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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1735 (Whole Sediment Acute Toxicity of Invertebrates, freshwater)
GLP compliance:
yes (incl. certificate)
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 analyzed.


OVERLYING WATER CHARACTERISTCS

The water used during the definitive exposure was characterized 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 characterization, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterized 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 behavior 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 (SDa)

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

Recoveryd

Day 0a

Day 7b

Day 28c

Meand

Control

< 2.0e

< 2.0

< 2.0

NAf

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

 

 

 

 

 

 

QCg#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.
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:
other: OECD Guideline 225
GLP compliance:
yes (incl. certificate)
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 characterized 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. Synchronized 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 synchronized 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 synchronization 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 synchronization 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 minimize 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 characterization, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterized 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 characterized 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 oligochates per concentration and 60 oligochates 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, but 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 behavior (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 utilized 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 analyzed 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)

Meand

Percent of Nominald

Day 0a

Day 7b

Day 28c

Control

< 4.4

< 4.4

< 4.1

NAe

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.
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:
OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)
GLP compliance:
yes (incl. certificate)
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. 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 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 characterized 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 characterization, the sediment was wet-pressed through a 2.0 mm sieve to remove large particles. The sediment used in this study was characterized 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 characterized 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 centimeter (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 behavior 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 Nominald

Day 0a

Day 7b

Day 28c

Control

< 4.6

< 4.5

< 4.1

NAe

(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.

Description of key information

28-day NOEC: 130 mg/kg dw (135 mg/kg dwt normalised to 5% OC), Hyalella azteca

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:
135 mg/kg sediment dw

Additional information

Long-term sediment toxicity studies are not required for Annex VIII substances under the REACH Regulation. However, due to the high sediment adsorption potential of the substance and the potential for persistence in the sediment, the compartment has been assessed.

No sediment toxicity data are available for the registration substance, Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9).

A category approach is applied to this endpoint and is detailed in the Siloxane Category report (PFA, 2017at). 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.

 

A total of twenty-four sediment toxicity studies for siloxanes and nineteen results from studies of standard duration in standard test species have been reviewed in detail. 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.

READ-ACROSS JUSTIFICATION

No data are available for the effects of the registration substance on sediment organisms. Data are read-across from low functionality siloxanes of a similar molecular weight, decamethylcyclopentasiloxane (D5, CAS 541-02-6) and dodecamethylcyclohexasiloxane (D6, CAS 540-97-6).

The registered substance, (Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (CAS 352230-22-9)) and read-across substances decamethylcyclopentasiloxane (D5, CAS 541-02-6) and dodecamethylcyclohexasiloxane (D6, CAS 540-97-6) are members of the Reconsile Siloxanes Category.

D5 is a cyclic siloxane with 5 silicon atoms with methyl branches and alternated by oxygen. D6 has 6 silicon atoms with methyl branches and alternated by oxygen. The registration substance is a multi-constituent substance containing two main constituents 3,3-diphenylhexamethyltrisiloxane (55-70%) and 3,3,5,5-tetraphenylhexamethyltetrasiloxane (15-25%). The main constituent has three silicon atoms with 2 phenyl groups and 6 methyl groups. The second constituent has 4 silicon atoms, with 4 phenyl groups and 6 methyl groups. The registration and read-across substances have similar physicochemical properties (low water solubility, high log Kow and slow hydrolysis rates), are not readily biodegradable and have high potential for adsorption to sediment.

See IUCLID Section 6.0, CSR Section 7.0, Ecotoxicological Information for further discussion and justification of read-across.

A result for effects in natural sediment on the invertebrate amphipod Hyalella azteca is read across from D5. The result from that test is a 28 d 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.

The following results are read-across from D6 in studies using natural sediment:

A 28-d EC50 value of >420 mg/kg dry weight and a NOEC of ≥420 mg/kg dry weight (≥656 mg/kg dw normalised to 5% OC) have been determined for effects on numbers and biomass of Lumbriculus variegatus. A 28-d EC50 value of >620 mg/kg dry weight and a NOEC of ≥620 mg/kg dry weight (≥969 mg/kg dw normalised to 5% OC) have been determined for effects on emergence and development rate of Chironomus riparius.

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

A NOEC value of 130 mg/kg dw sediment for effects on Hyalella azteca is used as the key value for the CSA, normalised to 135 mg/kg dw.