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Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-05-22 to 2016-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
- Sampling interval: day 0, 7 , 28

PORE WATER- not sampled

OVERLYING WATER - not sampled
Vehicle:
yes
Remarks:
acetone
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Details of spiking: At day -3, 1.2 kg (equivalent to 0.90 L) wet weight of sediment was weighed in individual glass jars, followed by 2.7 L of fortified well water to give an approximate water volume to sediment volume ratio of 3:1. The jar contents were shaken manually to suspend the sediment immediately prior to dosing. Each jar was then dosed with 3.0 mL of the appropriate dosing stock solution. After settling overnight, the overlying water was decanted and 1.534 g of both Urtica powder and alpha cellulose (0.25% of the total sediment dry weight each) were weighed out and manually mixed into the sediment for each concentration as a supplemental food source for the test organisms. The final sediment dry weight following this addition was 0.6167 kg per concentration. The sediment was then allocated to the replicate test vessels.
- Controls: solvent, negative
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution):
- Stock solution: 10 mg/mL
- Dosing stock solutions 6.20, 3.10, 1.54, 0.780, 0.400 mg/mL
- Evaporation of vehicle before use: No

Test organisms (species):
Lumbriculus variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Lumbriculus variegatus
- Source: laboratory cultures maintained at Smither Viscient


ACCLIMATION
- Acclimation period: 13 days following synchronisation
- Acclimation conditions (same as test or not): organisms were placed in an aquarium with test sediment and laboratory well water under flow-through conditions. Renewal of overlying water in holding aquarium with the fortified well water used in the test at a rate of approximately half a volume replacement per day.
- Type and amount of food: 12 mL of finely ground suspension of flaked fish food (100 mg/mL)
- Feeding frequency: weekly
- Health during acclimation (any mortality observed): none
Study type:
laboratory study
Test type:
static
Water media type:
freshwater
Type of sediment:
natural sediment
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
92-160 mg/L as CaCO3
Test temperature:
18 to 22°C
pH:
overlying water: 7.1 to 8.4
Dissolved oxygen:
5.1 to 9.0 mg/L
Ammonia:
≤0.10 to 3.9 mg/L as N
Conductivity:
510-580 µS/cm
Nominal and measured concentrations:
nominal: 1.9, 3.8, 7.5, 15 and 30 mg/kg
measured: dosed sediment after mixing and prior to allocation to test vessels 1.7, 2.5, 6.0, 12 and 25 mg/kg
measured: mean measured concentrations during exposure period: 1.3, 2.7, 6.3, 12 and 19 mg/kg
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 600 mL clear glass beakers
- Sediment volume: 75 mL
- Sediment wet weight: 115g (average)
- Overlying water volume: 300 mL
- Depth of sediment and overlying water: sediment 1.5 cm, water 6 cm (ratio 1:4)
- Aeration: yes
- Aeration frequency and intensity: 1 to 3 bubbles per second
- Replacement of evaporated test water, if any:

EXPOSURE REGIME
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control / vehicle control: total of 6 control
- Feeding regime: added at start of test
- Type and preparation of food: Urtica powder and alpha cellulose added to sediment during sediment preparation

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): fortified well water
- Alkalinity: 88 mg/L as CaCO3
- Conductivity: 560 to 620 µS/cm

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Glen Charlie Pond, Wareham, Massachusetts. (SV Batch no. 042115-M-1-1)

HANDLING OF NATURAL SEDIMENT
- Storage conditions: under refrigerated conditions at approximately 4ºC

CHARACTERIZATION OF SEDIMENT
- Particle size distribution
- % sand: 91%
- % silt: 8%
- % clay: 1%
- %solids: 51.13%
- Sediment sieved: yes, wet pressed through a 2.0 mm sieve prior to use and characterisation
- pH dry matter and/or whole sediment: 5.6
- Ammonia content of pore water: 3.9 mg/L as N
- Total organic carbon (%): 2.5%
- Proof of absence of chemical contaminants: representative sample analysed for presence of pesticides, PCBs and toxic metals. None detected at concentrations considered to have an adverse impact on the test.

OTHER TEST CONDITIONS
- Light quality: fluorescent bulbs
- Photoperiod: 16 hours light, 8 hours darkness
- Light intensity: 350 to 490 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: nominal L5 concentrations chosen for the definitive study were 1.9, 3.8, 7.5, 15 and 30 mg/kg, based on sediment dry weight

PRELIMINARY TESTING/EXPOSURE
- range-finding concentrations: nominal concentrations of 0.025, 0.17, 1.1, 7.5 and 50 mg/kg
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 3
- control / vehicle control: negative and solvent control sediments were maintained
- results: Statistical analysis determined no significant reduction in the mean number of surviving oligochaetes or in the biomass among oligochaetes exposed to any treatment level compared to the solvent control.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 19 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
total number of worms
Remarks:
oligochaete biomass
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 19 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
total number of worms
Remarks:
oligochaete biomass
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 19 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
total number of worms
Remarks:
oligochaete biomass
Details on results:
- mean number of oligochaetes recovered in the control and solvent control was 30 and 27, respectively
- mean biomass per replicate in the control and solvent control was 37 and 30 mg, respectively
- control and solvent control data met the minimum performance criteria of the guideline (i.e., population increase of ≥ 1.8)

At exposure termination (day 28), the mean number of surviving oligochaetes observed among the oligochaetes exposed to the 1.3, 2.7, 6.3, 12 and 19 mg/kg treatment levels was 32, 24, 28, 25 and 24, respectively. Statistical analysis (Bonferroni’s Adjusted t-Test) determined a significant difference in the mean number of surviving oligochaetes in the 2.7 mg/kg treatment level compared to the pooled control (28 oligochaetes). Due to the lack of a dose response and lack of significant reductions at higher dose levels, the significant reduction determined for the 2.7 mg/kg was determined not to be treatment related.

Mean biomass per replicate in the 1.3, 2.7, 6.3, 12 and 19 mg/kg treatment levels was 33, 31, 31, 30 and 29 mg, respectively. Statistical analysis (Bonferroni’s Adjusted t-Test) determined no significant difference in mean biomass in any of the treatment levels tested compared to the pooled control (34 mg).
Validity criteria fulfilled:
yes
Conclusions:
No effects on survival or biomass have been reported when testing the registered substance at a loading rate of 19 mg/kg dwt sediment with Lumbriculus variegatus. Therefore 28-day NOEC and LC50 values of ≥19 and >19 mg/kg have been determined respectively in a sediment containing 2.5% organic carbon.
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
5th of June 2019 to 27th of January 2020
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)
Version / remarks:
Adopted 13th of April 2004
Deviations:
yes
Remarks:
pH of the overlying water were between 5.7 to 8.0 (rather than 6.0-8.0); addition of 1.1% rather than 1.0 % of nettle leaf powder (Urtica dioica) to the sediment, ammonia concentration was analysed post-experiment rather than prior to experiment.
GLP compliance:
yes
Remarks:
Followed GLP with the exception that routine food, water and sediment contaminant screening analyses were conducted using standard validated U.S. EPA procedures and thus, not considered as having an impact on the study results.
Analytical monitoring:
yes
Details on sampling:
Dosing stock solutions were sampled at the approximate time of application to the sediment to confirm that the stock solutions were prepared correctly.
A sample of dosed sediment from each treatment level and the controls was removed and analysed for L5 concentration after the sediment slurry had settled overnight, the overlying water had been removed and the sediment had been manually mixed.

