4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February to 13 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA Office of Water. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates. Test Method 100.4

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples of the sediment and overlying water were collected at day 0, 14, and 28.

Vehicle:

yes

Remarks:

Acetone was used as a solvent.

Details on sediment and application:

3.2 kg sphagnum peat, 12.8 kg of kaolin clay, and 48 kg fine sand (based on dry weight). Peat was conditioned for 7 days in laboratory well water prior to use to increase pH.
A 2.0 µg/mL primary stock solution was prepared by dissolving 0.00401 g of test substance in 2000 mL of acetone (CAS No. 67-64-1). The resulting primary stock solution was observed to be clear and colorless with no visible undissolved material. This stock solution was used to prepare the dosing stock solutions (0.00400, 0.0104, 0.0264, 0.0656, 0.164, and 0.408 µg/mL). All dosing stock solutions were observed to be clear and colorless with no visible undissolved material following preparation.
A jar rolling technique was used to apply the test substance to the sediment, and to mix the negative control and solvent control sediments. A 10-mL volume of each dosing stock solution was applied to 0.0500 kg of fine silica sand and the solvent was allowed to evaporate off for 40 minutes, leaving the test substance adhered to the sand. Since the solvent was allowed to evaporate completely to dryness there was, in effect, no solvent introduced into the exposure system. The dry sand containing the test substance, was then added to 3.0 kg of wet sediment (2.0429 kg total dry weight based on a percent solids value of 66.43% and including the 0.050 kg of fine silica sand) in individual 4-L glass jars. Therefore, the exposure treatment levels were 0.020, 0.051, 0.13, 0.32, 0.80, and 2.0 µg/kg dry weight. Each jar was then mixed on a rolling mill for hours and then placed in a refrigerator for a 13-day equilibration period.
A solvent control was prepared in a similar manner to the treated sediment but only 10 mL of acetone was added to the sand substrate without test substance. A negative control was also included in the study however it did not contain acetone or test substance and only consisted of 3.25 kg of unadulterated wet sediment.

Test organisms (species):

Hyalella azteca

Details on test organisms:

Source: Smithers Viscient culture which was periodically supplemented with adult amphipods from Environmental Consulting and Testing (ETC), Superior, Wisconsin
Feeding: Combination of yeast, cereal leaves, and flaked fish food suspension (YTC) fed daily during holding, acclimation and exposure period

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

42 d

Exposure phase:

total exposure duration

Hardness:

56 to 76 mg/L as CaCO3

Test temperature:

22 to 24 °C

pH:

6.6 to 7.4

Dissolved oxygen:

2.9 to 7.6 mg/L in overlying water

Salinity:

Total alkalinity of 20 to 32 mg/L as CaCO3

Ammonia:

≤0.10 to 0.81 mg/L as Nitrogen

Conductivity:

470 to 520 µS/cm

Nominal and measured concentrations:

Nominal: 0.020, 0.051, 0.13, 0.32, 0.80, and 2.0 µg/kg sediment dry weight

Details on test conditions:

Test vessels: 300 mL glass beakers
Test medium: artificial sediment with moisture content of 33.57%, based on a percent solids value of 66.43%
Replication: 10 organisms per replicate; due to inadvertent addition, some replicates resulted in up to 12 amphipods.
16 replicates per test concentration or solvent control (A through O); 19 replicates per negative control (A through R)
12 replicates (A through L) for biological response
3 replicates (M through O) for chemical analysis
3 negative control replicates (P through R) were for assessing pore water quality characteristics
One extra replicate was maintained per treatment level and control for contingency
Lighting: 16 hours light at illumination intensity of 290 to 530 lux to 8 hours darkness

