtained from laboratory cultures maintained at Springborn Smithers. Prior to test initiation, amphipods were maintained in 20-L glass aquaria containing approximately 15 L of culture water under flow-through conditions. The culture water was from the same source as water used as overlying water during the test.

Amphipods (8 days old) used in the exposure were collected from reproducing adult amphipods removed from the main culture tanks 9 days prior to test initiation. The adult amphipods were placed in 9.5-L aquaria (isolation tanks) containing approximately 8 L of water. Juvenile amphipods (< 24 hours old) produced by these isolated adults were then removed from the isolation tanks and pipetted into 1-L beakers containing approximately 0.80 L of laboratory dilution water. The juvenile amphipods were reared under static conditions for 8 days with gentle oil-free aeration. During the holding period, dissolved oxygen ranged from 8.3 to 8.6 mg/L and temperature was maintained at 22 ºC. No mortality was observed in the test population 48 hours prior to test initiation.

During holding and acclimation, the freshwater amphipods were fed 2.5 mL of a combination of yeast, cereal leaves and flaked fish food suspension (YCT) and 2.5 mL of Ankistrodesmus falcatus, a unicellular green algae, every other day. During the 28-day exposure, each replicate test vessel received 1.5 mL of YCT daily. Representative samples of the food source were analysed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations considered toxic in any of the samples analysed. Based on these analyses, food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

Hardness ranged from 44-52 mg/L as CaCO3 and alkalinity 10-20 mg/L as CaCO3

Test temperature:

22 to 24 ºC

pH:

6.7-7.1

Dissolved oxygen:

40 - 100% saturation ( 3.4 - 8.4 mg/L)

Salinity:

Not applicable

Ammonia:

Day 0: 0.44-0.81 mg/L as nitrogen

Day 28: <0.10 mg/L as nitrogen

Nominal and measured concentrations:

Nominal concentrations: 0(Control), 0 (solvent control), 16, 31, 63, 125, 250 and 500 mg a.i./kg

Mean measured concentrations in treatments 18, 28, 62, 130, 230 and 460 mg a.i./kg

The results are interpreted with reference to mean measured concentrations

Details on test conditions:

TEST SYSTEM

- Test container (material, size): The test chambers used in the static-renewal test were 300-mL glass vessels which were chemically cleaned prior to use and rinsed several times using dilution water. Each test vessel had a hole cut on the top edge of the beaker which was covered with 40-mesh Nitex® screen for drainage.

- Sediment volume: Each vessel contained 100 mL (approximately 4-cm layer) of sediment (equivalent to 127 g wet weight per vessel or 69.2 g dry weight per vessel). The total overlying/sediment volume was maintained at approximately 275 mL.

- Overlying water volume: 175 mL. The overlying water was renewed by adding two volume additions (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system.

- Aeration: no


EXPOSURE REGIME
- No. of organisms per container (treatment): 10

- No. of replicates per treatment group: 12 replicates total. Eight replicates (A through H) were used to evaluate the biological response of the test organisms. The remaining four replicates (I through L) were maintained for the purpose of chemical analysis.

- No. of replicates per control / vehicle control: 12

- Feeding regime: During the 28-day exposure, each replicate test vessel received 1.5 mL of YCT daily. Representative samples of the food source were analysed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected at concentrations considered toxic in any of the samples analysed.


OVERLYING WATER CHARACTERISTCS

The water used during the definitive exposure was characterised as having a total hardness and total alkalinity as calcium carbonate (CaCO3) of 54 mg/L and 20 mg/L, respectively, a pH of 7.1 and a specific conductivity of 230 µmhos/cm. Representative samples of the dilution water source were analysed for the presence of pesticides, PCBs and metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected in any of the water samples analysed in agreement with ASTM guidelines (2002). In addition, representative samples of the dilution water source were analysed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water source was 0.69 mg/L for December 2008.


SOURCE OF NATURAL SEDIMENT

The sediment (Springborn Smithers Batch No. 091908) used during this study was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to use and characterisation, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterised by Agvise Laboratories, Northwood, North Dakota, as having a percent organic carbon of 4.8%, a particle size distribution of 83% sand, 12% silt, and 5% clay, a pH of 6.0 and a percent moisture at 1/3 bar (water holding capacity) of 28.6%. The percent solids was measured by Springborn Smithers Laboratories to be 54.30%. A sample of the sediment pore water was generated from the sediment batch prior to testing and yielded a measured concentration of 2.4 mg/L as nitrogen (ammonia). This concentration is well below levels of concern for this organism. A representative sample of the sediment source was analysed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that would be considered to have an adverse impact on the results of the test.


OTHER TEST CONDITIONS

- Lighting: The test area was illuminated with fluorescent bulbs at an intensity range of 840 to 870 lux

- Photoperiod: 16 hours light and 8 hours darkness


EFFECT PARAMETERS MEASURED: All vessels were examined at test initiation and at 24-hour intervals thereafter, until test termination (day 28). Observations of mortality and abnormal behaviour were made and the physical characteristics of the test samples were recorded. At test termination (day 28), the total number and growth of surviving amphipods was determined in each test vessel. The remaining sediment was sieved to remove all surviving amphipods. Growth of the surviving amphipods were determined by pooling all surviving amphipods from each replicate in a tared weighing tin and drying at 60 ± 5 ºC for approximately 24 hours. The pooled amphipods were then weighed on a calibrated analytical balance to the nearest 0.01 mg. Growth as dry weight per amphipod for each replicate was calculated using the following equation:

G = TW/NS

Where:
G = Growth as dry weight per amphipod
TW = Total weight of surviving amphipods
NS = Number of surviving amphipods


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

230 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

310 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 210-360

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 130 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Reported statistics and error estimates:

The linear interpolation method within TOXSTAT® Version 3.5 was used to determine the LC50 value for survival.

During this study, no concentration tested resulted in ≥ 50% reduction in growth, therefore the EC50 value was empirically estimated to be greater than the highest mean measured sediment concentration that was statistically analysed.

At the termination of the study, data obtained on amphipod survival and growth (dry weight) were statistically analysed to identify significant treatment-related effects. The lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant effect (No-Observed-Effect Concentration, NOEC) were determined. Analyses were performed using the mean replicate organism response in each treatment group rather than individual response values. All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks', Chi-Square Test and Bartlett's Test, in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests.

