Prometryn

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jun 2015 to 10 Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Draft

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables"

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the in-life phase of the definitive study, sediment, pore water and overlying water samples were removed and analyzed for the test substance concentration on test days 0 and 10. On day 0 and 10, samples were removed and analyzed from replicate vessels I and J, respectively, for each treatment level and control. Replicate vessels K were maintained for contingency purposes, and were not sampled for chemical analysis during the exposure. Replicate K in the 50 mg/kg treatment level, however, was used to measure overlying water quality on day 10. Overlying water from each replicate vessel was removed 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 removed from the centrifuge tube and its volume measured. Sediment samples were collected from each centrifuge tube following centrifugation and removal of the pore water sample.

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

STOCK SOLUTION PREPARATION
A 10 mg/mL primary stock solution was prepared by dissolving 0.5000 g of the test substance in 50 mL of acetone. The primary stock solution was observed to be clear with a slight white hue and no visible undissolved test substance following preparation. Five individual dosing stock solutions were prepared in acetone for application of the test substance to the sediment. The preparation scheme is provided in Table 1 in "Any other information on materials and methods incl. tables". All stocks were observed to be clear and colorless with no visible undissolved test substance following preparation.

APPLICATION OF TEST SUBSTANCE TO SEDIMENT
- A jar-rolling technique was used to apply the test substance to the sediment (Ditsworth et al., 1990). A 10 mL aliquot of each dosing stock solution was applied to 0.050 kg of fine silica sand and mixed with a metal spatula for approximately two minutes. The acetone solvent was then allowed to evaporate for 30 minutes, leaving the test substance adhered to the sand. Since the solvent was allowed to completely evaporate to dryness, there was, in effect, no solvent introduced into the exposure system. Following evaporation, the entire sand/test substance mixture was added to 2.0 kg of wet sediment (0.9236 kg total dry weight based on 43.68% solids and including the aforementioned 0.050 kg of fine silica sand) in individual jars (e.g., 4 L glass jar). The application scheme is provided in Table 2 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 four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the jars were stored upright at 2 to 8 ºC in the dark. Based on the physicochemical characteristics of the test material and recommendations of the OCSPP 850.1735 draft guideline, the dosed sediments were allowed to equilibrate for a 27-day period in the refrigerator. The OCSPP 850.1735 draft guideline states that an approximate one-month aging period of dosed sediments should be sufficient for pore water and sediment concentrations to reach equilibrium, provided that the test compound is known to be persistent. Once a week during the 27-day equilibration period and prior to distribution of the sediments into the replicate test vessels, the jars were mixed on the rolling mill as previously mentioned for an additional two hours at room temperature to ensure the sediment was homogeneous.
The negative control sediment was prepared as described above using only untreated sediment (no test substance, solvent or additional 0.050 kg of fine silica sand). A solvent control was prepared in the same manner as the treated sediment by adding 10 mL of acetone, containing no test substance, to 0.050 kg of fine silica sand and the solvent was allowed to evaporate. The sand was then added to 2.0 kg wet weight of sediment and processed in the same manner as the treated sediments. The control and solvent control vessels were maintained under the same conditions as the treatment vessels.

Test organisms (species):

Hyalella azteca

Details on test organisms:

TEST ORGANISM
- Common name: Amphipod
- Source: Laboratory cultures maintained at the test facility
- Details on identification: The amphipods used during this study have been morphologically identified as Hyalella azteca at the original source of the culture and the identification has recently been confirmed through genomic screening.

TEST ORGANISM COLLECTION
- Prior to exposure initiation, adult amphipods were maintained in 38L glass aquaria containing approximately 31 L of culture water under flow-through conditions. The source of both the culture water and the overlying water used during the test was laboratory well water.
- Reproducing adult amphipods were removed from the main culture tanks nine days prior to exposure initiation. The adult amphipods were placed in 9.5 L aquaria (isolation tanks) containing approximately 8 L of water.
- Neonate amphipods (≤ 24 hours old) produced by these isolated adults were then removed from the isolation tanks on the following day and pipetted into 1 L beakers containing approximately 0.80 L of laboratory dilution water. Each 1 L beaker was stocked with approximately 230 neonate amphipods.