Sixteen replicates were included with each test concentration and the solvent control. Nineteen replicates were established for the negative control. Twelve replicates (A through L) were used to evaluate the biological response of the test organisms. Three replicates (M through O) were established and designated for chemical analysis. The three additional negative control replicates (P through R) were maintained for the purpose of measuring representative pore water quality characteristics (pore water ammonia and pH). One additional replicate was maintained for each treatment level and control as a contingency, in case one of the primary analytical replicates was compromised during the exposure. Each vessel contained 100 mL (approximately 4-cm layer) of sediment and 175 mL of overlying water. The total overlying water plus sediment volume was maintained at approximately 275 mL.
Amphipods (8 days old) were impartially added to an intermediate set of beakers by adding no more than two amphipods to each beaker until all beakers contained two amphipods. This process was repeated until all intermediate beakers contained 10 amphipods. Although the protocol stated that each replicate will only contain 10 amphipods, on exposure day 28, it was noted that several replicates were observed to contain more than 10 amphipods. Replicate G of the 0.020 µg/kg treatment level contained 11 amphipods. Replicates B and L of the 0.051 µg/kg treatment level contained 12 and 11 amphipods, respectively. Replicates A and B of the 0.13 µg/kg treatment level contained 11 amphipods. Replicates F and G of the 0.80 µg/kg treatment level contained 11 and 12 amphipods, respectively. Replicate C of the 2.0 µg/kg treatment level contained 11 amphipods. In instances where more than 10 organisms were recovered, survival was considered 100%, which was consistent with performance in associated replicates of each applicable treatment level. There was no discernible difference in growth or reproduction for those replicates which were loaded with >10 amphipods. The additional organisms did not impact the assessment of any individual endpoint, and therefore, this deviation did not have a negative impact on the results or interpretation of this study.
Daily observations of organism behaviour (i.e., adverse effects) and mortality were made, and the physical characteristics of the overlying water and sediment were recorded. On exposure day 37, biological observations and observations of the sediment-water system were inadvertently not recorded. All other records of study maintenance on this test day were complete, indicating that this deviation was attributable to a recording omission, and not indicative of the exposure system not physically being observed. Since daily observations are not used in the determination of endpoints, with the exception of test days 28, 35, and 42, this deviation did not have a negative impact on the results or interpretation of this study.
On day 28, four of the 12 replicate vessels (C, D, I, and J) were randomly selected using a computer generated random number table and sacrificed to determine amphipod growth (length). Amphipod survival was determined for all 12 replicates. Amphipods in the remaining eight replicates were also removed by sieving and survival was assessed and recorded. Surviving adult amphipods were transferred into 300-mL water-only exposure vessels containing a thin layer of fine silica sand as a substrate and were returned to the exposure system. Offspring present were discarded following enumeration. Reproduction and survival of these adult amphipods were again measured on exposure days 35 and 42 by removing and counting the adults and offspring in each replicate vessel. On day 35, the contents of each replicate were rinsed into a sorting tray with laboratory well water. Offspring were enumerated and discarded. Surviving adults were returned to their respective replicate vessels containing clean laboratory well water and silica sand. At exposure termination (day 42), adult amphipods and any offspring present were again enumerated by rinsing the contents of each vessel into a sorting tray. The number of adult males and females were determined after exposure termination, prior to length determination. Mature males were identified by the enlarged second gnathopod. Those amphipods not identified as males were recorded as female amphipods. In addition, the number of gravid females (identified by the presence of eggs within the brood pouch) recovered on test day 42 in each replicate was recorded. The day 42 growth (measured as length) of surviving, preserved amphipods was determined after exposure termination and measured

Reference substance (positive control):

no

Key result

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

0.77 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

2.1 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

2.1 µg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Validity criteria fulfilled:

yes

Conclusions:

The 42-day NOEC was 0.77 µg/kg sediment dry weight.

Executive summary:

The effects of the substance on the survival, growth, and reproduction of the freshwater amphipod Hyalella azteca under intermittent static-renewal conditions were evaluated under GLP to EPA 100.4. Organisms were exposed to nominal concentrations of 0.020, 0.051, 0.13, 0.32, 0.80, and 2.0 µg/kg sediment dry weight (corresponding to 0.019, 0.051, 0.13, 0.31, 0.77, and 2.1 µg/kg sediment dry weight), alongside a negative control and a solvent (acetone) control. 

Based on data obtained during this study, the 28-day growth endpoint, and mean measured sediment concentrations (µg/kg sediment dry weight) the NOEC and LOEC values were determined to be 0.77 and 2.1 µg/kg sediment dry weight, respectively.  Additionally, the NOEC and LOEC values for the 42-day growth endpoint were determined to be 0.77 and 2.1 µg/kg sediment dry weight, respectively when compared to the pooled control, and were determined to be 2.1 and >2.1 µg/kg sediment dry weight, respectively when compared to the negative control.

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov 2018 to 20 Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA/600/R-01/020 (Methods for Assessing the Chronic Toxicity of Marine and Estuarine Sediment-Associated Contaminants with the Amphipod Leptocheirus plumulosus)

Version / remarks:

2001

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sediment samples from each treatment level and control were removed and analyzed for the test substance concentration during the mixing/equilibration period. The sediment was sampled after seven days of equilibration. In addition, subsamples of the dosing stock solutions used to dose the sediments were analyzed for test substance concentration on the day of dosing. Results of these stock solution and pretest analyses were used to confirm that appropriate quantities of the test substance had been applied during the dosing process and were homogenously distributed prior to exposure initiation.
- During the in-life phase of the study, sediment, pore water, and overlying water samples were removed and analyzed for the test substance concentration on test days 0, 14, and 28. On days 0, 14, and 28, samples were removed and analyzed from replicate vessels G, I, and J, respectively, for each treatment level and control. Overlying water from each replicate vessel was first decanted and its volume measured. Pore water was then collected by removing the entire sediment sample from each test vessel and centrifuging for 30 minutes at approximately 10,000 g in multiple 50 mL polypropylene centrifuge tubes. The resulting pore water (supernatant) was removed from the centrifuge tube and its volume measured. Following centrifugation and removal of the pore water sample, sediments were mixed to ensure homogeneity and then subsamples were collected from the centrifuge tubes for analysis. The entire remainder of all separated, unprocessed samples was transferred to an appropriate Nalgene container and archived in a freezer for possible future use.

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

STOCK SOLUTION PREPARATION
- A nominal 20.0 µg/mL primary stock solution was prepared by placing 0.00401 g of the test substance in a 200 mL volumetric flask and bringing it to volume with acetone. The resulting stock solution was observed to be clear and colorless with no visible undissolved material.
- A 1.00 µg/mL secondary stock solution was prepared by diluting 2.50 mL of the 20.0 µg/mL primary stock solution to a final volume of 50.0 mL with acetone. The resulting stock solution was observed to be clear and colorless with no visible undissolved material.

SEDIMENT DOSING
- Dosing stock solutions were prepared by adding the appropriate volume of the 1.00 µg/mL secondary stock solution to 25 mL volumetric flasks and bringing to volume with acetone. Upon preparation, all dosing stock solutions were observed to be clear and colorless with no visible undissolved test substance.

APPLICATION OF TEST SUBSTANCE TO SEDIMENT
- A jar-rolling technique was used to apply the test substance to the sediment, and to mix the negative control and the solvent control sediment. For each treatment level, a 10 mL volume of the appropriate dosing stock solution was applied to 0.0500 kg of fine silica sand and the solvent was allowed to evaporate for 40 minutes to complete dryness. Since the solvent was allowed to completely evaporate, there was, in effect, no solvent introduced into the exposure system. The dry sand, containing the test substance, was then added to 2.25 kg of wet sediment (0.6928 kg dry weight based on a percent solids of 30.79%) in individual 4 L glass jars. The total mass of sediment spiked on a dry weight basis for each treatment level and control was 0.7428 kg (0.0500 kg sand for test substance application and 0.6928 kg dry weight sediment). The test substance was applied for each treatment level as described in Table 1 in 'Any other information on materials and methods incl. tables'.
- The jars were sealed and positioned horizontally on the rolling mill. Each jar was then rolled for 4 hours at room temperature at approximately 15 rpm. Following 4 hours of rolling, the jars were stored upright in a refrigerator, where they were allowed to equilibrate for 13 days. Once during the 13-day equilibration period and again prior to addition into the replicate test vessels, the jars were mixed on the rolling mill for an additional 2 hours at room temperature to ensure the sediment was homogeneous. The equilibration time of spiked sediments was based on a non-GLP sediment-pore water equilibration study conducted prior to preliminary and definitive sediment toxicity testing with the test substance.
- A solvent control sample was prepared in a similar manner as the treated sediment by adding 10 mL of acetone, containing no test substance, to 0.0500 kg of sand and the solvent was allowed to evaporate off. The sand was then added to 2.25 kg of wet sediment and processed in a similar manner as the treated sediments.
- A negative control sample was included in the exposure and consisted of only 2.25 kg of unadulterated wet sediment (no addition of test substance, solvent, or fine sand). The negative control was also equilibrated and stored with the treated and solvent control sediments.

Test organisms (species):

Leptocheirus plumulosus

Details on test organisms:

TEST ORGANISM
- Age of of animals at beginning of acclimation: 1 to 2 days old
- Age of animals at beginning of exposure: 7 to 8 days old
- Dry weight of animals at beginning of exposure: 0.025 mg d.w./ amphipod (On the day of exposure initiation, a measurement of dry weight was made on three subsets of 20 juvenile amphipods from the same population used to initiate the exposure in order to determine growth rate (mg gained per amphipod per day) at the end of the exposure).

ACCLIMATION
- Acclimation period: 5 days
- Acclimation conditions: Two 11-L plastic tubs containing approximately 9 L of 20‰ seawater and a layer of marine sediment from the same source as that which was used in the exposure.
- Number of animals in each tub: Approximately 950 amphipods
- The overlying water within each tub was renewed on the second and fourth full day of holding.
- Salinity: 21 to 23‰
- Temperature: 23 to 24 °C
- Dissolved oxygen: 6.2 to 6.8 mg O2/L (82 to 90% air saturation)
- Type and amount of food: Approximately 1 mL of 100 mg/mL flaked fish food suspension to each holding vessel
- Health during acclimation (any mortality observed): No mortality was observed in the test population upon receipt nor during the acclimation period.