Table 1. Mean percent survival and mean dry weight during the 28-day static-renewal exposure of freshwater amphipods (Hyalella azteca) to D5 applied to sediment.

 

Mean Measured Sediment Concentration (mg a.i./kg)	Test Day 28
	Mean Percent Survival (SD^a)	Mean Dry Weight Per Larvae in mg (SD)
Control	98 (5)	0.49 (0.07)
Solvent Control	84 (31)	0.54 (0.12)
18	94 (5)	0.51 (0.06)
28	83 (27)	0.36 (0.08)^b
62	98 (7)	0.42 (0.07)
130	93 (7)	0.46 (0.04)
230	64 (37)^c	0.39 (0.08)^d
460	19 (16)^c	0.40 (0.18)^d

 

^aSD = Standard Deviation.

^bSignificantly reduced compared to the negative control, based on Dunnett’s Test. However, due to the lack of statistical significance at the higher treatment levels tested (i.e., 62 and 130 mg a.i./kg), this effect was not considered to be biologically relevant.

^cSignificantly reduced compared to the negative control, based on Steel’s Many-One Rank Test.

^dThis treatment level was excluded from statistical analysis for growth due to the survival effect observed.

Table 2. Concentrations of D5 measured in sediment samples during the 28‑day static-renewal exposure of freshwater amphipods (Hyalella azteca) to D5 applied to sediment.

 

Nominal Sediment Concentration (mg a.i./kg)	Measured Sediment Concentration (mg a.i./kg)				Mean Percent Recovery^d
	Day 0^a	Day 7^b	Day 28^c	Mean^d
Control	< 2.0^e	< 2.0	< 2.0	NA^f	NA
Solvent Control	< 2.0	< 2.0	< 2.0	NA	NA
16	14	22	19	18	110
31	33	23	29	28	91
63	66	63	57	62	99
125	120	150	140	130	110
250	170	220	300	230	92
500	390	500	490	460	92
QC^g#1	3.00	2.34	1.53
2.00	(150)^h	(117)	(76.4)
QC#2	51.4	63.0	49.0
50.0	(103)	(126)^h	(98.0)
QC#3	535	520	550
500	(107)	(104)	(110)

 

^aAnalytical samples were removed from replicate I.

^bAnalytical samples were removed from replicate J.

^cAnalytical samples were removed from replicate K.

^dMean measured and percent recovery values were calculated using the actual analytical results and not the rounded values (two significant figures) presented in this table.

^eConcentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for this analysis was set at 2.0 mg a.i./kg, the lowest validated concentration.

^fNA = Not Applicable.

^gQC = Quality Control sample. Percent of nominal is presented in parentheses below the measured results.

^hPercent recovery for this QC sample is outside of the acceptable range (i.e., 70.0 to 120%,Appendix 2).

 

Validity criteria fulfilled:

yes

Conclusions:

A 28-day LC50 of 310 mg/kg dry weight has been determined for the effects of the test substance on mortality of Hyalella azteca when exposed via sediment. A 28-day NOEC of 130 mg/kg and a LOEC of >130 mg/kg have been determined for effects on growth rate.

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010-06-14 to 2010-07-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 225

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Stock solutions used to dose the exposure solutions were analysed at the approximate time of dosing. In addition, a sample of sediment from each treatment level and control were analysed prior to test initiation to confirm that the appropriate levels were achieved.

During the in-life phase of the definitive study, sediment samples were removed and analysed for D6 concentration on test days 0 (test initiation), test day 7 and test day 28 (test termination). On test days 0, 7 and 28, samples were removed and analysed from replicate vessels E, F and G, respectively, for each treatment level and vessels G, H and I for the controls. Overlying water from each replicate vessel was decanted and discarded. Pore water samples were collected by removing the entire sediment sample and centrifuging for 15 to 30 minutes at approximately 10,000 g. The resulting pore water was removed from the centrifuge tube and discarded. Sediment samples were collected with a stainless steel spatula from the centrifuge tubes, following centrifugation and removal of the pore water samples.

In addition, three quality control (QC) samples were prepared at each sampling interval and were stored and analysed with the set of study samples. These QC samples were prepared in sediment at concentrations of D6 similar to the treatment level range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of sediment samples.

Vehicle:

yes

Details on sediment and application:

PREPARATION OF TREATMENT SOLUTIONS

A 200 mg/mL primary stock solution was prepared by placing 5.0003 g of D6 into a 25-mL volumetric flask and bringing it to volume with acetone (CAS No. 67-64-1). The resulting stock solution was observed to be clear and colourless with no visible undissolved test substance. Further treatment solutions were prepared by dilution. All solutions were observed to be clear and colourless with no visible undissolved test substance following preparation.

PREAPARATION OF TREATED SEDIMENTS

For each dose level, a 2.5-kg aliquot of wet sediment (1.3383 kg dry weight based on a percent solids of 53.53%) along with 7.5 L of fortified well water (sediment:volume ratio of 3:1) were placed in individual glass jars. Immediately prior to dosing, the contents of each jar were shaken to suspend the sediment. An 8.3-mL volume of each dosing stock solution was then added to each jar. Each jar was shaken again after dosing to distribute the test substance. Following settling for approximately 24 hours, the water was decanted from the sediment and 3.35 g each of urtica powder and alpha cellulose were mixed into the sediment of each test group as a food source for the oligochaetes. This weight of urtica powder and alpha cellulose was equal to 0.25% of the dry weight for the sediment batch. The final dry weight of the sediment plus added food was 1.345 kg. Following mixing by hand, the treated sediments were allocated to the replicate test vessels for each exposure level.

A solvent control sample was prepared in the same manner as the treated sediment by adding 8.3 mL of acetone, containing no test substance, to 2.5 kg of wet sediment suspended in 7.5 L of fortified well water. The negative control sediment consisted of 2.5 kg of unadulterated wet sediment suspended in 7.5 L of fortified well water without the addition of solvent or test substance. Both the solvent and negative control sediments were also allowed to settle for approximately 24 hours after shaking. The water was then decanted from the control sediments and the sediments were allocated to the appropriate replicate test vessels.