NEONATE AMPHIPODS HOLDING CONDITIONS
- Culture period: 8 days
- Culture type: Static condition with gentle, oil-free aeration
- Dissolved oxyge: 7.5 - 8.4 mg/L (87 - 98% air saturation)
- Temperature: 23 °C

FEEDING
- Adult amphipods: They were fed a combination of yeast, cereal leaves and flaked fish food suspension (YCT) on a daily basis.
- Isolated amphipods for testing (9 days before the test): They were fed a small amount of 100 mg/mL flaked fish food suspension and Ankistrodesmus falcatus, a unicellular green algae, were added to the isolation vessels as a supplemental food source.
- During the test: Each replicate test vessel received 1.0 mL of YCT daily.
- Representative samples of the food source 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, longevity of the culture population and survival of the juvenile test organisms, the food sources were considered to be of acceptable quality since analyte concentrations were below levels of concern (EPA, 1997).
- Health during acclimation (any mortality observed): No mortality was observed in the test population 48 hours prior to exposure initiation.

Study type:

laboratory study

Test type:

semi-static

Water media type:

freshwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Hardness:

- Day 0: 40 - 52 as CaCO3 mg/L
- Day 10: 48 - 52 as CaCO3 mg/L

Test temperature:

23 ± 1 ºC

pH:

6.4 - 7.0

Dissolved oxygen:

3.6 - 7.2 mg O2/L ( (40 - 84 % of saturation)

Ammonia:

- Day 1: 0.25 - 0.32 as nitrogen mg/L
- Day 10: ≤ 0.11 as nitrogen mg/L

Conductivity:

- Day 0: 300 - 310 µS/cm
- Day 10: 330 - 340 µS/cm

Nominal and measured concentrations:

- Nominal concentration: 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight
- Mean measured concentration: 5.1, 9.9, 20, 41 and 74 mg/kg sediment dry weight, respectively. An complete overview of the measure concentrations in sediment, pore water and overlying water at different time intervals is presented in Tables 3, 4 and 5 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test container: 300 mL glass vessels (chemically cleaned prior to use and rinsed several times using tap water)
- Each test vessel had an opening on the top edge of the beaker which was covered with 40-mesh screen for drainage.
- Sediment volume: 100 mL (approximately 4 cm layer)
- Weight of wet and dry sediment: 145 g wet weight per vessel or 63.3 g dry weight per vessel
- Overlying water volume: 175 mL
- The total overlying/sediment volume: Approximately 275 mL
- Test vessels were randomly positioned in a water bath containing circulating water designed to maintain the test temperature

EXPOSURE REGIME
- No. of organisms per container: 10
- No. of organisms per concentration: 80 amphipods per concentration or control for the replicates maintained for monitoring the
biological response. The additional 3 replicates were maintained under the same conditions and contained test organisms during the exposure, with the exception of one additional replicate which was sampled on day 0 prior to the addition of organisms. The additional replicates were not used to evaluate the biological response of the test organisms.
- No. of replicates per treatment group: 11 (8 replicates were used to evaluate the biological response (survival and growth) of the test organisms. The remaining 3 replicates were maintained for the purpose of chemical analysis.).
- No. of replicates per control / vehicle control: 11
- Age of organisms at initial test: 8 days old

RENEWAL OF OVERLYING WATER
- Details on volume additions: The overlying water was initially renewed by adding two volume additions (i.e., 350 mL) per test vessel per day using an intermittent delivery system in combination with a calibrated water-distribution system (Zumwalt et al., 1994). 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 7 times per day, providing approximately 350 mL per vessel every 24 hours. Due to an observed trend of decreasing dissolved oxygen measurements in the test vessels on day 7 (minimum of 3.78 mg/L equivalent to 44% air saturation value), the flow rate in the system was doubled so that the water delivery system cycled approximately 14 times per day, providing approximately 700 mL per vessel every 24 hours (i.e., approximately four overlying volume replacements per vessel per day). All subsequent dissolved oxygen measurements were within an acceptable range. The trend of decreasing dissolved oxygen is commonly observed during intermittent-renewal sediment testing and is often attributable to increasing biomass within the test vessels.
- The calibration of the overlying water renewal system was checked prior to exposure initiation and confirmed at exposure termination. During the definitive exposure, the renewal system was visually inspected at least twice daily. A complete check of intermittent delivery system function was made once daily.

OVERLYING WATER CHARACTERISTCS
- Type of water: Laboratory well water
- Total hardness: 44 to 56 mg/L as CaCO3
- Alkalinity: 19 to 20 mg/L as CaCO3
- pH: 6.6 to 7.3
- Conductivity: 240 to 320 µS/cm
- Representative samples of the overlying water source 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 water samples analyzed, in agreement with EPA (1997) standard practice.
- Amphipod cultures are maintained in water from the same source as the overlying water utilized during 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 during the conduct of bioassays.

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Natural, freshwater sediment was collected from Glen Charlie Pond, Wareham, Massachusetts
- Prior to use and characterization, the sediment was pressed through a 2.0-mm sieve to remove large particles and indigenous organisms.
- Mean organic carbon: 2.5%
- Particle size distribution: 91% sand, 8% silt and 1% clay
- pH: 5.6
- Moister: At 1/3 bar (water holding capacity) of 16.5%
- Solids value: 43.68%
- Water ammonia (as nitrogen): 1.4 mg/L
- 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 EPA (1997) standard practice.