FEEDING DURING TEST PERIOD
- Food type: A diet consists of a flaked fish food suspension prepared in natural, filtered seawater.
- Feeding frequency: Once daily by adding 1.0 mL of flaked fish food suspension prepared at the appropriate concentration to each replicate vessel
- Amount of feed: On test days 0 through 6, each exposure vessel was fed 9.0 mg of flaked fish food. On days 7 through 13, 14 mg of flaked fish food was added to each vessel. On days 14 through 20, 20 mg of flaked fish food was added to each vessel. On days 21 through 27, 30 mg of flaked fish food was added to each vessel.
- Quality of the food: Representative samples of the food sources used during testing were analyzed periodically for the presence of pesticides, PCBs and toxic metals. None of these compounds have been detected at concentrations considered toxic in any of the samples analyzed. Based on these analyses, food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern (ASTM, 2007).

Study type:

laboratory study

Test type:

semi-static

Water media type:

saltwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Test temperature:

24 - 25 °C

pH:

- 7.2 - 8.0 (overlying water)
- 6.6 - 7.6 (pore water)

Dissolved oxygen:

- 3.8 - 7.8 mg O2/L
- 50 - 100% of saturation

Salinity:

- 18 - 20‰ (overlying water)
- 21 - 26 ‰ (pore water)

Ammonia:

- Day 0: 0.61 - 0.71 mg/L as nitrogen (overlying water)
- Day 28: 0.13 - 0.29 mg/L as nitrogen (overlying water)
- Day 0: 6.5 - 7.5 mg/L as nitrogen (pore water)
- Day 28: 0.85 - 2.2 mg/L as nitrogen (pore water)

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 0 (solvent control), 0.75, 1.5, 3.0, 6.0, and 12 µg/kg sediment dry weight
- Measured concentration (sediment): < LOQ (negative control), < LOQ (solvent control), 0.21, 0.38, 0.79, 1.7, and 3.0 µg/kg, respectively. See Table 2 to Table 4 in 'Any other information on materials and methods incl. tables'

Details on test conditions:

TEST SYSTEM
- Test container: 1 L clear glass jars (chemically cleaned prior to use and rinsed several times using tap water)
- Each jar had a hole cut at a height of 12 cm which allowed for drainage during the renewal of overlying water. The hole was covered with 40-mesh nylon screen to retain any test organisms that may have entered the water column.
- Sediment volume and depth: 120 mL (approximately 2 cm layer) of sediment
- Weight of sediment in each test container: Averaged 164 g (50.6 g dry weight)
- Overlying water volume and depth: 600 mL of overlying water (approximately 10 cm depth)
- The total overlying water/sediment volume was maintained at approximately 720 mL.
- Aeration: Yes
- Aeration method: Aeration with oil-free air was provided to each replicate with a constant, gentle flow of bubbles from a 1 mL glass pipette

EXPOSURE REGIME
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 11 (6 replicates for evaluationg the biological response and 5 replicates for chemical analysis and pore water quality measurements)
- No. of replicates per control / vehicle control: 11 (6 replicates for evaluationg the biological response and 5 replicates for chemical analysis and pore water quality measurements)

ALLOCATION OF SIDEMENT TO EXPOSURE VESSELS
- One day prior to exposure initiation (day -1), the treated and control sediments were allocated to the replicate vessels and overlying water was added. Overlying water was gently added to each vessel and then each vessel was randomly placed in the water bath used to house the exposure. 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 test vessels were then placed under the renewal system and aeration was initiated. The vessels were allowed to equilibrate overnight under exposure conditions, prior to the addition of test organisms.

RENEWAL OF OVERLYING WATER
- Details on volume additions: During the exposure, the overlying water was 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. The intermittent delivery system
was calibrated to provide 500 mL of water per cycle to the water-distribution system, which subsequently provided 100 mL of diluted seawater per cycle to each replicate test vessel. The water delivery system cycled approximately 12 times per day, providing approximately 1200 mL per vessel every 24 hours.
- Flow-rate: Approximately two overlying volume replacements per vessel per day
- 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.