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM

- Source: The oligochaetes used during this study were obtained from the main laboratory cultures maintained at Springborn Smithers.

- Culture conditions: The oligochaetes from this culture were maintained in an aquarium under flow-through conditions. The water used in the main culture is laboratory well water from a 100-meter bedrock well and has been characterised as soft water with typical ranges as total hardness of 30 to 60 mg/L as CaCO3, alkalinity of 25 to 45 mg/L as CaCO3, pH of 6.9 to 7.7, and a specific conductance of 350 to 450 micromhos/cm. Oligochaetes were fed approximately 15 mL of finely ground suspension (100 mg/mL) of flaked fish food weekly.

- Test organism production: Thirteen to fourteen days prior to test initiation, worms from the culture were removed and artificially fragmented using a scalpel to remove the anterior ends and placed in an aquarium with test sediment and laboratory well water under flow-through conditions. Synchronised oligochaetes were then held under test conditions (i.e., water and test sediment) for a period of thirteen to fourteen days to regenerate new heads. During this period, the synchronised population was acclimated to test conditions by renewing the overlying water in the aquarium with dilution water (fortified well water) at the rate of approximately half a volume replacement per day. The synchronisation of worms is performed to avoid “uncontrolled” regeneration and reproduction that may contribute to high variation in the test results. Measured water quality parameters during the synchronisation period prior to test initiation yielded a dissolved oxygen concentration range of 7.8 to 9.0 mg/L, a pH range of 7.5 to 8.0 and a temperature range of 21 to 23 ºC. The photoperiod in the culture area was 16 hours light and 8 hours dark.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

180 mg/L as CaCO3

Test temperature:

18 to 22 °C

pH:

5.6 to 7.9

Dissolved oxygen:

7.1 to 9.2 mg/L

Salinity:

Not applicable

Ammonia:

4.4 to 9.4 mg/L (as N)

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Vehicle control), 63, 130, 250, 500 and 1000 mg/kg dry weight

Mean measured concentrations in the treated sediments ranged from 41 to 54% of nominal concentrations and defined the treatment levels tested as 34, 53, 120, 250 and 420 mg/kg dry weight.

Details on test conditions:

TEST SYSTEM

- Test vessels: The test vessels used were 600-mL clear glass beakers. The sediment and overlying water were added to the test vessels two days prior to test initiation. A 75-mL (1.5-cm layer) aliquot of sediment was added to each test vessel. The wet weight of the sediment in each jar averaged 96 g (51 g dry weight). A turbulence reducer, consisting of a plastic disk, was used to minimize the disruption of the sediment layer during the introduction of 300 mL (6 cm) of overlying water. The total medium volume (sediment/water) was maintained at 375 mL and the ratio of sediment to water was 1:4. The initial water level in each test vessel was marked in order to evaluate evaporation. Each test vessel was covered with a clear plastic plate to minimise evaporation and gentle aeration was provided through a glass pipette fixed approximately 2 to 3 cm above the sediment surface. Aeration was delivered at a rate of 1 to 3 bubbles per second.

- Sediment: The sediment (Springborn Smithers Batch No. 072009) used during this study was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to use and characterisation, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterised by Agvise Laboratories, Northwood, North Dakota, as having a percent organic carbon of 3.2%. The particle size distribution conducted by Agvise Laboratories resulted in an average particle size distribution of 88% sand, 10% silt and 2% clay. A sediment pH of 6.1 was also determined at Agvise Laboratories. A percent solids value of 53.53% was determined by Springborn Smithers Laboratories. In addition, a sample of sediment pore water was analysed prior to test initiation for ammonia concentration. The total ammonia concentration of the sediment pore water sample was 22 mg/L as nitrogen (ammonia). A representative sample of the sediment source was analysed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that would be considered to have an adverse impact on the results of the test.

- Overlying water: Overlying water used during this study was laboratory well water. The water used during the definitive exposure was characterised as having total hardness and total alkalinity as calcium carbonate (CaCO3) of 180 and 86 mg/L, respectively, a pH of 8.3, and a specific conductivity of 600 micromhos per centimeter (µmhos/cm).

- Replication: Four replicate test vessels (A through D) were established for each treatment level and six vessels (A through F) were established for the control and solvent control. Ten additional replicate test vessels (E through N for treatment levels and G through P for the controls) were maintained for the purpose of chemical analysis of the sediment and oligochaete tissues. Each replicate vessel contained ten oligochaetes, a total of 40 oligochaetes per concentration and 60 oligochaetes in the control and solvent control for the biological response replicates. The additional replicates were maintained under the same conditions and contained ten test organisms, however, were not used to evaluate the biological response of the test organisms.

- Test initiation: The spiked sediment and overlying water were added to the test vessels three days prior to addition of the test organisms (day -3). At test initiation, oligochaetes were added impartially to intermediate vessels by adding no more than two oligochaetes to each vessel until all vessels contained two oligochaetes. This procedure was repeated until each vessel contained ten oligochaetes. The test was initiated when each intermediate vessel of oligochaetes was added to each respective test vessel. The ten oligochaetes were released simultaneously into the overlying water of each test vessel just above the sediment surface. A total of 40 oligochaetes for the treatment levels and 60 oligochaetes for the control and solvent control were exposed to the test conditions.

- Observations: Replicate test vessels A through D for treatment levels and A through F for the control and solvent control were examined at test initiation and daily thereafter, until test termination (day 28). Daily observations of mortality and abnormal behaviour (e.g., leaving sediment) were made and recorded.

On test day 28 of the exposure, the replicate vessels from each exposure level (four replicates) and controls (six replicates) were terminated to determine the number of oligochaetes recovered and the biomass of the recovered oligochaetes. Instances of dead oligochaetes were also recorded. However, dead oligochaetes often decompose in the vessels and are difficult to recover. If fewer than ten oligochaetes were observed and no dead oligochaetes were recovered, the missing individuals were assumed to have died. Surviving worms were assigned to one of three groups:

a) Large, complete worms (adults) without regenerated body regions.
b) Complete worms with regenerated, lighter-coloured body regions.
c) Incomplete worms (i.e., recently fragmented worms with non-regenerated body regions.