ALLOCATION OF SEDIMENT TO TEST VESSELS
- One day prior to exposure initiation (day -1), the treated and control sediments were allocated to each treatment and control vessel (100 mL per vessel). Overlying water was gently added to each vessel and then each vessel was placed under the renewal system. 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 on test day -1.

EXPOSURE INITIATION
- Amphipods were added impartially to an intermediate set of beakers by adding no more than two amphipods to each beaker until all beakers contained two amphipods. This procedure was repeated until each beaker contained ten amphipods. The exposure was initiated when each intermediate beaker of ten amphipods was added to each respective test vessel. Measurement of dry weight was performed on a subpopulation of twenty amphipods at exposure initiation and the dry weight was determined to be 0.0185 mg dry weight per amphipod.

OTHER TEST CONDITIONS
- Light source: Fluorescent bulbs
- Photoperiod: 16 hours/ 8 hours (light/dark)
- Light intensity: 460 to 530 lux (measured once during the exposure)

WATER PARAMETER
- At exposure initiation and termination, temperature, dissolved oxygen concentration and pH were measured in the overlying water of each replicate vessel of each treatment level and control used for biological monitoring (A through H) during the 10-day exposure. On test days 1 through 9, dissolved oxygen and temperature were measured daily in one alternating replicate vessel from 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.
- Total hardness, alkalinity, conductivity and ammonia concentration of the overlying water were monitored at exposure initiation and exposure termination in each treatment level and control solution from a composite sample (replicates A through H).

EFFECT PARAMETERS MEASURED:
- Following the addition of amphipods, all vessels were examined at exposure initiation and daily thereafter, until exposure termination (day 10). Observations of mortality and abnormal behavior were made and the physical characteristics of the test solutions were recorded. At exposure termination, the sediment from each biological replicate was sieved using fine mesh fish nets (approximately 0.25 mm opening) to remove all surviving amphipods. 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 67 to 70 ºC for 24 hours. The pooled amphipods were then weighed on a calibrated analytical balance to the nearest 0.01 mg.

RANGE FINDING STUDY:
- Prior to initiation of the definitive exposure, a preliminary range-finding study was conducted where amphipods were exposed to the test substance under nominal concentrations of 0.39, 1.6, 6.3, 25 and 100 mg/kg sediment dry weight, a control and solvent (acetone) control. Sediments were prepared as similar procedures as the definitive exposure and were equilibrated for approximately 30 days prior to the preliminary exposure. Four replicate vessels, each containing ten amphipods (eight days old), were established for each treatment level and the controls.
- Results: At exposure termination, 100, 98, 98, 98 and 100% survival was observed among amphipods exposed to the 0.39, 1.6, 6.3, 25 and 100 mg/kg sediment dry weight treatment levels, respectively. During the same period, survival of amphipods in the negative control and solvent control averaged 95 and 98%, respectively. Dry weight among surviving amphipods exposed to the 0.39, 1.6, 6.3, 25 and 100 mg/kg sediment dry weight treatment levels averaged 0.23, 0.23, 0.24, 0.24 and 0.23 mg, respectively. During the same period, dry weight among amphipods in the negative control and the solvent control averaged 0.25 and 0.24 mg, respectively.
- Based on these results and in consultation with the Study Sponsor, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight were selected for the definitive exposure.

Reference substance (positive control):

no

Key result

Duration:

10 d

Dose descriptor:

EC50

Effect conc.:

> 74 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality and growth

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

74 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality and growth

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

> 74 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: mortality and growth

Details on results:

An overview of the results in provided in Tables 6, 7 and 8 in “Any other information on results incl. tables”.

Following 10 days of exposure, amphipod survival in the negative control and solvent control averaged 99 and 98%, respectively. During the same period, amphipod growth (dry weight) in the negative control and solvent control averaged 0.16 and 0.14 mg per amphipod, respectively, indicating a mean 8.6 and 7.6-fold increase in amphipod weight, respectively, based on the initial mean dry weight of 0.0185 mg per amphipod. Amphipod survival and growth observed in both the negative control and solvent control during this period met the minimum standard criteria established by the draft OCSPP Guideline (i.e., 80% control survival and measurable growth in the controls). As demonstrated by the negative control and solvent control organism performance, the exposure system provided test conditions that were appropriate for promoting acceptable survival and growth of Hyalella azteca.