SEDIMENT
- Concentrations: 2.2; 4.4; 8.8; 18 and 35 mg/kg sediment dry weight. Following centrifugation and removal of the pore water sample, sediment samples were collected from the centrifuge tubes with a stainless steel spatula.
- Sampling interval: Day -2 (pre-test day), 0, 7 and 28
- Sample storage before analysis: not reported

PORE WATER
- Concentrations: 2.2; 4.4; 8.8; 18 and 35 mg/kg sediment dry weight. Pore water samples were collected by removing the entire sediment sample from each test vessel and centrifuging for 30 minutes at approximately 10,000 g. The resulting pore water was decanted from the centrifuge tube and its volume measured.
- Sampling interval: Day 0, 7 and 28
- Sample storage before analysis: pore water samples were collected by removing the entire sediment sample from each test vessel and centrifuging followed by collection of sediment samples from the centrifuge tubes.

OVERLYING WATER
- Concentrations: 2.2; 4.4; 8.8; 18 and 35 mg/kg sediment dry weight. Overlying water from each replicate vessel was decanted and its volume measured.
- Sampling interval: Day 0, 7 and 28
- Sample storage before analysis: not specified

The remainder of all samples that was not used for processing was stored in a freezer for possible future analysis.
Vehicle:
yes
Remarks:
acetone
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
Natural, freshwater sediment was used in the exposure. The natural, freshwater sediment (Smithers Batch No. 111418) was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to being used in the study, the freshwater sediment was wet pressed through a 2-mm sieve to remove large particles, and was characterised by Agvise Laboratories, Northwood, North Dakota, as having a mean organic carbon content of 3.1%, a pH of 5.6, and a water holding capacity (at 1/3 bar) of 20.1%. The particle size distribution conducted by Agvise Laboratories resulted in average particle size distribution of 89% sand, 11% silt, and 0% clay. A percent solids value of 36.44% and pore water ammonia concentration of 5.77 mg/L (as mg nitrogen/L) was determined by Smithers.

A 20.0 mg/mL primary stock solution was prepared by dissolving 1.03812 g of L5 (1.00189 g as active ingredient) in 50 mL of acetone (CAS No. 67-64-1). The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following preparation. The primary stock solution was used to prepare dosing stock solutions as described in the following table.
Primary Stock Concentration (mg/mL) Volume of Primary Stock Used (mL) Final Dilution Volume with Acetone (mL) Nominal Dosing Stock Concentration (mg/mL)
20.0 1.20 25 0.960
20.0 2.41 25 1.93
20.0 4.82 25 3.86
20.0 9.85 25 7.88
20.0 19.2 25 15.4

All dosing stock solutions were observed to be clear and colourless with no visible undissolved test substance following preparation.

- Details of spiking: At day -3, 1.19 kg (equivalent to 0.95 L) wet weight of sediment was weighted in individual glass jars, followed by the addition of 2.85 L of laboratory well water to give an appproximate water-volume to sediment-volume ratio of 3:1. Each jar was manually shaken to suspend the sediment and thereafter, dosed with 1.0 mL of the appropriate dosing stock solution. The test substance was applied to the sediment below the sediment/water suspension. After settling overnight, the overlying water was decanted and 4.34 g of Urtica dioica powder (1% of the total sediment dry weight each) was weighed out and manually mixed into the sediment for each concentration as a supplemental food source for the test organisms. The addition of the food occurred following dosing to ensure it was adequately mixed into the sediment and equally incorporated for each control and treatment level. Adding the food prior to dosing could result in food being suspended in the overlying water during the dosing procedure and never reincorporating into the sediment. The final sediment dry weight following this addition was 0.4380 kg per concentration. The sediment was then allocated to the replicate test vessels. The method of test substance application described here was intended to eliminate possible test material losses via volatilization during the dosing procedure and maximise adsorption of the test material to the sediment. This method also mitigated the possibility of greatly exceeding sorption capacity of the sediment, which could be achieved using conventional dosing methods.
- Equilibration time: two days prior to addition of the test organisms.
- Equilibration conditions: The dosed sediment and overlying water were added to the test vessels two days prior to addition of the test organisms and were allowed to equilibrate under exposure conditions until initiation.
- Controls: A solvent control sample was prepared in a similar manner as the treated sediment by adding 1.0 mL of acetone, containing no test substance, to a jar containing 1.19 kg (wet weight) of sediment and 2.85 L of laboratory well water. The sediment and water were suspended and allowed to settle overnight. After settling, the overlying water was decanted, and 4.34 g of Urtica dioica powder was added to the jar and the sample was processed in the same manner as the treated sediments. The negative control sediment was prepared by suspending 1.19 kg (wet weight) of untreated sediment with 2.85 L of laboratory well water in a jar and processing in the same manner as the treated/solvent control sediments. Consistent with the solvent control and treatment sediments, 4.34 g of Urtica dioica powder was also added to the negative control sediment after the suspension had settled overnight and the overlying water had been decanted. After processing, the solvent control and negative control sediments were then allocated to the replicate test vessels. The solvent control and negative control vessels were maintained under the same conditions as the treatment vessels.
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 1 ml acetone per 1.19 kg (equivalent to 0.95 L) wet weight of sediment and 2.85 L overlying water.
- Evaporation of vehicle before use: No
Test organisms (species):
Chironomus riparius
Details on test organisms:
TEST ORGANISM
- Common name: Chironomus riparius
- Strain/clone: cultured at Smithers.
- Justification for species other than prescribed by test guideline: commonly used as an aquatic benthic invertebrate in sediment toxicity tests.
- Source: laboratory cultures maintained at Smithers.
- Details on collection: Hatched midge larvae were transferred to a shallow glass bowl containing cultured water (laboratory well water) and Ankistrodesmus falcatus, a unicellular green algae, to serve as a substrate. Midge larvae were reared under static conditions in laboratory well water with gentle, oil-free aeration.
- Age of parental stock (mean and range, SD): not specified
- Breeding conditions: temperature ranged from 20 to 21 °C and the dissolved oxygen ranged from 8.9 to 9.5 mg/L.
- Handling of egg masses and larvae: The larvae were reared in the culture bowls for three days after hatching to provide first-instar larvae (three days old) for use during the exposure to the test item.
- Age of animals at beginning of exposure: 3 day old larvae
- Feeding during test: 4.34 g of Urtica dioica powder (1% of the total sediment dry weight each) was weighed out and manually mixed into the sediment for each concentration as a supplemental food source for the test organisms. The food was added at the beginning of the test and no further feeding was carried out as requested by the regulator.
- Food type: During the dosing, Urtica dioica powder was mixed into the sediments.
- Amount: Urtica dioica powder: 1% of the total sediment dry weight.
- Frequency: after the dosing, Urtica dioica powder was added to avoid food being suspended.

ACCLIMATION
- Acclimation period: not specified. The water used during the test was from the same source as the culture water and test conditions were the same as culture conditions.
- Acclimation conditions (same as test or not): Same as test.
- Type and amount of food: during rearing, finely ground flaked fish food suspended in laboratory well water (1.0 mL of a 10 mg/mL suspension)
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no
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:
CaCO3: 74 mg/L
Test temperature:
18 to 21 ºC
pH:
5.7 to 8.0
Overlying water pH 7.2
Natural sediment pH 5.6
Dissolved oxygen:
6.4 to 8.9 mg/L
Salinity:
N/a
Ammonia:
Pore water ammonia 5.77 mg/L
Conductivity:
480 µS/cm
Nominal and measured concentrations:
Nominal: 2.2, 4.4, 8.8, 18, and 35 mg/kg sediment dry weight
Initial Measured: 1.4, 3.2, 4.3, 11, and 12 mg/kg sediment dry weight
Mean Measured: 1.3, 3.7, 6.2, 11, and 17 mg/kg sediment dry weight
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 600-mL glass beakers, 8 cm diameter
- Sediment volume: 75 mL
- Weight of wet sediment with and without pore water: 111 g wet weight per vessel.
- Overlying water volume: 300 mL
- Depth of sediment and overlying water: approximately 1.5 cm sediment and 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.
- Aeration: yes
- Aeration frequency and intensity: 1 to 3 bubbles/second.
- Replacement of evaporated test water, if any: not specified

EXPOSURE REGIME
- No. of organisms per container: 20
- No. of replicates per treatment group: Four replicate exposure vessels (A through D) were established for each treatment level and control to monitor the biological results. Four additional replicate vessels (E through H) were established for each treatment level and control for analytical measurements to determine exposure concentrations of L5 in the overlying water, pore water, and sediment.
- No. of replicates per control / vehicle control: Four replicate exposure vessels (A through D) were established for each treatment level and control to monitor the biological results. Four additional replicate vessels (E through H) were established for each treatment level and control for analytical measurements to determine exposure concentrations of L5 in the overlying water, pore water, and sediment.
- Feeding regime: added at start of test.
- Type and preparation of food: Urtica dioica powder (finely ground flaked fish food suspended in laboratory well water during rearing)
- Amount of food: After settling overnight, the overlying water was decanted and 4.34 g of Urtica dioica powder (1% of the total sediment dry weight each) was weighed out and manually mixed into the sediment for each concentration as a supplemental food source for the test organisms.