- Tissue analysis: On test day 28, oligochaetes were removed from six designated replicate vessels per treatment level and the controls by sieving the contents of each replicate vessel through a fine mesh net (0.25 mm). Five replicates were utilised for the control group because one of the designated replicates was inadvertently omitted with respect to the addition of oligochaetes at test initiation. The sieved oligochaetes were then transferred to a shallow glass tray and separated from any remaining sediment. The number of oligochaetes recovered per replicate was recorded.

The biomass was determined by pooling the surviving oligochaetes from each replicate vessel in tared weighing tins and then drying in an oven at 100 ± 5 ºC overnight (18 to 24 hours). Following drying, the tins were cooled to room temperature and then weighed to the nearest 0.01 mg on a calibrated analytical balance.

In addition, three quality control (QC) samples in oligochaete tissue were prepared on day 28 and were stored and analysed with the set of study samples. QC samples were prepared at nominal concentrations of D6 in tissue of 3.65, 36.1 and 386 mg/kg. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of tissue samples.

Tissue samples were analysed for D6 using gas chromatography with mass selective detection (GC/MSD) procedures based on methodology validated at Springborn Smithers. The method validation study established an average recovery of 103% ± 3.17% from oligochaete tissue. The quality control sample acceptance range for subsequent studies with D6 was set at 70 to 120%. Conditions and procedures used throughout the analysis of exposure and QC samples during this study were similar to those used in the method validation study.

- Water quality: Water quality measurements made during the study were performed in the replicate exposure vessels (A through D or A through F). Measurements of dissolved oxygen concentration, temperature and pH were made on the day the test organisms were added (day 0) and at test termination (day 28) in each exposure vessel. In addition, dissolved oxygen concentration, temperature and pH were measured daily in one replicate vessel of each treatment level and the controls during the 28-day exposure. Daily water quality measurements were made on alternating replicates. The temperature was continuously monitored in an auxiliary vessel in the water bath throughout the study. Total hardness, alkalinity, specific conductivity, and total ammonia of the test solutions were determined at test initiation and at test termination in a composite sample from the highest treatment level (replicates F, G and N on day 0 and replicates A through D for the remainder of the study) and the solvent control (replicates H, I and P on day 0 and A through F). In addition, total ammonia was determined three times per week in the solvent control and the 1000 mg/kg nominal treatment level.

- Range finding test: A range finding test was conducted using the same test procedures at nominal concentrations of 0.1, 1, 10, 100 and 100 mg/kg dw. There were no observed differences in the numbers of oligochaetes recovered or their biomass at the end of the test.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 420 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Number of oligochaetes recovered and biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 420 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Number of oligochaetes recovered and biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 420 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Number of oligochaetes recovered and biomass

Details on results:

- Other biological observations: Measured tissue concentrations at day 28 were 10, 18, 33, 50 and 72 mg/kg, respectively, in tissue samples from oligochaetes exposed to the 63, 130, 250, 500 and 1000 mg/kg nominal treatment levels.

Reported statistics and error estimates:

At the termination of the study, data obtained on oligochaetes reproduction (the total number of oligochaetes recovered at test termination) and biomass were statistically analysed to identify significant treatment-related effects. The lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant effect (No-Observed-Effect Concentration, NOEC) were determined. All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks’ Test (Weber et al., 1989) and Bartlett's Test (Sokal and Rohlf, 1981), in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests.

CETIS-Comprehensive Environmental Toxicity Information SystemTM (Ives, 2009) Version 1.7 software was used to perform the computations for the definitive data set. The results were used to establish, at the 95% level of certainty, the lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant difference (No-Observed-Effect Concentration, NOEC) from the pooled control data.

Table 1. Results of analysis of sediment for exposure concentrations of the test substance.   

Nominal Concentration (mg/kg dw)	Measured Concentration(mg/kg dw)			Mean^d	Percent of Nominal^d
	Day 0^a	Day 7^b	Day 28^c
Control	< 4.4	< 4.4	< 4.1	NA^e	NA
Solvent Control	< 4.4	< 4.4	< 4.1	NA	NA
63	31	37	34	34	54
130	62	51	47	53	41
250	140	110	120	120	49
500	270	220	260	250	50
1000	490	460	320	420	42

 

^a      Analytical samples were removed from replicate E, with the exception of the control and solvent control which were removed from replicate G.

^b      Analytical samples were removed from replicate F, with the exception of the control and solvent control which were removed from replicate H.

^c      Analytical samples were removed from replicate G, with the exception of the control and solvent control which were removed from replicate I.

^d      Mean measured and percent recovery values were calculated using the actual analytical results and not the rounded values (two significant figures) presented in this table.

^e      NA = Not Applicable.

 

Table 2. Test results

Mean measured sediment concentration (mg/kg dw)	Mean number of oligochaetes per replicate (SD)	Mean biomass per replicate per concentration in mg (SD)
Control	21 (3)	32 (3)
Solvent Control	21 (4)	32 (2)
Pooled Control	21 (3)	32 (3)
34	24 (2)	30 (2)
53	22 (3)	31 (4)
120	19 (4)	29 (8)
250	19 (5)	29 (1)
420	21 (1)	27 (4)

 

Validity criteria fulfilled:

yes

Conclusions:

A 28-d EC50 value of >420 mg/kg dry weight and a NOEC of ≥420 mg/kg dry weight have been determined for the effects of the test substance on numbers and biomass of Lumbriculus variegatus recovered from treated sediments. The results are expressed relative to mean measured exposure concentrations.

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010-06-07 to 2010-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Stock solutions used to dose the exposure solutions were analysed at the approximate time of dosing. In addition, a sample of sediment from each treatment level and control were analysed prior to test initiation to confirm that the appropriate levels were achieved.

During the in-life phase of the definitive study, sediment samples were collected from each treatment level and control on day 0 (replicate E), day 7 (replicate F) and day 28 (replicate G). Overlying water from each replicate vessel was decanted and discarded. Pure water samples were collected by removing the entire sediment sample and centrifuging for 15 to 30 minutes at approximately 10,000 g. The resulting pore water was removed from the centrifuge tube and discarded. Sediment samples were collected with a stainless steel spatula from the centrifuge tubes, following centrifugation and removal of the pore water samples.