OVERLYING WATER CHARACTERISTCS
- Type of water: The source of overlying water used during this study was natural, filtered seawater (salinity range of 30 to 32‰ and pH range of 7.9 to 8.0)
- Location: Seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts from about 4 meters offshore at a depth of approximately 0.5 meters.
- Filtration: The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a polyethylene holding tank. In the laboratory, the seawater was diluted with on-site laboratory well water to a salinity of 20 ± 2‰, filtered through 20 µm and 5 µm polypropylene core filters, and through 50 µm and 1 µm bag type filters.
- Salinity: 20 ± 2‰
- pH: 7.4 to 7.6
- TOC: Representative samples of the overlying water source were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the overlying water source was 1.9 and 1.5 mg/L for the months of November and December 2018, respectively. These analyses confirm the acceptability of this water source for ecotoxicity testing.
- Representative samples of the overlying water source were analyzed for the presence of pesticides, PCBs, and metals. None of these compounds were detected in any of the water samples analyzed in accordance with ASTM guidelines (2007).

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: The natural, marine sediment used during this study was collected from Sequim Bay, Sequim, Washington. Prior to characterization and use in testing, the sediment was wet pressed through a 0.25 mm sieve to remove large particles and indigenous organisms. A sample of the sieved sediment was characterized as having a mean (N = 3) percent organic carbon of 3.4%, a particle size distribution of 17% sand, 58% silt, and 25% clay, a pH of 7.5, and a water holding capacity (1/3 bar) of 77.7%. A percent solids value of 30.79% was also determined. The ammonia concentration in the sediment-pore water was measured 6 days prior to application of the test substance (20 days prior to exposure initiation) and was found to have a measured concentration of 9.1 mg/L as nitrogen. The ammonia level was within the expected range based on historical data for marine sediment from this source and was below the limit of 60 mg/L specified in the test method.
- Contaminations: Representative samples of the sediment were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the samples analyzed, in agreement with ASTM (2007) standard practice.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness (with 30 minutes transition periof from light to dark and vice versa)
- Light intensity and source: 510 to 890 lux; fluorescent bulbs

WATER QUALITY MEASUREMENTS
- At exposure initiation and termination, dissolved oxygen concentration, salinity, temperature, and pH were measured in the overlying water of each replicate vessel of each treatment level and control used for biological monitoring during the 28-day exposure.
- On test days 1 through 27, dissolved oxygen, salinity, pH, and temperature 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 temperature controlled water bath used to house the test vessels throughout the study.
- Ammonia concentration (as nitrogen) of the overlying water was monitored at exposure initiation and termination in each treatment level and control from a composite sample of all biological replicates.
- At exposure initiation (replicate H) and exposure termination (replicate K), salinity, pH, and ammonia concentrations (as nitrogen) were measured in a pore water sample of each treatment level and control.

EFFECT PARAMETERS MEASURED:
- All vessels established for monitoring biological response were examined at exposure initiation and daily thereafter, until exposure termination (day 28). Observations of organism mortality and abnormal behavior were made and the physical characteristics of the sediment/water system were recorded.
- At exposure termination (day 28), the surviving adult and young amphipods were collected from each test vessel by sieving the sediment through a 0.50 mm and 0.25 mm sieve or fine mesh net. Adult amphipods recovered at exposure termination were defined as those individuals that were retained on the 0.50-mm sieve. Young amphipods were defined as those individuals that were retained on the 0.25 mm sieve. At exposure termination, adult amphipods were preserved in buffered formalin solution to allow for identification of the gender of each adult at a later date (i.e., two days following exposure termination). The gender of the surviving adults was determined by identifying a notched palm on the distal segment of the gnathopod for males or the presence of brood plates or eggs in the brood pouch for females. After determination of gender, growth was determined by pooling the surviving adult amphipods (separated by gender) from each replicate vessel and drying at 63 °C for approximately 24 hours in an oven. Amphipods were rinsed gently with deionized water to remove any residual preservative prior to being dried in the oven. The pooled, dry amphipods were then weighed on an analytical balance to the nearest 0.01 mg. Growth rate for both males and female individuals was calculated using the following equation: mg gain/amphipod/day = (mg/amphipod at termination – mg/amphipod at initiation) / 28 days.
- At exposure termination, the offspring were removed from the 0.25 mm sieve and transferred to a labeled sample jar. Approximately 3.0 mL of concentrated Rose Bengal solution was added to the sample jar to stain the offspring for enumeration. The offspring were preserved
in a 70% ethanol solution until they were counted (within eight days of exposure termination). Reproduction was determined as the number of young per surviving female amphipod in each replicate vessel.

RANGE FINDING STUDY
- Study design: Prior to initiating the definitive study, an initial preliminary exposure was conducted under intermittent-renewal conditions. The test substance was applied to the sediment in the same manner and similar test procedures as the definitive test.
- Test nominal concentrations: 0.0076, 0.051, 0.34, 2.3 and 15 µg/kg sediment dry weight, and included a negative control and a solvent control.
- Results used to determine the conditions for the definitive study: An overview of the results is provided in Table 6 in "Any other information on results incl. tables". Based on these results and consultation with the Study Sponsor, nominal concentrations of 0.75, 1.5, 3.0, 6.0, and 12 µg/kg sediment dry weight were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1.7 µg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival, male growth rate, female growth rate and reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1.7 µg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: male growth rate, female growth rate and reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

3 µg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

An overview of the results in provided in Table 7 and Table 8 in 'Any other information on results incl. tables'.