The dosed sediment and overlying water were added to the test vessels two days prior to addition of the test organisms (day -2) and were allowed to equilibrate under exposure conditions until initiation. At exposure initiation (day 0), 20 midge larvae (three days old) were impartially added to each replicate test vessel (A through D). The additional replicates (E through H) established for chemical analysis of the overlying water, pore water, and sediment were maintained under the same test conditions as replicates A through D and each contained midge with the exception of replicate E, which was sacrificed at exposure initiation prior to addition of midge larvae; observations of midge emergence were not made on these vessels. At the time of addition of the midge larvae, aeration of the water was suspended for 24 hours to allow the test organisms to settle on the sediment surface. The following day (day 1), aeration was resumed at 1 to 3 bubbles per second.

RENEWAL OF OVERLYING WATER
- Details on volume additions: not specified
- Flow-rate: static test

OVERLYING WATER CHARACTERISTCS
- Type of water: laboratory well water, characterised as soft water.
- Source of water: laboratory well water
- Location: not specified
- Description of sampling site: not specified
- Filtration: not specified
- Alkalinity: 20 mg/L as CaCO3
- Salinity: n/a
- Conductivity: 480 µS/cm
- Particulate matter: not specified
- Total organic carbon: 3.1% based on the average result of triplicate measurements
- Chemical oxygen demand: not specified
- Unionized ammonia: not specified
- Residual chlorine: not specified
- Total organic chlorine compounds and PCBs: None of these compounds were detected at concentrations that are considered toxic.
- Total organophosphorous compounds: not specified
- Total organic chlorine: not specified

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Natural, freshwater sediment was collected at Glen Charlie Pond, Wareham, Massachusetts.
- Contamination history of site: Representative samples of the sediment were analysed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analysed, in agreement with ASTM (2007) standard practice.

HANDLING OF NATURAL SEDIMENT
- Time of collection: not specified
- Core depth: not specified
- Water depth: not specified
- Storage conditions: not specified
- Storage duration (prior to test): not specified

CHARACTERIZATION OF SEDIMENT
- Particle size distribution :
- % sand: 89
- % silt: 11
- % clay: 0
- % solids: 36.44
- Sediment sieved: yes, through a 2 mm sieve
- pH pore water: 5.6
- Ammonia content of pore water: 5.77 mg/L
- Total organic carbon (%): 3.1
- water holding capacity (at 1/3 bar) (%): 20.1
- Proof of absence of chemical contaminants: yes, representative samples.

OTHER TEST CONDITIONS
- Light quality: fluorescent bulbs
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 500 to 1000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Replicate test vessels A through D were examined at exposure initiation and daily thereafter, until test termination (day 28). Observations of midge emergence and abnormal behaviour were made, and the physical characteristics of the water/sediment system were recorded. During the period of expected emergence (typically starting at day 13 to 16 and lasting until day 25), 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.
The development rate of male and female midge in each exposure vessel was determined. Mean development time represents the mean time span between the addition of test organisms (day 0) and the emergence of the experimental midges.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study : yes
- Test concentrations: 0.018, 0.12, 0.79, 5.3, and 35 mg/kg.
- Results used to determine the conditions for the definitive study: No effects on emergence or development rate.
Reference substance (positive control):
yes
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 12 other: mg/kg
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence and male/female combined developmental rate
Remarks on result:
other: no effects at the highest concentration tested
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 12 other: mg/kg
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: emergence and male/female combined developmental rate
Remarks on result:
other: no effects at the highest concentration tested
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 17 other: mg/kg
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: emergence and male/female combined developmental rate
Remarks on result:
other: no effects at the highest concentration tested
Remarks:
no effects at the highest concentration tested
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
> 17 other: mg/kg
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: emergence and male/female combined developmental rate
Remarks on result:
other: no effects at the highest concentration tested
Details on results:
- Mortality of test animals at end of exposure period: not specified
- Total mass of test animals at beginning of test: not specified
- Changes in body weight: 58 of live adults (% of initial weight) at end of exposure period: not specified
- No. of offspring produced: not specified
- No. of emerged male and female midges (per vessel and per day): see table 1.
- No. of pupae failing to emerge (per vessel and per day): not specified
- Percent emergence per replicate and test concentration: see table 1
- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled): not specified
- Mean individual dry weight of larvae (per larvae and per instar): not specified
- Number of living worms per replicate: not specified
- Number of dead worms per replicate: not specified
- Total biomass per replicate: not specified
- Morphological abnormalities: not specified
- Behavioural abnormalities: not specified
- Other biological observations: not specified
Results with reference substance (positive control):
A reference test was conducted concurrently with the definitive test to evaluate the sensitivity of C. riparius to a known toxicant (lindane) in natural sediment.
The test was carried out using the same procedures and the same natural sediment batch as in the definitive test with L5.
Nominal lindane concentrations: 0.020, 0.078, 0.31, 1.3, and 5.0 mg/kg sediment dry weight.
Effect Concentrations:
Midge emergence
LOEC 0.31 mg/kg sediment dry weight
NOEC 0.078 mg/kg sediment dry weight

Development rate:
LOEC 0.31 mg/kg sediment dry weight
NOEC 0.078 mg/kg sediment dry weight

CONCLUSION: Lindane was chosen as the reference toxicant for this exposure as it has been used successfully in ring-tests and validation studies (OECD, 2010). The purpose of the lindane reference exposure was to demonstrate equivalent toxicity between artificial and natural sediments. Based on nominal sediment concentrations, the results of the reference exposure with natural freshwater sediment and lindane indicates that toxicity in this sediment was consistent with published NOEC/LOEC values for lindane (Weltje, 2010).