In addition, three quality control (QC) samples were prepared at each sampling interval and were stored and analysed with the set of study samples. These QC samples were prepared in sediment at concentrations of D6 similar to the treatment level range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of sediment samples.

Vehicle:

yes

Details on sediment and application:

PREPARATION OF TREATMENT SOLUTIONS

A 200 mg/mL primary stock solution was prepared by placing 4.9996 g of D6 into a 25-mL volumetric flask and bringing it to volume with acetone (CAS No. 67-64-1). The resulting stock solution was observed to be clear and colourless with no visible undissolved test substance. Further treatment solutions were prepared by dilution. All solutions were observed to be clear and colourless with no visible undissolved test substance following preparation.

PREAPARATION OF TREATED SEDIMENTS

Application of the test substance (day -3) to the sediment occurred prior to the allocation of the sediment to the test vessels. Sediment jars were prepared by adding 1.5 kg of wet sediment (0.8154 kg dry weight based on a percent solids of 54.36%) followed by 4.5 L of laboratory well water and shaking to suspend sediment prior to dosing. Each test concentration was prepared individually by adding the appropriate dosing stock solution directly to each jar and shaking again following dosing to distribute the test substance.

Following settling for approximately 24 hours, water was decanted from the treated sediments and the sediments were allocated to the replicate test vessels for each exposure level. A solvent control sample was prepared in a similar manner as the treated sediment by adding 5.0 mL of acetone, containing no test substance, to 1.5 kg of wet sediment suspended in 4.5 L of laboratory well water. The negative control sediment consisted of 1.5 kg of unadulterated wet sediment suspended in 4.5 L of laboratory well water without the addition of solvent or test substance. Both the solvent and negative control sediments were also allowed to settle for approximately 24 hours after shaking. The water was then decanted from the control sediments and the sediments were allocated to the appropriate replicate test vessels.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM

- Source: The midges used during this study were obtained from laboratory cultures maintained at Springborn Smithers. The culture water was laboratory well water and was characterised as soft water.

- Feeding: During rearing, the midge larvae were fed a finely-ground suspension of flaked fish food (i.e., 10 mg/mL). Midge larvae were fed daily during the culturing and rearing period.

- Test organism production: Prior to test initiation, egg masses were removed from culture aquaria and each individual egg mass was placed in a 30-mL plastic cup with approximately 25 mL of culture water. The egg masses were observed daily until hatching was complete (approximately 24 to 48 hours after release of egg masses by the female midges). Hatched midge larvae were transferred to a shallow glass bowl containing 1 L of culture water (laboratory well water) and 5 mL of the algae, Ankistrodesmus falcatus, to serve as a substrate. Midge larvae were reared under static conditions in laboratory well water. During the rearing of the midge larvae, the temperature ranged from 20 to 21 ºC and the dissolved oxygen ranged from 9.1 to 9.4 mg/L. The larvae were reared in the culture bowls for three days after hatching to provide first-instar larvae (3 days old) for use during the exposure to D6. No mortality of midge larvae was observed 48 hours prior to test initiation.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

68 mg/L as CaCO3

Test temperature:

20 to 22 °C

pH:

4.2 to 7.3

Dissolved oxygen:

7.2 to 8.9 mg/L

Salinity:

Not applicable

Ammonia:

3.4 to 24 mg/L as N. Elevated ammonia levels at the end of the test are most likely due to increased microbial activity caused by continuous food addition to the test vessels coupled with a lack of midge larvae feeding activity. Since organism performance in these treatment levels exceeded the acceptability criteria, these elevated ammonia levels did not significantly impact the results or interpretation of the study.

Nominal and measured concentrations:

Nominal concentrations: 0 (Control), 0 (Vehicle control), 63, 130, 250, 500 and 1000 mg/kg dry weight.

Mean measured concentrations in treated sediments: 53, 88, 150, 320 and 620 mg/kg dry weight.

The results are interpreted with reference to mean measured concentrations.

Details on test conditions:

TEST SYSTEM

- Test vessels: The test vessels used were 600-mL clear glass beakers. The sediment and overlying water were added to the test vessels two days prior to test initiation. A 75-mL (approximately 1.5-cm layer) aliquot of sediment was added to each test vessel. The wet weight of the sediment in each test vessel averaged 120 g (65.2 g dry weight). A turbulence reducer, consisting of a modified plastic disk, was used to minimize the disruption of the sediment layer during the introduction of 300 mL (approximately 6 cm) of overlying water. The total medium volume (sediment/water) was maintained at 375 mL and the ratio of sediment to water was 1:4. The initial water level in each test vessel was marked in order to evaluate evaporation. Each test vessel was covered with a clear plastic plate to minimize evaporation and trap emerging adult midges.

- Sediment: The sediment (Springborn Smithers Batch No. 072009) used during this study was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to use and characterisation, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterised by Agvise Laboratories, Northwood, North Dakota, as having a percent organic carbon of 3.2%. The particle size distribution conducted by Agvise Laboratories resulted in an average particle size distribution of 88% sand, 10% silt and 2% clay. A sediment pH of 6.1 was also determined at Agvise Laboratories. A percent solids value of 54.36% was determined by Springborn Smithers Laboratories. In addition, a sample of sediment pore water was analysed prior to test initiation for ammonia concentration. The total ammonia concentration of the sediment pore water sample was 22 mg/L as nitrogen (ammonia). A representative sample of the sediment source was analysed for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that would be considered to have an adverse impact on the results of the test.

- Overlying water: Overlying water used during this study was laboratory well water. The water used during the definitive exposure was characterised as having total hardness and total alkalinity as calcium carbonate (CaCO3) of 68 and 20 mg/L, respectively, a pH of 7.1, and a specific conductivity of 420 micromhos per centimetre (µmhos/cm).

- Replication: Four replicate exposure vessels (A through D) were established for each treatment level and control to monitor the biological results (i.e., percent emergence and development rate) of the exposed midges. Four additional replicate vessels (E through H) were established for each treatment level and control to determine exposure concentrations of D6 in the sediment.