SURVIVAL
Following 28 days of exposure, amphipod survival in the negative control and solvent control averaged 92 and 93%, respectively (pooled control = 92%). Amphipod survival observed in the negative control and solvent control groups during this period met the minimum standard criteria established by EPA test method 600/R-01/020 (i.e., ≥ 80% mean survival) and demonstrated that the exposure system provided test conditions that were appropriate for promoting acceptable survival of Leptocheirus plumulosus.
At test termination (test day 28), survival observed among amphipods in the 0.21, 0.38, 0.79, 1.7, and 3.0 µg/kg sediment dry weight treatment levels was 92, 93, 93, 90, and 54%, respectively. Statistical analysis (Dunnett's Multiple Comparison Test) determined a significant reduction in survival among amphipods exposed to the 3.0 µg/kg treatment level compared to the survival of the negative control and pooled control. Due to the statistically significant reduction in survival, the 3.0 µg/kg treatment level was excluded from subsequent statistical analysis for NOEC and LOEC determinations for sublethal endpoints.
Based on mean measured sediment concentrations, the NOEC and LOEC for survival were determined to be 1.7 and 3.0 µg/kg sediment dry weight, respectively.

GROWTH
Following 28 days of exposure, individual male amphipod growth rate in the negative control and solvent control averaged 0.10 and 0.10 mg/day, respectively (pooled control = 0.10 mg/day). Individual female amphipod growth rate in the negative control and solvent control averaged 0.072 and 0.067 mg/day, respectively (pooled control = 0.069 mg/day). Amphipod growth observed in the negative control and solvent control groups during this period met the minimum standard criteria established by EPA test method 600/R-01/020 (i.e., measurable growth) and demonstrated that the exposure system provided test conditions that were appropriate for promoting acceptable growth of Leptocheirus plumulosus.
Growth rate observed among individual male amphipods in the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 0.11, 0.095, 0.012, 0.093 and 0.087 mg/day, respectively. Due to the statistically significant reduction in survival, the 3.0 µg/kg treatment level was excluded from analysis of growth rate. Statistical analysis (Dunnett's Multiple Comparison Test) determined no significant reduction in male growth rate among amphipods exposed to any remaining treatment level compared to the negative control and the pooled control.
Growth rate observed among individual female amphipods in the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 0.072, 0.058, 0.074, 0.063, and 0.059 mg/day, respectively. Due to the statistically significant reduction in survival, the 3.0 µg/kg treatment level was excluded from analysis of growth rate. Statistical analysis (Dunnett’s Multiple Comparison Test) determined a significant reduction in female growth rate among amphipods exposed to the 0.38 µg/kg treatment level compared to the growth rate of the negative control and the pooled control; no other significant reductions were observed among the remaining treatment levels. The reduction at 0.38 µg/kg is not considered to be treatment-related due to the lack of a significant reduction at the next highest treatment level and the observation of the highest reproductive output among all treatment levels and controls. Higher reproductive output may have resulted in a lower dry weight at termination due to recent brood release, and additionally indicates that the reduction in growth observed is not biologically meaningful. The NOEC and LOEC for both male and female growth rate were determined to be 1.7 and > 1.7 µg/kg sediment dry weight, respectively.

REPRODUCTION
Following 28 days of exposure, amphipod reproduction in the negative control and solvent control averaged 27 and 25 offspring per female amphipod, respectively (pooled control = 26 offspring per female amphipod). Amphipod reproduction observed in the negative control and solvent control groups during this period met the minimum standard criteria established by EPA test method 600/R-01/020 (i.e., measurable reproduction) and demonstrated that the exposure system provided test conditions that were appropriate for promoting acceptable reproduction of Leptocheirus plumulosus.
Reproduction among amphipods exposed to the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 30, 32, 30, 22 and 18 offspring per female amphipod, respectively. Due to the statistically significant reduction in survival, the 3.0 µg/kg treatment level was excluded from subsequent analysis of reproduction. Statistical analysis (Dunnett's Multiple Comparison Test) determined no significant reduction in reproduction among amphipods exposed to any remaining treatment level compared to the negative control and the pooled control.
The NOEC and LOEC for reproduction were determined to be 1.7 and >1.7 µg/kg sediment dry weight, respectively.