Table 1 - The Percentage of Emergence and Development Rate:

Initial Measured Concentration in Sediment
(mg/kg dry weight)
Mean Measured Concentration in Sediment
(mg/kg dry weight
Replicate  Percent Emerged  Combined Development Rate 
Negative control  Negative control  A 95 0.066
    B 80 0.0648
    C 90 0.0646
    D 85 0.0651
    Mean (SD) 88 (6)  0.0651 (0.0006) 
Solvent control  Solvent control  A 100 0.0676
    B 95 0.068
    C 85 0.069
    D 90 0.0681
    Mean (SD) 93 (6)  0.0682 (0.0006) 
Pooled control Pooled control      
1.4 1.3 A 90 0.069
    B 95 0.0707
    C 100 0.0674
    D 100 0.0684
    Mean (SD) 96 (5)  0.0689 (0.0014) 
3.2 3.7 A 80 0.0677
    B 100 0.067
    C 80 0.0676
    D 100 0.0626
    Mean (SD) 90 (12)  0.0662 (0.0024) 
4.3 6.2 A 100 0.0686
    B 85 0.0672
    C 90 0.0669
    D 100 0.0683
    Mean (SD) 94 (8)  0.0678 (0.0008) 
11 11 A 85 0.0664
    B 90 0.062
    C 90 0.0644
    D 95 0.065
    Mean (SD) 90 (4)  0.0645 (0.0018) 
12 17 A 90 0.07
    B 100 0.0644
    C 100 0.0652
    D 90 0.0653
    Mean (SD) 95 (6)  0.0662 (0.0025) 

Table 2 - Measured Dosing Stock Solution Concentrations prior to Exposure Initiation

Nominal Stock concentration (mg/mL) Measured Stock Concentration, mg/mL (% of Nominal Stock Concentration)
0.96 1.02 (106)
1.93 2.06 (107)
3.86 4.27 (111)
7.88 8.26 (105)
15.4 16.8 (109)

Table 3 - Measured Sediment Concentrations

Nominal sediment concentration (mg/kg sediment dry weight) Pretest (Day -2)  Day 0b  Day 7c  Day 28d  Mean 
Negative Control  <0.20e  <0.20  <0.20  <0.20  NA 
Solvent Control  <0.20  <0.20  <0.20  <0.20  NA 
2.2 1.3 (57) 1.4 (64) 1.3 (60) 1.2 (54) 1.3 (59)
4.4 3.3 (75) 3.2 (72) 4.8 (110) 3.2 (72) 3.7 (85)
8.8 5.4 (62) 4.3 (49) 8.1 (92) 6.3 (72) 6.2 (71)
18 12 (66) 11 (61) 8.6 (48) 13 (71) 11 (60)
35 16 (46) 12 (35) 21 (59) 18 (52) 17 (48)
QCg #1 1.00/2.55h  0.518 (51.8)i 0.929 (36.4)i 0.861 (86.1) 1.06 (107)  
QC #2 9.00/23.0  6.31 (70.1) 16.9 (73.3) 7.68 (85.4) 8.48 (94.2)  
QC #3 35.0/89.3   36.5 (104) 83.1 (93.1) 31.9 (91.2) 35 (99.9)  

a       Percent recoveries were calculated using the original unrounded results and not the rounded numbers presented in this table.

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

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

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

e       Concentrations expressed as less than values were below the limit of quantitation (LOQ).  The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.  

f       NA = Not Applicable

g       QC = Quality Control sample

h       Nominal QC fortified concentrations were 1.00, 9.00, and 35.0 mg/kg sediment dry weight at days -2, 7, and 28; and 2.55, 23.0, and 89.3 mg/kg sediment dry weight on day 0.

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

Table 4 -Measured Pore Water Concentrations

Nominal sediment concentration (mg/kg sediment dry weight) Day 0a Day 7b Day 28c Mean 
Negative Control  <0.00040d  <0.00040  <0.00040  NAe 
Solvent Control  <0.00040  <0.00040  <0.00040  NA 

2.2

0.013 0.014 0.0045 0.011
4.4 0.031 0.022 0.012 0.022
8.8 0.27 0.39 0.04 0.23
18 0.34 0.64 0.27 0.41
35 1.1 0.81 0.4

0.76

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       Concentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the        dilution factor of the controls.  

e       NA = Not Applicable

Table 5 - Measured Overlying Water Concentrations

Nominal sediment concentration (mg/kg sediment dry weight) Day 0a  Day 7b  Day 28c 
Negative Control  <0.00020d  <0.00020  <0.00020 
Solvent Control  <0.00020  <0.00020  <0.00020 
2.2 0.0049 <0.00020  0.0013
4.4 0.0051 0.00064 0.0017
8.8 0.0096 0.00082 0.0039
18 0.013 0.0022 0.024
35 0.029 0.0045 0.0095
QCg#1 1.00/2.55h  0.000305 0.000362 0.000336
QC #2 9.00/23.0  0.00844 (84.4) 0.0103 (103) 0.0086 (86.0)
QC #3 35.0/89.3   0.0386 (96.4) 0.0425 (106) 0.0364 (90.9)

QC = Quality Control sample (mg/L).

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       Concentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls.

Validity criteria fulfilled:
yes
Conclusions:
A 28cday sediment toxicity study with the test item dodecamethylpentasiloxane was conducted according to OECD TG 218 and in compliance with GLP. No effects on the emergence and combined developmental rate were observed. A NOEC value of ≥12 mg/kg was determined based on initial measured concentrations, as well as a LOEC value of >12 mg/kg. Additionally, based on the mean measured sediment dry weight concentrations NOEC and LOEC values of ≥17 mg/kg and >17 mg/kg were determined for emergence and combined developmental rate.
Executive summary:

A 28 day sediment toxicity study with the test item dodecamethylpentasiloxane was conducted according to OECD TG 218 and in compliance with GLP. No effects on the emergence and combined developmental rate were observed. A NOEC value of ≥12 mg/kg was determined based on initial measured concentrations, as well as a LOEC value of >12 mg/kg. Additionally, based on the mean measured sediment dry weight concentrations NOEC and LOEC values of ≥17 mg/kg and >17 mg/kg were determined for emergence and combined developmental rate.

Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
10 March to 22 April 2020 (dates encompass test substance application on day -2 to completion of sediment and aqueous analysis at test termination); In-life dates were 12 March to 9 April 2020
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
ASTM E1706 (Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrate
Version / remarks:
ASTM Standard E1706-19
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
SEDIMENT
- Concentrations: Dosing stock, dosed sediment from each treatment level, prior to allocation of organisms, and each treatment level and the controls during the in-life phase of the study. day 0, day 7 and day 28
- Sampling interval: Day 0, day 7 and day 28
- Sample storage before analysis: Not reported

PORE WATER
- Concentrations: Each treatment level and the controls
- Sampling interval: Day 0, day 7 and day 28
- Sample storage before analysis: Not reported

OVERLYING WATER
- Concentrations: Each treatment level and the controls
- Sampling interval: Day 0, day 7 and day 28
- Sample storage before analysis: Not reported

Dosing stock solutions were sampled at the approximate time of application to the sediment to confirm that the stock solutions were prepared correctly. In addition, a sample of dosed sediment from each treatment level and the controls was removed and analysed for L5 concentration after the sediment slurry had settled overnight, the overlying water had been removed, and the sediment had been manually mixed. Results of these analyses were used to judge whether sufficient quantities of L5 had been applied to the sediment, and what portion of the applied test substance was associated with the sediment prior to allocation to the exposure system.

During the in-life phase of the definitive study, sediment, overlying water, and pore water samples were removed from one replicate exposure vessel for each treatment level, negative control, and solvent control on day 0 (replicate I), day 7 (replicate J), and on day 28 (replicate K). Replicate L was maintained during the test for contingency purposes and was not sampled.

Overlying water from each replicate vessel was decanted and its volume measured. Pore water samples were collected by removing the entire sediment sample from each test vessel and centrifuging for 30 minutes at approximately 10,000 g. The resulting pore water was decanted from the centrifuge tube and its volume measured. Following centrifugation and removal of the pore water sample, sediment samples were collected from the centrifuge tubes with a metal spatula. The remainder of all samples that was not used for processing was stored in a freezer for possible future analysis.

In addition, six quality control (QC) samples (three aqueous and three sediment 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 natural, freshwater sediment (based on sediment dry weight) and laboratory well water at relevant concentrations of L5 with respect to the treatment level range. Results of the analyses of the QC samples were used to judge the accuracy and quality control maintained during the analysis of exposure samples. An additional set of QC samples (aqueous and sediment) was prepared in the same manner for frozen archival with the test samples with no further additions or processing.
Vehicle:
yes
Remarks:
acetone
Details on sediment and application:
PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates: Natural, freshwater sediment was used in the exposure. The natural, freshwater sediment (Smithers Batch No. 112119-M-1-1) was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to being used in the study, the freshwater sediment was wet pressed through a 2-mm sieve to remove large particles and amended with clean silica sand to achieve an acceptable TOC. Following processing, the sediment was characterised by Agvise Laboratories, Northwood, North Dakota, as having a mean organic carbon content of 3.2%, a pH of 5.7, and a water holding capacity (at 1/3 bar) of 30.8%. The particle size distribution conducted by Agvise Laboratories resulted in average particle size distribution of 88% sand, 7% silt, and 5% clay. A percent solids value of 39.18% and pore water ammonia concentration of 0.93 mg/L (as mg nitrogen/L) was determined by Smithers.