- Test initiation: Application of the test substance (day -3) to the sediment occurred prior to the allocation of the sediment to the test vessels. Sediment jars were prepared by adding 1.5 kg of wet sediment (0.8154 kg dry weight based on a percent solids of 54.36%) followed by 4.5 L of laboratory well water and shaking to suspend sediment prior to dosing. Each test concentration was prepared individually by adding the appropriate dosing stock solution directly to each jar and shaking again following dosing to distribute the test substance.

Following settling for approximately 24 hours, water was decanted from the treated sediments and the sediments were allocated to the replicate test vessels for each exposure level. A solvent control sample was prepared in a similar manner as the treated sediment by adding 5.0 mL of acetone, containing no test substance, to 1.5 kg of wet sediment suspended in 4.5 L of laboratory well water. The negative control sediment consisted of 1.5 kg of unadulterated wet sediment suspended in 4.5 L of laboratory well water without the addition of solvent or test substance. Both the solvent and negative control sediments were also allowed to settle for approximately 24 hours after shaking. The water was then decanted from the control sediments and the sediments were allocated to the appropriate replicate test vessels.

At test initiation (day 0), twenty midge larvae (3 days old) were impartially added to each replicate test vessel (A through D). The additional replicates (E through G) established for chemical analysis of the sediment were maintained under the same test conditions as replicates A through D, but observations of midge emergence were not made on these vessels. Midges were added impartially to an intermediate beaker by adding no more than five midges to each vessel until all beakers contained five midges. This procedure was repeated until each intermediate beaker contained 20 midges. The test was initiated when each intermediate beaker of 20 midges was added to each respective test vessel. At the time of addition of the midge larvae, aeration of the water was suspended for 24 hours. The following day (day 1), aeration was resumed at 1 to 3 bubbles per second.

- Feeding: At test initiation (day 0), 1.0 mL of finely ground flaked fish food suspension (10 mg/mL) was added to each test vessel. Test midges were fed 1.0 mL of finely ground flaked fish food suspension (10 mg/mL) daily, on test days 0 through 10. On days 11 through termination, midges were fed 2.0 mL of finely ground flaked fish food suspension (10 mg/mL) daily.

- Observations: Replicate test vessels A through D were examined at test initiation and daily thereafter, until test termination (day 28). Observations of midge emergence and abnormal behaviour were made and the physical characteristics of the test solutions were recorded. During the period of expected emergence (typically starting at day 10 and lasting until day 28), a daily check of emerged midges was made. The sex and number of adult midges that emerged daily were recorded. Male midges were identified by their plumose antennae.

- Water quality: Water quality measurements made during the study were performed in the four replicate exposure vessels (A through D) established for monitoring the biological performance of the exposed midges. Measurements of dissolved oxygen concentration, temperature and pH were made on the day the test organisms were added (day 0) and at test termination (day 28) in each exposure vessel. In addition, dissolved oxygen concentration, pH and temperature were measured daily in an alternating replicate vessel of each treatment level and the controls during the remainder of the 28-day exposure. The temperature was continuously monitored in an auxiliary vessel in the water bath throughout the study. Total hardness, alkalinity, specific conductivity, and total ammonia of the test solutions were determined at test initiation and at test termination in a composite sample (replicates E through H on day 0 and replicates A through D on day 28) from the highest treatment level and the control solution.

- Range finding test: Prior to initiating the definitive study, a preliminary range-finding exposure was conducted at Springborn Smithers Laboratories at nominal D6 concentrations of 0.10, 1.0, 10, 100 and 1000 mg/kg, a control and a solvent (acetone) control. Three replicates of twenty midges (3 days old) were exposed to each treatment level and control. Following 28 days of exposure, the mean percent emergence among midges exposed to the nominal concentrations tested (0.10, 1.0, 10, 100 and 1000 mg/kg) was 98, 97, 95, 95 and 88%, respectively. During the same period, mean percent emergence among the midges exposed to the control and solvent control was 98 and 97%, respectively (pooled control = 98%). The mean development rate (male/female midge combined), after 28 days of exposure, for the nominal concentrations tested (0.10, 1.0, 10, 100 and 1000 mg/kg) was 0.0666, 0.0660, 0.0668, 0.0641 and 0.0575, respectively. During the same period, mean development rate among the midges exposed to the control and solvent control was 0.0633 and 0.0632, respectively (pooled control = 0.0632). Based on these results and consultation with the Study Sponsor, the following nominal D6 concentrations were selected for the definitive study: 63, 130, 250, 500 and 1000 mg/kg.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 620 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Midge emergence and development rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 620 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Midge emergence and development rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 620 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Midge emergence and development rate

Reported statistics and error estimates:

At the termination of the study, data obtained on midge emergence and male and female development rate were statistically analysed to identify significant treatment-related effects. The lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant effect (No-Observed-Effect Concentration, NOEC) were determined. All statistical analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks’ Test (Weber et al., 1989) and Bartlett's Test (Sokal and Rohlf, 1981), in which the 99% level of certainty was applied. The 99% level of certainty is preferred for these qualifying tests.

CETIS-Comprehensive Environmental Toxicity Information SystemTM (Ives, 2009) Version 1.7 software was used to perform the computations. The results were used to establish, at the 95% level of certainty, the lowest test concentration that showed a statistically significant effect (Lowest-Observed-Effect Concentration, LOEC) and the highest test concentration that showed no statistically significant difference (No-Observed-Effect Concentration, NOEC) from the pooled control data.

Table 1. Results of analysis of test sediments

 

Nominal Concentration (mg/kg dry weight)	Measured Concentration(mg/kg dry weight)			Mean (Standard Deviation)^d	Percent of Nominal^d
	Day 0^a	Day 7^b	Day 28^c
Control	< 4.6	< 4.5	< 4.1	NA^e (NA)	NA
Solvent Control	< 4.6	< 4.5	< 4.1	NA (NA)	NA
63	37	74	48	53 (19)	84
130	86	96	82	88 (7.1)	68
250	120	160	160	150 (22)	59
500	270	380	320	320 (58)	64
1000	520	800	530	620 (160)	62

 

^a      Analytical samples were removed from replicate E of the additional exposure vessels. 

^b      Analytical samples were removed from replicate F of the additional exposure vessels.