Reported statistics and error estimates:

See statistical analysis in 'Any other information on materials and methods incl. tables'

Table 6. Results of the preliminary exposure test

Nominal Concentration (µg/kg sediment dry weight)	Mean Percent Survival	Mean Male Growth Rate/Amphipod/Day (mg)	Mean Female Growth Rate/Amphipod/Day (mg)	Mean Number of Offspring per Surviving Female Amphipod
Negative Control	74 (7)	0.072 (0.0075)	0.056 (0.0095)	17 (11)
Solvent Control	93 (6)	0.072 (0.0042)	0.072 (0.011)	16 (3.4)
0.0076	71 (21)	0.078 (0.013)	0.057 (0.0094)	23 (15)
0.051	80 (14)	0.074 (0.0076)	0.063 (0.0089)	17 (3.6)
0.34	58 (35)	0.060 (0.021)	0.057 (0.0060)	19 (1.9)
2.3	80 (9)	0.068 (0.0089)	0.056 (0.00082)	15 (4.1)
15	0 (0)	NAa	NA	NA

 

Table 7. Mean Percent Survival, Mean Growth Rate, and Number of Offspring per Surviving Female Amphipod at Test Termination of the 28-Day Exposure with the test substance and Amphipods (Leptocheirus plumulosus)

Mean Measured Sediment Concentration (µg/kg sediment dry weight)	Mean Percent Survival (SD^a)	Mean Male Growth Rate/Amphipod/Day (SD) (mg)	Mean Female Growth Rate/Amphipod/Day (SD) (mg)	Mean Number of Offspring per Surviving Female Amphipod (SD)
Negative Control	92 (7)	0.10 (0.0039)	0.072 (0.0043)	27 (5.0)
Solvent Control	93 (7)	0.10 (0.014)	0.067 (0.0098)	25 (5.6)
Pooled Control	92 (7)	0.10 (0.010)	0.069 (0.0078)	26 (5.1)
0.21	92 (7)	0.11 (0.013)	0.072 (0.0086)	30 (7.2)
0.38	93 (5)	0.095 (0.013)	0.058 (0.0063)^b	32 (3.9)
0.79	93 (6)	0.12 (0.022)	0.074 (0.0096)	30 (6.2)
1.7	90 (4)	0.093 (0.011)	0.063 (0.0055)	22 (5.1)
3.0	54 (16)^c	0.087 (0.018)	0.059 (0.021)	18 (14)

a SD = Standard Deviation

b Statistically significant reduction compared to the negative control and pooled control based on Dunnett’s Multiple Comparison Test; however, it is not considered to be treatment-related, nor biologically meaningful, due to the lack of significant reduction at the next highest treatment level which yielded the observation of highest reproductive output among all treatment levels and controls.

c Statistically significant reduction compared to the negative control and pooled control, based on Dunnett’s Multiple Comparison Test. This treatment level was excluded from subsequent statistical analysis for sublethal NOEC and LOEC determinations.

 

Table 8. Established Endpoints for the Exposure of Amphipods (Leptocheirus plumulosus) to the test substance Applied to Sediment

Based on Mean Measured Sediment Concentrations (µg/kg sediment dry weight)
Endpoint	Survival	Male Growth Rate	Female Growth Rate	Reproduction
Lowest-Observed-Effect Concentration (LOEC)	3.0^a	> 1.7	> 1.7	> 1.7
No-Observed-Effect Concentration (NOEC)	1.7	1.7	1.7	1.7

a Due to the effect in survival at 3.0 µg/kg, this treatment was excluded from subsequent sublethal NOEC/LOEC determinations.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the NOEC for survival, male growth rate, female growth rate and reproduction was determined to be 1.7 µg/kg sediment dry weight.

Executive summary:

This study was performed to determine the potential effects of the test substance on the estuarine, sediment-dwelling amphipod, Leptocheirus plumulosus, under intermittent-renewal conditions for 28 days following EPA guideline 600/R-01/020. The study was in compliance with GLP criteria. Eleven replicates were established for the negative control, solvent control, and each treatment level. Nominal concentrations for the treatment levels tested were 0.75, 1.5, 3.0, 6.0 and 12 µg/kg sediment dry weight (mean measured 0.21, 0.38, 0.79, 1.7, and 3.0 µg/kg sediment dry weight). Six replicates (A through F) were used to evaluate biological response of the test organisms. The five remaining replicates (G through K) were maintained for chemical analysis and for monitoring of water quality in the pore water (total ammonia, pH, and salinity). A sample of overlying water, pore water, and sediment of each treatment and control were removed from analytical replicates on test days 0, 14, and 28 for analysis of test substance concentration. All test vessels contained 20 amphipods, with the exception of replicates G and H, which were sacrificed at exposure initiation for the purpose of analytical and pore water quality measurements. Test vessels were maintained in a temperature-controlled water bath at 25 ± 1 °C with a 16-hour light:8-hour dark photoperiod at a light intensity range of 510 to 890 lux. The overlying water was 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. All vessels were examined at exposure initiation and once daily thereafter, until test termination (day 28). Observations of mortality and abnormal behavior were made and the physical characteristics of the sediment and overlying water were recorded. At test termination (day 28), the total number of surviving amphipods was determined in each test vessel by sieving the sediment through a 0.50 mm and 0.25 mm sieve or appropriate sized fine-mesh net to retain all surviving amphipods. Adult amphipods were defined as those individuals that were retained on the 0.50 mm sieve and offspring were defined as those amphipods which were retained on the 0.25 mm sieve. Adult amphipods were sexed and dry weight was determined post-termination. Offspring were preserved and enumerated over a 2 day period, 6 days post termination.

Following 28 days of exposure, amphipod survival in the negative control and solvent control averaged 92 and 93%, respectively (pooled control = 92%). At test termination (test day 28), survival observed among amphipods in the 0.21, 0.38, 0.79, 1.7, and 3.0 µg/kg sediment dry weight treatment levels was 92, 93, 93, 90, and 54%, respectively. After 28 days of exposure, individual male amphipod growth rate in the negative control and solvent control averaged 0.10 and 0.10 mg/day, respectively (pooled control = 0.10 mg/day). Individual female amphipod growth rate in the negative control and solvent control averaged 0.072 and 0.067 mg/day, respectively (pooled control = 0.069 mg/day). Growth rate observed among individual male amphipods in the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 0.11, 0.095, 0.012, 0.093 and 0.087 mg/day, respectively. Growth rate observed among individual female amphipods in the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 0.072, 0.058, 0.074, 0.063, and 0.059 mg/day, respectively. The amphipod reproduction after 28 days of exposure in the negative control and solvent control averaged 27 and 25 offspring per female amphipod, respectively (pooled control = 26 offspring per female amphipod). Reproduction among amphipods exposed to the 0.21, 0.38, 0.79, 1.7 and 3.0 µg/kg sediment dry weight treatment levels averaged 30, 32, 30, 22 and 18 offspring per female amphipod, respectively.

Based on the findings, the NOEC for survival, male growth rate, female growth rate and reproduction was determined to be 1.7 µg/kg sediment dry weight.

Description of key information

Freshwater sediment, 42-d NOEC = 0.77 µg/kg dry soil, Hyalella azteca, growth rate, EPA 100.4, Bradley 2019

Marine sediment, 28-d NOEC = 1.7 µg/kg dry soil, Leptocheirus plumulosus, mortality, male growth rate, female growth rate and reproduction, EPA 600/R-01/020, Bradley 2019

Key value for chemical safety assessment

EC50 or LC50 for marine water sediment:

0.77 µg/kg sediment dw

EC10, LC10 or NOEC for marine water sediment:

1.7 µg/kg sediment dw

Additional information

Freshwater

The effects of the substance on the survival, growth, and reproduction of the freshwater, sediment-dwelling amphipod Hyalella azteca under semi-static conditions were studied for a period of 42 days. Organisms were exposed to nominal concentrations of 0.020, 0.051, 0.13, 0.32, 0.80, and 2.0 µg/kg sediment dry weight (corresponding to mean measured concentrations of 0.019, 0.051, 0.13, 0.31, 0.77, and 2.1 µg/kg sediment dry weight), alongside a negative control and a solvent (acetone) control. Over the full 42-day study period, the growth endpoint was most sensitive with NOEC and LOEC values of 0.77 and 2.1 µg/kg sediment dry weight, respectively, when compared to the pooled control and 2.1 and >2.1 µg/kg sediment dry weight, respectively, when compared to the negative control. The endpoints for mortality and reproduction were less sensitive with 42-day NOEC values of 2.1 µg/kg sediment dry weight.

Marine water

The effects of the substance on the survival, growth rate and reproduction of the estuarine, sediment-dwelling amphipod Leptocheirus plumulosus under semi-static conditions were investigated for 28 days. Amphipods were exposed to nominal concentrations of 0.75, 1.5, 3.0, 6.0 and 12 µg/kg sediment dry weight (corresponding to mean measured concentrations of 0.21, 0.38, 0.79, 1.7, and 3.0 µg/kg sediment dry weight), alongside a negative control and a solvent (acetone) control. Over the study period of 28 days, the NOEC for survival, male growth rate, female growth rate and reproduction was determined to be 1.7 µg/kg sediment dry weight.