Representative samples of the sediment were analysed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analysed, in agreement with ASTM (2007) standard practice.

- Method of mixing: NA

- Details of spiking: A 4.99 mg/mL primary dosing stock solution was prepared by dissolving 0.25887 g of L5 (0.24955 g as active ingredient) in 50 mL of acetone (CAS No. 67-64-1). The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following preparation. The primary dosing stock solution was used to prepare four secondary dosing stock solutions as described in the following table.
Primary Stock Concentration (mg/mL) Volume of Primary Stock Used (mL) Final Dilution Volume with Acetone (mL) Nominal Dosing Stock Concentration (mg/mL)
4.99 1.57 25 0.313
4.99 3.14 25 0.627
4.99 6.26 25 1.25
4.99 12.9 25 2.57
All dosing stock solutions were observed to be clear and colorless with no visible undissolved test substance following preparation.

Application of the test substance (day -2) to the sediment occurred prior to the allocation of the sediment to the exposure vessels (day -1). Each test concentration was prepared by first weighing out 1.20 kg (equivalent to 1.0 L) wet weight of sediment (0.4702 kg sediment dry weight) in individual, duplicate, glass jars, followed by the addition of 3.0 L of laboratory well water, to give an approximate water-volume to sediment-volume ratio of 3:1. The sediment and water volumes were intended to eliminate any headspace in each glass jar. Because L5 is highly volatile, while also being minimally water soluble, elimination of head space maximized the potential for sediment sorption during the dosing procedure. Each jar was manually shaken to suspend the sediment and was then dosed with 3.33 mL of the appropriate dosing stock solution. The test substance was applied to the sediment below the sediment/water suspension and using a glass pipette as described in Table 1 in Section "Any other information on materials and methods incl. tables":

Following dosing, the jars were sealed and manually shaken again to re-suspend the sediment, and thus distribute the test substance. After settling overnight, the overlying water was decanted and 4.70 g of Urtica dioica powder (1% of the total sediment dry weight per jar) was weighed out and manually mixed into the sediment (0.4749 kg total sediment dry weight) for each jar as a supplemental food source for the test organisms. The addition of the food occurred following dosing to ensure it was adequately mixed into the sediment and equally incorporated for each control and treatment level. Adding the food prior to dosing could result in food being suspended in the overlying water during the dosing procedure and never reincorporating into the sediment. The sediment from both jars for each treatment level were thoroughly mixed together by hand prior to allocation to the replicate test vessels. The method of test substance application described here was intended to eliminate possible test material losses via volatilisation during the dosing procedure and maximise adsorption of the test material to the sediment. This method also mitigated the possibility of greatly exceeding sorption capacity of the sediment, which could be achieved using conventional dosing methods.

A solvent control sample was prepared in a similar manner as the treated sediment by adding 3.33 mL of acetone, containing no test substance, to each duplicate jar containing 1.20 kg (wet weight) of sediment and 3.0 L of laboratory well water. The sediment and water were suspended and allowed to settle overnight. After settling, the overlying water was decanted, and 4.70 g of Urtica dioica powder was added to each jar and the samples were processed in the same manner as the treated sediments. The negative control sediment was prepared by suspending 1.20 kg (wet weight) of untreated sediment with 3.0 L of laboratory well water in each duplicate jar and processing in the same manner as the treated/solvent control sediments. Consistent with the solvent control and treatment sediments, 4.70 g of Urtica dioica powder was also added to the negative control sediment after the suspension had settled overnight and the overlying water had been decanted. After processing, the solvent control and negative control sediments were then allocated to the replicate test vessels. The solvent control and negative control vessels were maintained under the same conditions as the treatment vessels.

- Equilibration time: 1 day
- Equilibration conditions: As test
- Controls: Negative control and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 3.3 ml acetone stock solution per 1.2 kg wet weight sediment and 3.0 L water
- Evaporation of vehicle before use: No
Test organisms (species):
Hyalella azteca
Details on test organisms:
TEST ORGANISM
- Common name: Hyalella azteca (amphpod)
- Strain/clone: not reported
- Justification for species other than prescribed by test guideline: N/A
- Source: Smithers cultures
- Details on collection: Amphipods (8 days old at exposure initiation) used in the exposure were collected from reproducing adult amphipods removed from the main culture tanks 9 days prior to exposure initiation. The adult amphipods were placed in 9.5 L aquaria (isolation tanks) containing approximately 8 L of laboratory well water. Neonate amphipods (<24 hours old) produced by these isolated adults were then removed the following day and pipetted into four, 1 L beakers containing approximately 0.80 L of well water. Each beaker contained approximately 300 amphipods.
- Age of parental stock (mean and range, SD): Not reported
- Breeding conditions: Amphipods were maintained in 38 L glass aquaria containing approximately 31 L of culture water under flow-through conditions. The culture water was laboratory well water from the same source as the overlying water used during the test.

At exposure initiation, measurement of dry weight was performed on four subsets of 20 amphipods from the same population used to initiate the exposure. The average dry weight was determined to be 0.038 mg dry weight per amphipod.

- Handling of egg masses and larvae: Neonate amphipods (<24 hours old) produced by the isolated adults were removed and pipetted into four, 1 L beakers containing approximately 0.80 L of well water.
- Age of animals at beginning of exposure: 7 to 8 days old at initiation of the exposure.
- Feeding during test
- Food type: During the test, the amphipods were fed once by the addition of nettle leaf powder (Urtica dioica) to the sediment. The powder was mixed with the sediment following application of the test substance prior to allocation to the test vessels, at a rate of 1.0% sediment dry weight. This method of feeding is being utilised due to the physicochemical properties of the test material (Log Pow >5) and regulatory guidance.
- Amount: Once at the beginning of the test 1.0% sediment dry weight.
- Frequency: Once at the beginning of the test.

ACCLIMATION
- Acclimation period: The neonate amphipods were reared under static conditions for 8 days with gentle, oil-free aeration. During the holding period, dissolved oxygen ranged from 7.4 to 8.3 mg/L, and temperature ranged from 22 to 23 °C.
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: While being maintained in culture prior to testing, the freshwater amphipods were fed daily a combination of yeast, cereal leaves, and flaked fish food suspension (YCT) once daily. A small amount of Ankistrodesmus falcatus, a unicellular green algae, and flaked fish food suspension (100 mg/mL) was added to the isolation beakers at the start of the isolation period to provide a supplemental food source.
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): The test organisms appeared healthy and no mortality was observed in the test population prior to exposure initiation.
Study type:
laboratory study
Test type:
other: renewal of overlying water but not renewal of test substance
Water media type:
freshwater
Type of sediment:
natural sediment
Limit test:
no
Duration:
28 d
Exposure phase:
total exposure duration
Hardness:
Day 0: 84 to 88 mg/L as CaCO3
Day 28: 36 to 72 mg/L as CaCO3
Test temperature:
21 - 23 °C
pH:
Range 6.3 - 7.6
Dissolved oxygen:
Range 2.5 - 6.8 mg/l
Salinity:
n/a
Ammonia:
Day 0: ≤0.10 mg/L as N
Day 28: 0.67 to 0.97 mg/L as N
Conductivity:
Day 0: 620-640 µS/cm
Day 28: 530 to 550 µS/cm
Nominal and measured concentrations:
Nominal concentrations: Negative Control, Solvent Control, 2.2, 4.4, 8.8, 18, and 35 mg/kg sed dw.
Initial measured: Negative Control, Solvent Control, 2.0, 4.5, 9.8, 19, 34 mg/kg sed dw.
Mean measured: Negative Control, Solvent Control, 2.2, 4.4, 9.1, 19, 33 mg/kg sed dw.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): The test vessels used in this study were 300-mL glass beakers which were chemically cleaned prior to use. The sediment and overlying water were added to the test vessels 1 day prior to exposure initiation. Each test vessel had two openings near the top edge of the beaker to allow for drainage during the renewal of overlying water. The openings were covered with 40-mesh nylon screen to retain any test organisms that may be present in the water column. Each vessel contained 100 mL (approximately 4-cm layer) of sediment (approximately equivalent to 135 g wet weight per vessel) and 175 mL of overlying water. A turbulence reducer, consisting of a modified plastic disk, was used to minimize the disruption of the sediment layer during the introduction of overlying water. The total overlying water plus sediment volume was maintained at approximately 275 mL. The test vessels were labeled to identify the nominal test concentration, study number, and the replicate designation.