^c         Analytical samples were removed from replicate G of the additional exposure vessels.

^d      Mean, standard deviation and percent of nominal were calculated using the original unrounded results and not the rounded (two significant figures) presented in this table.

^e      NA = Not Applicable.

 

 

Table 2. Test results

 

Mean Measured Sediment Concentration (mg/kg)	Test Day 28
	Mean % emerged	Mean Male/Female Combined Development Rate
Control	95	0.0670
Solvent Control	91	0.0707
Pooled Control	93	0.0689
53	93	0.0701
88	93	0.0720
150	95	0.0724
320	96	0.0719
620	90	0.0667

 

Validity criteria fulfilled:

yes

Conclusions:

A 28-d EC50 value of >620 mg/kg dry weight and a NOEC of >/= 620 mg/kg dry weight have been determined for the effects of the test substance on emergence and development rate of Chironomus riparius exposed to treated sediments. The results are expressed relative to mean measured exposure concentrations.

Reference 4

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2008-03-28 to 2008-04-29

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is disregarded on the basis that it is thought that the artificial sediment with peat based carbon source and high pH values interfered with the test system to exhibit toxicity that is mediated by the interaction of the substance with components of artificial sediment with peat based carbon source at high pH. In many different studies across the range of siloxanes with sediment toxicity data available, the observations indicate in general no effects, or organic carbon normalised NOEC ≥30 mg/kg dwt. This would be in line with a general narcotic mode of action, observed for other structural classes and effects in pelagic species. In some cases, however, a significantly higher toxicity for a substance is exhibited, resulting in very low NOEC values. Furthermore, in each case where significantly higher toxicity was observed, an alternative study is available in which much lower toxicity was measured in the same species for the identical substance using natural sediment and with lower pH (<8). Consideration of the possible contributing factors that could have caused high toxicity in these studies, led to an understanding of the importance of certain factors of the test design, particularly the use of artificial sediment with a peat-based carbon source, and elevated pH in the test system.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SEDIMENT

- Sampling interval: Sediment samples were collected from the analytical replicates from each test concentration and control shortly after the introduction of the organisms on Day 0, on Day 7 and at test termination on Day 28.

Vehicle:

no

Details on sediment and application:

SEDIMENT

- Formulated sediment: The sediment used in the study was a formulated sediment based on the recommendations of OECD Guideline 218. The sediment was composed of approximately 10% sphagnum peat moss, 20% silt and clay (kaolin clay) and 70% industrial quartz sand. The sand and clay were mixed in a PK Twinshell mixer for 20 minutes without the peat, since the peat was added later. The targeted organic carbon content of the final mixture was 5.0 ± 1.0%. The dry soil was stored under ambient conditions until used. The final pH of the sediment was 7.1. The percent organic carbon of the sediment was found to be 2.7.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM

- Source: Egg masses were obtained from Environmental Consulting and Testing, Superior, Wisconsin. The organisms were held for four days prior to the start of the test at approximately the same temperature and in water from the same source as the water used during the test. At test initiation, the midges were collected from the culture and impartially added one and two at a time to test chambers. All transfers were made below the air/water interface using wide-bore pipettes.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

132-160 mg/L as CaCO3

Test temperature:

18.5-21.0 ºC

pH:

pH ranged from 8.2 to 8.5

Dissolved oxygen:

≥7.3 mg/L

Salinity:

not applicable

Ammonia:

<0.17 mg/L

Nominal and measured concentrations:

Nominal concentrations in mg/Kg: 0 (Control), 156, 259, 432, 720, 1200 and 2000

Arithmetic mean measured concentrations in mg/Kg in the treated sediments: 22, 45, 94, 173, 376 and 684

The mean measured concentrations in the treated sediments are equivalent to 14, 18, 22, 24, 31 and 34% of nominal.

The results are interpreted with reference to the mean measured concentrations.

Details on test conditions:

TEST SYSTEM

- Test container (material, size): Test chambers were 2000-mL glass beakers containing approximately 2 cm of sediment and 8 cm of overlying water

- Aeration: yes

- Overlying water renewal: None

- Aeration frequency and intensity: Loose plastic covers were placed over each test chamber. Each test chamber was gently aerated through a glass pipette that did not extend to a depth closer than 2 cm from the surface of the sediment. Air was bubbled into the test chamber at a rate greater than 1 bubble per second but not so great as to disturb the sediment. .

EXPOSURE REGIME

- No. of organisms per container (treatment): Four replicates were tested in each treatment group with 20 midges in each replicate for a total of 80 midges per treatment group.

- Type and preparation of food: A 28-day ration of food (280 mg Tetramin flake food) was dry mixed into the sediment prior to the addition of the overlying water and 50.5 hours before adding the test organisms.

OVERLYING WATER CHARACTERISTCS

- Dilution water source: Well Water

- Dilution water chemistry: hardness 132-148 mg/L as CaCO3 , alkalinity 180-186 mg/L as CaCO3 and conductivity 300-310 mhos/cm

OTHER TEST CONDITIONS

- Lighting (quality, intensity, and periodicity): fluorescent lighting with wavelengths similar to natural lighting, intensity was 434 lux at the surface of the water at test initiation and 530 lux on Day 17, photoperiod was 16 hours light:8 hours dark with a 30-minute transition period.

The sediment contained 70% industrial quartz sand, 10% sphagnum peat moss and 20% silt. Textural class: sandy loam. Organic carbon: 2.7%.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

37 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 22-94

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

22 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks:

and development time

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

< 22 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development rate

Remarks:

and development time

Reported statistics and error estimates:

The 28-Day EC50 was calculated using the computer software of C.E. Stephan. The program is designed to calculate the LC50 value and 95% confidence interval by probit analysis, the moving average method, or binomial probability with nonlinear interpolation. In this study, the binomial method was used to calculate the EC50 value. The EC50 value was calculated using the mortality data collected at the end of the test. The no-observed-effect-concentration (NOEC) and the lowest-observed-effect-concentration (LOEC) were determined using Dunnett's test and the mean development times, emergence ratios and development rates.