Eight replicate exposure vessels (A through H) were established for each treatment level and control to monitor the biological results (i.e., survival and growth) of the exposed amphipods. Four additional replicate vessels (I through L) were established for each treatment level and control for analytical measurements to determine exposure concentrations of L5 in the overlying water, pore water, and sediment.

- Sediment volume: 100 mL (an approximate 4 cm layer) of sediment (approximately equivalent to 135 g wet weight per vessel) and 175 mL of overlying water. The overlying water/sediment volume will thus be maintained at approximately 275 mL.
- Weight of wet sediment with and without pore water: 135 g wet weight per vessel
- Overlying water volume: 175 ml
- Depth of sediment and overlying water: 4 cm sediment
- Aeration: no
- Aeration frequency and intensity: n/a
- Replacement of evaporated test water, if any: n/a

EXPOSURE REGIME
- No. of organisms per container (treatment): 10 (80 pr treatment group)
- No. of replicates per treatment group: 12: Eight replicate exposure vessels (A through H) were established for each treatment level and control to monitor the biological results (i.e., survival and growth) of the exposed amphipods. Four additional replicate vessels (I through L) were established for each treatment level and control for analytical measurements to determine exposure concentrations of L5 in the overlying water, pore water, and sediment.
- No. of replicates per control / vehicle control: 12: Eight replicate exposure vessels (A through H) were established for each treatment level and control to monitor the biological results (i.e., survival and growth) of the exposed amphipods. Four additional replicate vessels (I through L) were established for each treatment level and control for analytical measurements to determine exposure concentrations of L5 in the overlying water, pore water, and sediment.
- Feeding regime: During the test, the amphipods were fed once by the addition of nettle leaf powder (Urtica dioica) to the sediment. The powder was mixed with the sediment following application of the test substance prior to allocation to the test vessels, at a rate of 1.0% sediment dry weight. This method of feeding is being utilised due to the physicochemical properties of the test material (Log Pow >5) and regulatory guidance.
- Type and preparation of food: nettle leaf powder (Urtica dioica) added directly to the sediment.
- Amount of food: A rate of 1.0% sediment dry weight

RENEWAL OF OVERLYING WATER
- Details on volume additions: During the exposure, the overlying water was initially renewed by adding two volume additions of water per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system (Zumwalt et al., 1994). During the second daily inspection of the water-distribution system on test day 0, the renewal rate was doubled to add four volume additions of water per vessel per day. This change was made due to low dissolved oxygen levels in the overlying water. The intermittent delivery system was calibrated to provide 1 L of water per cycle to the water-distribution system, which subsequently provided 50 mL of water per cycle to each replicate test vessel. The water delivery system initially cycled approximately seven times per day, providing approximately 350 mL per vessel every 24 hours. During the second visual check of the test system on day 0, dissolved oxygen levels were observed to be dropping. Therefore, the water delivery system cycled approximately 14 cycles per day, providing approximately 700 mL per vessel every 24 hours for the duration of the exposure.

The calibration of the overlying water-renewal system was checked prior to exposure initiation and confirmed at exposure termination. During the test, the renewal system was visually inspected twice daily. A complete check of intermittent delivery system function was made once daily.

- Flow-rate: 14 cycles per day

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Laboratory well water
- Source of water (if non-standard medium) : The source of the overlying water used during this study was a mixture of unadulterated on-site well water and de-chlorinated Town of Wareham well water and was characterised as soft water with a typical total hardness of ≤160 mg/L as CaCO3. The on-site well water was obtained from a 100-meter bedrock well. The Town of Wareham well water was de-chlorinated by use of ultraviolet (UV) treatment and activated carbon filtration. The two sources of water passed individually through separate 1-µm polypropylene bag filters, a degasser, and then were mixed in a 5700-L polyethylene tank. The resulting water was continuously circulated through a degassing chamber to ensure proper equilibration of dissolved gases with the laboratory atmosphere. The water was then pumped through PVC piping to the laboratory.

The pH, total hardness, alkalinity, and conductivity of this water was measured and recorded weekly. Total hardness and alkalinity was determined according to APHA et al. (2017) standard practice. The water used during the definitive exposure was characterised as having total hardness and total alkalinity ranges as calcium carbonate (CaCO3) of 72 to 74 mg/L and 20 to 22 mg/L, respectively, a pH range of 6.9 to 7.4, and a conductivity range of 430 to 530 µS/cm.

Representative samples of the overlying water source were analysed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds were detected at concentrations that are considered toxic in any of the water samples analysed, in agreement with ASTM (2007) standard practice.

In addition, representative samples of the overlying water source were analysed monthly for total organic carbon (TOC) concentration. TOC of the dilution water should be equal to or less than 2 mg/L. The TOC concentration of the overlying water source was 0.82 and 0.72 mg/L for March and April 2020, respectively. Amphipod cultures have historically been continuously maintained in water from the same source as the overlying water utilised in this study and have successfully survived and reproduced over multiple generations. The acceptable performance of the amphipod cultures, in combination with the previously mentioned analyses confirmed the acceptability of this overlying water for use in toxicity testing.

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Natural, freshwater sediment was used in the exposure. The natural, freshwater sediment (Smithers Batch No. 112119-M-1-1) was collected from Glen Charlie Pond, Wareham, Massachusetts. Prior to being used in the study, the freshwater sediment was wet pressed through a 2-mm sieve to remove large particles and amended with clean silica sand to achieve an acceptable TOC. Following processing, the sediment was characterised by Agvise Laboratories, Northwood, North Dakota, as having a mean organic carbon content of 3.2%, a pH of 5.7, and a water holding capacity (at 1/3 bar) of 30.8%. The particle size distribution conducted by Agvise Laboratories resulted in average particle size distribution of 88% sand, 7% silt, and 5% clay. A percent solids value of 39.18% and pore water ammonia concentration of 0.93 mg/L (as mg nitrogen/L) was determined by Smithers.

- Contamination history of site: Representative samples of the sediment were analysed periodically for the presence of pesticides, PCBs, and toxic metals by Eurofins Lancaster Laboratories Environmental, Lancaster, Pennsylvania. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analysed, in agreement with ASTM (2007) standard practice.

HANDLING OF NATURAL SEDIMENT
- Time of collection:
- Core depth:
- Water depth:
- Storage conditions:
- Storage duration (prior to test):


OTHER TEST CONDITIONS
- Light quality: The test area was illuminated with fluorescent bulbs
- Photoperiod: 16 hours light and 8 hours darkness. The photoperiod of the light banks used to illuminate the exposure system was staggered to turn on and off with a difference of 30 minutes to simulate a diurnal transition period from light to dark and vice versa.
- Light intensity: Intensity range of 520 to 1000 lux.

Dissolved oxygen concentration, temperature, and pH were measured in the overlying water of each replicate vessel of each treatment level and control used for biological monitoring at exposure initiation (day 0) and exposure termination (day 28). On the remaining test days, dissolved oxygen, temperature, and pH were measured in one alternating replicate of each treatment level and control. In addition, the temperature was continuously monitored in an auxiliary vessel in the water bath used to house the test vessels throughout the study using a traceable minimum/maximum thermometer. Minimum/maximum readings were recorded daily. Total hardness, alkalinity, conductivity, and total ammonia (as nitrogen) concentration of the overlying water were monitored at exposure initiation and termination in each treatment level and controls. Overlying water samples consisted of a composite sample of all available biological replicates for each respective treatment and control group.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of mortality and abnormal behavior were made and the physical characteristics of the sediment/water system were recorded. At exposure termination, the entire contents (sediment and overlying water) from each biological replicate were sieved using fine-mesh fish nets (approximate 0.25-mm opening) to remove all surviving amphipods, according to Smithers SOP 22.068.05. The total number of surviving amphipods and growth (expressed as dry weight) was determined for each test replicate. Growth of the surviving amphipods was determined by pooling all surviving amphipods from each replicate and drying at approximately 62 to 63 °C for approximately 24 hours. The pooled amphipods were then weighed on a calibrated analytical balance to the nearest 0.01 mg.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0.018, 0.12, 0.79, 5.3, and 35 mg/kg, and a solvent (acetone) control.
- Results used to determine the conditions for the definitive study: No effects at the highest concentration tested - at the sorption capacity of the sediment.
Reference substance (positive control):
yes
Remarks:
4,4’-DDD
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 34 mg/kg sediment dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 34 mg/kg sediment dw
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 33 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 33 mg/kg sediment dw
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Results with reference substance (positive control):
- Results with reference substance valid? Yes
Reference substance: 4,4’-DDD analytical standard, Batch No. BCBS3969V, CAS No. 72-54-8, reported to have a purity of 99.9%
- Relevant effect levels: Reference values; complete mortality at a concentration of 400 mg/kg OC, a reduction in growth at 120 mg/kg OC, and a NOEC of 36 mg/kg OC.
- Limit test: No
- Dose-response test: Yes: Negative Control, Solvent Control, 0.94, 1.9, 3.8, 7.5, 15 mg/kg; corresponding organic carbon (OC) normalized concentrations of 29, 59, 120, 230, and 470 mg/kg OC, respectively.

- ECx: SURVIVAL GROWTH
NOEC 59 59
LOEC 120 >59
LC50 / EC50 >470 >59

Based on OC normalised concentrations.

Due to the survival effects at the 120, 230, and 470 mg/kg OC normalized concentrations, these concentrations were subsequently dropped from analysis of the growth data per guidance recommendation.
Reported statistics and error estimates:
Wilcoxon’s Rank Sum Two-Sample Test and Equal Variance Two-Sample t-Test were used to compare the performance of the negative control organisms with that of the solvent control organisms in order to determine if there were any statistically significant positive or negative differences for survival and growth, respectively. No significant difference was observed between the negative control and the solvent control data for survival; therefore, analysis of the treatment level data was performed against the pooled control for this endpoint. A significant difference between the negative control and solvent control data for growth was observed; therefore, analysis of the treatment level data was performed against the solvent control for this endpoint.
Shapiro-Wilk’s Test for normality was conducted to compare the observed sample distribution with a normal distribution for all endpoints. Analysis of amphipod growth (dry weight) met the assumption of normal distribution. Analysis of survival data did not meet the assumption of normal distribution.
As a check on the assumption of homogeneity of variance implicit in parametric statistics, data for each endpoint were analyzed using Bartlett’s Equality of Variance Test. Data for both amphipod growth (dry weight) and survival met the assumption for homogeneity of variance.
Data for amphipod growth (dry weight) met the assumptions for normality and homogeneity; therefore, Dunnett’s Multiple Comparison, a parametric statistical procedure, was used to establish treatment effects. Data for survival did not meet the assumption for normality; therefore, Wilcoxon’s Test with Bonferroni-Holm’s Adjustment was used to establish treatment effects. The aforementioned statistical tests were selected as the appropriate parametric or non-parametric methods, as a step-wise monotonic response was not observed for any endpoint.
CETIS Version 1.9 (Ives, 2019) was used to perform the statistical computations.

Table 1 - 28-Day Toxicity Test Exposing Amphipods (Hyalella azteca) to L5 - Measured Dosing Stock Solution Concentrations prior to Exposure Initiation

Nominal Stock Concentration (mg/mL)

 Measured Stock Concentration, mg/mL (Percent of Nominal Stock Concentration)a

0.313

 0.276 (88.1)

0.627

 0.563 (89.7)

1.25

 1.06 (84.4)

2.57

 2.16 (83.9)

4.99

 4.33 (86.8)

aPercent recoveries were calculated using the actual analytical results and not the rounded values (three significant figures) presented in this table.

Table 2 - 28-Day Toxicity Test Exposing Amphipods (Hyalella azteca) to L5 - Measured Sediment Concentrations

Nominal Sediment Concentration (mg/kg sediment dry weight)

 

Sediment Measured Concentration, mg/kg (% of nominal concentrationa) Pretest (Day -1)

Sediment Measured Concentration, mg/kg (% of nominal concentrationa) Day 0b

Sediment Measured Concentration, mg/kg (% of nominal concentrationa) Day 7c

Sediment Measured Concentration, mg/kg (% of nominal concentrationa) Day 28d

Negative Control

 <0.12e (NAf)

 <0.12 (NA)

 <0.12 (NA)

 <0.13 (NA)

Negative Control

 <0.12e (NAf)

 <0.12 (NA)

 <0.12 (NA)

 <0.13 (NA)

2.2

2.1 (96)

 2.0 (92)

 2.0 (93)

 2.4 (110)

4.4

 5.8 (130)

 4.5 (100)

 4.3 (97)

 4.5 (100)

8.8

 10 (120)

 9.8 (110)

 9.3 (110)

 8.3 (95)

18

 22 (120)

 19 (100)

 18 (100)

 19 (100)

35

 40 (110)

 34 (97)

 34 (98)

 31 (88)

QCg #1 1.00

 0.908 (90.8)

 0.987 (98.7)

 0.851 (85.1)

 0.925 (92.5)

QC #2 9.00

 7.49 (83.3)

 8.54 (94.9)

 8.24 (91.6)

 8.70 (96.6)

QC #3 35.0

 36.4 (104)

 35.9 (103)

 33.9 (96.9)

 34.1 (97.3)

aPercent recoveries were calculated using the original unrounded results and not the rounded numbers presented in this table.

bAnalytical samples were removed from replicate I of the additional exposure vessels.

cAnalytical samples were removed from replicate J of the additional exposure vessels.

dAnalytical samples were removed from replicate K of the additional exposure vessels.

eConcentrations expressed as less than values were below the limit of quantitation (LOQ). The LOQ for each analysis is dependent upon the regression, the area of the low standards and the dilution factor of the controls.

fNA = Not Applicable

gQC = Quality Control sample

 

Table 3 - 28-Day Toxicity Test Exposing Amphipods (Hyalella azteca) to L5 - Measured Pore Water Concentrations

Nominal Sediment Concentration (mg/kg sediment dry weight)