   Table 1. Results of analysis of sediment exposure concentrations

 

Nominal Test Concentration (mg/Kg)	Mean Measured Concentration (mg/Kg)	Mean Percent of Nominal
Negative Control	--	--
156	22	14
259	45	18
432	94	22
720	173	24
1200	376	31
2000	684	34

 

 

Table 2. Test results

 

Mean Measured Concentration (mg/Kg)	Number Exposed	Percent Emergence	Mean Development Time (Days)	Emergence ratio	Development rate
Negative Control	80	84	17.7	0.84	0.0593
22	80	66	21.0*	0.66	0.0501*
45	80	44*	21.8*	0.44*	0.0485*
94	80	26*	25.2*	0.26*	0.0410*
173	80	28*	24.5*	0.28*	0.0422*
376	80	19*	24.1*	0.19*	0.0427*
684	80	1.3*	28.0*	0.013*	0.0364*

^*There was a statistically significant difference (p<0.05) from the negative control using Dunnett’s t-test.

Validity criteria fulfilled:

yes

Conclusions:

A 28-Day EC50 value of 37 mg/kg dry weight has been determined for the effects of the sediment incorporated test substance on mortality of Chironomus riparius. A NOEC of <22 mg/kg dry weight for effects on development rate and development time has been determined in the same test.

Description of key information

A 28-day EC₅₀ value of >130 mg/kg sediment dry weight and a NOEC of 130 mg/kg sediment dry weight (135 mg/kg dw normalised to 5% OC) have been determined.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

135 mg/kg sediment dw

Additional information

A category approach is applied to this endpoint and is detailed in the Siloxane Category report (PFA, 2017). The hypothesis for read-across of sediment ecotoxicity evidence within the Siloxanes Category is that no structure-based or property-based pattern is evident from the category dataset of existing studies, although patterns are identifiable associated with extrinsic aspects of test design to which effects may be attributed. The approach will be revisited in the event that reliable new data become available. With this in mind, a single overall interpretation is made across the category. To fulfil the requirements of REACH, a conservative approach is made by reading across on a nearest-neighbour basis the reliable data within the category.

 

In the context of the RAAF, Scenario 6 is expected to apply to this endpoint. It is considered that effects observed in benthic organisms are associated primarily with extrinsic factors associated with test design and not to structural similarities as such. 

Over twenty-five sediment toxicity studies for siloxanes are available and have been reviewed in detail. The majority of these studies are of standard duration in standard test species. There is a general trend for studies using natural sediment, which all have pH <~8, to show no effects, or higher NOECs than corresponding studies with artificial sediment. No significant toxicity (NOEC <100 mg/kg) in any organism is found at pH near 7 with natural sediment. The data suggest that it is possible to read across sediment toxicity data between different siloxane structures, especially where natural sediment data are available, given that the studies which are not suspected to be confounded by extrinsic factors show relatively minimal effects across the dataset.

 

Data available for the substance:

The following results have been obtained in two tests performed with the registered substance (D6) using natural sediment:

A 28-day EC₅₀ value of >420 mg/kg dry weight and a NOEC of ≥420 mg/kg dry weight have been determined for effects on numbers and biomass of Lumbriculus variegatus in accordance with OECD TG 225 and in compliance with GLP (Springborn Smithers, 2010a).

A 28-day EC₅₀ value of >620 mg/kg dry weight and a NOEC of ≥620 mg/kg dry weight have been determined for effects on emergence and development rate of Chironomus riparius in accordance with OECD TG 218 and in compliance with GLP (Springborn Smithers, 2010b).

 

A test with Chironomus riparius exposed via artificial sediment gave a 28-day EC₅₀ value of 37 mg/kg dry weight for the effects of the registered substance (D6) on mortality and a NOEC of <22 mg/kg dry weight for effects on development rate and development time, in accordance with OECD TG 218 and in compliance with GLP (Wildlife International, 2010). However, consideration of the possible contributing factors that could have caused high toxicity in studies in artificial sediment led to an understanding of the importance of certain factors of the test design, particularly the use of artificial sediment with a peat-based carbon source and elevated pH in the test system. The result is therefore disregarded because it is thought that the artificial sediment with peat based carbon source and high pH values interfered with the test system to exhibit toxicity that is extrinsic to the actual toxicity of the substance.

In addition, a result for the same species is available from a test with natural sediment.

 

The results of all tests are expressed relative to mean measured exposure concentrations in the treated sediment.

 

Read-across data

No data are available for the effects of the registration substance on Hyalella azteca.

A result for effects in natural sediment on the invertebrate amphipod Hyalella azteca is read across from decamethylcyclopentasiloxane (D5), CAS 541-02-6. The result from that test is a 28-day EC₅₀ value of >130 mg/kg sediment dry weight for growth rate and a NOEC of 130 mg/kg sediment dry weight (135 mg/kg dw normalised to 5% OC) for growth rate conducted in accordance with EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater) and in compliance with GLP (Springborn Smithers, 2009).

 

The NOEC value normalised to 5% OC of 135 mg/kg dw sediment for effects of the read-across substance D5 on Hyalella azteca is used as the key value for the CSA.

 

 

READ-ACROSS JUSTIFICATION

The registered substance (D6) and read-across substance D5 (CAS 541-02-6) are members of the Reconsile Siloxanes Category. They are methylated siloxanes containing Si atoms linked by oxygen.

D6 and D5 are cyclic siloxanes. D6 is a cyclic siloxane with six dimethylated silicon atoms linked by six oxygen atoms. D5 is a directly analogous structure with five silicon and five oxygen atoms. The substances have similar physicochemical properties: high molecular weight (445 and 370 respectively), low water solubility (both insoluble, at 0.0051 and 0.017 mg/l respectively), high log K_ow (8.87 and 8.07 respectively) and high log K_oc (5.9 and 5.2 respectively). Both substances have negligible biodegradability, similar slow hydrolysis rates and have high potential for adsorption to sediment.

 

In the use of the data set for hazard assessment and derivation of predicted no-effect concentration (PNEC), the following approach is used:

- Where data are available for a substance with natural sediment and with artificial sediment for the same species, the natural sediment data will be given preference over data obtained with artificial sediment.

References

PFA, 2017, Siloxane Category Report for Environmental Endpoints, PFA.404.114.001.