 Pore Water Measured Concentration, mg/L Day 0a

 Pore Water Measured Concentration, mg/L Day 7b

 Pore Water Measured Concentration, mg/L Day 28c

 Mean

Negative Control

 <0.0040d

 <0.00040

 <0.00040

 NAe

Solvent Control

 <0.0040

 <0.00040

 <0.00040

 NA

2.2

 0.016

 0.0065

 0.0030

 0.0085

4.4

 0.033

 0.021

 0.0065

 0.020

8.8

 0.047

 0.039

 0.0088

 0.032

18

 0.066

 0.12

 0.022

 0.068

35

 0.40

 0.82

 0.16

 0.46

aAnalytical samples were removed from replicate I of the additional exposure vessels.

bAnalytical samples were removed from replicate J of the additional exposure vessels.

cAnalytical samples were removed from replicate K of the additional exposure vessels.

dConcentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls.

eNA = Not Applicable


Table 4 - 28-Day Toxicity Test Exposing Amphipods (Hyalella azteca) to L5 - Measured Overlying Water Concentrations

Nominal Sediment Concentration (mg/kg sediment dry weight)

 Overlying Water Measured Concentration, mg/L Day 0a

 Overlying Water Measured Concentration, mg/L Day 7b

 Overlying Water Measured Concentration, mg/L Day 28c

Negative Control

 <0.0020d

 <0.00020

 <0.00020

Solvent Control

 <0.0020

 <0.00020

 <0.00020

2.2

 0.0065

 <0.00020

 0.00026

4.4

 0.017

 <0.00020

 0.0012

8.8

 0.0087

 <0.00020

 0.0043

18

 0.045

 0.00094

 0.0014

35

 0.045

 0.00064

 0.0037

QCe #1 0.000333

 0.000534 (166)f

 0.000441 (132)f

 0.000302 (90.6)

QC #2 0.0100

 0.0118 (118)

 0.0101 (101)

 0.00707 (70.7)

QC #3 0.0400

 0.0447 (112)

 0.0460 (115)

 0.0265 (66.3)f

aAnalytical samples were removed from replicate I of the additional exposure vessels.

bAnalytical samples were removed from replicate J of the additional exposure vessels.

cAnalytical samples were removed from replicate K of the additional exposure vessels.

dConcentrations expressed as less than values were below the method detection limit (MDL). The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls.

eQC = Quality Control sample, in mg/L f Percent recovery for this QC sample is outside of the acceptable range (i.e., 70.0 to 120%, Appendix 6). See Section 3.2.2 for additional discussion.

Table 5 - 28-Day Toxicity Test Exposing Amphipods (Hyalella azteca) to L5 - Mean Percent Survival of Adult Amphipods and Mean Amphipod Growth (Dry Weight)

Initial Measured Sediment Concentration (mg/kg sediment dry weight)

 Mean Measured Sediment Concentration (mg/kg sediment dry weight)

 Test Day 28, Mean Percent Survival (SDa)

 Mean Dry Weight per Amphipod, in mg (SD)

Negative Control

 Negative Control

 95 (5)

 0.50 (0.11)

Solvent Control

 Solvent Control

 96 (5)

 0.32 (0.15)

Pooled Control

 Pooled Control

 96 (5)

 NAb (NA)

2.0

 2.2

 83c (9)

 0.26 (0.08)

4.5

 4.4

 84c (11)

 0.34 (0.19)

9.8

 9.1

 86c (7)

 0.39 (0.17)

19

 19

 85c (13)

 0.39 (0.18)

34

 33

 85c (13)

 0.26 (0.17)

aSD = Standard Deviation

bNA = Not Applicable; Treatment data were compared to the solvent control for this endpoint.

cSignificantly reduced compared to the pooled control, based on Wilcoxon’s Test with Bonferroni-Holm’s Adjustment. However, due to a flat, non-monotonic response observed for the treatment levels for the survival endpoint, in addition to all treatment levels exceeding acceptability criteria for control survival. (i.e., ≥80% survival), the significant reductions detected are not considered to be dose-dependent.

Validity criteria fulfilled:
yes
Conclusions:
A 28 day EC50 value of >34 mg/kg sediment dry weight and NOEC value of >=34 mg/kg sediment dry weight have been determined for the effects of the test substance on survival and growth of Hyalella azteca, based on initial measured concentrations. The EC50 and NOEC values based on mean measured concentrations are >33 mg/kg sediment dry weight and >=33 mg/kg sediment dry weight respectively.

Description of key information

WoE: 28 day NOEC ≥38 mg/kg dry weight normalised to 5% organic carbon, based on mean measured concentrations, for the effects of L5 on survival and biomass of Lumbriculus variegatus.

WoE: 28 day NOEC ≥27 mg/kg dry weight normalised to 5% organic carbon, based on mean measured concentrations, for the effects of L5 on emergence and combined developmental rate of Chironomus riparius.

WoE: 28 day NOEC ≥52 mg/kg dry weight normalised to 5% organic carbon, based on mean measured concentrations, for the effects of L5 on survival and growth of Hyalella azteca.

Key value for chemical safety assessment

Additional information

Measured long-term sediment toxicity data are available with L5 for three sediment dwelling species; Lumbriculus variegatus, Chironomus riparius and Hyalella azteca. The studies were all conducted in natural sediment and show no effects at the highest concentrations tested. The maximum test concentrations were selected for each sediment test based on the limit of test substance solubility in organic carbon. In each test, the sediment was dosed just above the limit of organic carbon solubility to allow for potential volatilisation losses of the test susbstance.

 

Sediment toxicity data

Lumbriculus variegatus

No effects on survival or biomass have been reported when testing the registered substance at a loading rate of 19 mg/kg dwt sediment with Lumbriculus variegatus under test guideline OECD 225, in compliance with GLP.

The study was conducted at nominal test substance concentrations of 1.9, 3.8, 7.5, 15 and 30 mg/kg sediment dry weight. The equivalent mean measured concencentrations were 1.3, 2.7, 6.3, 12 and 19 mg/kg sediment dry weight.

28 day NOEC and LC50 values of ≥19 and >19 mg/kg, respectively, have been determined in a sediment containing 2.5% organic carbon. The results normalised for a standard sediment of 5% organic carbon are equivalent to LC50 and NOEC values of >38 and ≥38 mg/kg sediment dry weight, respectively (Smithers Viscient, 2016).

 

Chironomus riparius

A 28 day sediment toxicity study with L5 was conducted according to OECD TG 218 and in compliance with GLP. No effects on the emergence and combined developmental rate of Chironomus riparius were observed in a natural sediment containing 3.1% organic carbon.

The study was conducted at nominal test substance concentrations of 2.2, 4.4, 8.8, 18, and 35 mg/kg sediment dry weight. The equivalent mean measured concencentrations were 1.3, 3.7, 6.2, 11, and 17 mg/kg sediment dry weight. 

A NOEC value of ≥12 mg/kg was determined based on initial measured concentrations and a NOEC value of ≥17 mg/kg was determined based on mean measured concentrations. The results normalised for a standard sediment of 5% organic carbon are equivalent to NOEC values of ≥19 mg/kg sediment dry weight for initial measured concentrations, and ≥27 mg/kg sediment dry weight for mean measured concentrations (Smithers, 2020a).

 

Hyalella azteca

A 28 day sediment toxicity study with L5 was conducted according to ASTM Standard E1706-19 and in compliance with GLP. No effects on the survival and growth of Hyalella azteca were observed in a natural sediment containing 3.2% organic carbon.

The study was conducted at nominal test substance concentrations of 2.2, 4.4, 8.8, 18, and 35 mg/kg sediment dry weight. The equivalent mean measured concencentrations were 2.2, 4.4, 9.1, 19, 33 mg/kg sediment dry weight.

A NOEC value of ≥34 mg/kg was determined based on initial measured concentrations and a NOEC value of ≥33 mg/kg was determined based on mean measured concentrations. The results normalised for a standard sediment of 5% organic carbon are equivalent to NOEC values of ≥53 mg/kg sediment dry weight for initial measured concentrations, and ≥52 mg/kg sediment dry weight for mean measured concentrations (Smithers, 2020b).