- Survival: At exposure termination (test day 10), survival observed among amphipods in the 5.1, 9.9, 20, 41 and 74 mg/kg treatment levels averaged 99, 94, 91, 98 and 94%, respectively. Statistical analysis (Steel’s Many-One Rank Sum Test) determined no significant difference in survival among amphipods exposed to any of the treatment levels tested compared to the negative control data (99%).
- Growth: At exposure termination (test day 10), growth observed among amphipods in the 5.1, 9.9, 20, 41 and 74 mg/kg treatment levels was 0.14, 0.13, 0.14, 0.16 and 0.15 mg per amphipod, respectively. Statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in growth among amphipods exposed to any of the treatment levels tested compared to the negative control (0.16 mg).

Reported statistics and error estimates:

See Statistical analyses in "Any other information on materials and methods incl. tables"

Table 6. Amphipod survival and growth (dry weight) in each replicate vessel at exposure termination of the 10-day static-renewal exposure of freshwater amphipods (Hyalella azteca) to the test substance applied to sediment.

Mean Measured Concentration (mg/kg sediment dry weight)		Test Day 10
	Replicate	Percent Survival	Dry Weight Per Amphipod (mg)
Control	A	100	0.14
	B	100	0.14
	C	100	0.12
	D	100	0.18
	E	100	0.17
	F	100	0.16
	G	90	0.19
	H	100	0.18
Solvent Control	A	100	0.17
	B	100	0.15
	C	90	0.11
	D	100	0.16
	E	100	0.14
	F	90	0.10
	G	100	0.14
	H^a	100	0.08
5.1	A	100	0.17
	B	100	0.14
	C	90	0.11
	D	100	0.20
	E	100	0.12
	F	100	0.14
	G	100	0.12
	H	100	0.13
9.9	A	90	0.11
	B	100	0.14
	C	90	0.16
	D	100	0.15
	E	100	0.14
	F	100	0.10
	G	90	0.16
	H	80	0.10
20	A	90	0.11
	B	90	0.14
	C	90	0.14
	D	100	0.16
	E	100	0.16
	F	80	0.10
	G	90	0.15
	H	90	0.14
41	A	100	0.14
	B	100	0.17
	C	100	0.18
	D	100	0.16
	E	100	0.14
	F	100	0.15
	G	100	0.17
	H	80	0.16
74	A	90	0.16
	B	90	0.13
	C	100	0.15
	D	90	0.22
	E	80	0.14
	F	100	0.16
	G	100	0.12
	H	100	0.11

a) Inadvertently initiated with 11 organisms. Percent survival considered to be 100% for use in statistical analysis. However, growth data has been omitted from statistical analyses and summary data due to potential influence of increased loading rate.

Table 7. Mean percent survival and mean dry weight during the 10-day static-renewal exposure of freshwater amphipods (Hyalella azteca) to the test substance applied to sediment.

Mean Measured Concentration (mg/kg sediment dry weight)	Test Day 10
	Mean Percent Survival (SD^a)	Mean Dry Weight Per Amphipod in mg (SD)
Control	99 (4)	0.16 (0.024)
Solvent Control	98 (5)	0.14 (0.027)
5.1	99 (4)	0.14 (0.030)
9.9	94 (7)	0.13 (0.025)
20	91 (6)	0.14 (0.021)
41	98 (7)	0.16 (0.014)
74	94 (7)	0.15 (0.034)

a) SD = Standard Deviation.

Table 8. Established endpoints for the 10-day static-renewal exposure of freshwater amphipods (Hyalella azteca) to the test substance applied to sediment.

Based on Mean Measured Concentrations (mg/kg sediment dry weight)
Endpoint	Amphipod Survival	Amphipod Growth
Lowest-Observed-Effect Concentration (LOEC)	> 74	> 74
No-Observed-Effect Concentration (NOEC)	74	74
10-Day LC50/EC50 (95% confidence intervals)	> 74 (NA^a)	> 74 (NA)

a) NA = Not Applicable. LC/EC50values were empirically estimated; therefore, corresponding 95% confidence intervals could not be determined.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 10-day EC50 of survivial and growth was empirically estimated to be > 74 mg/kg sediment dry weight, the highest mean measured concentration tested.

Executive summary:

The purpose of this study was to determine the effect of the test substance, applied to sediment, on the survival and growth (final dry weight) of the 8 days old benthic freshwater amphipod, Hyalella azteca. The test was conducted according to EPA 850.1735 guideline (draft) and in compliance with GLP. The definitive exposure was performed under intermittent, static-renewal conditions of overlying water for a period of 10 days, at nominal test concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight (5.1, 9.9, 20, 41 and 74 mg/kg sediment dry weight, respectively, mean measured concentration by LC/MS/MS). In addition, blank and solvent (10 mL acetone to 0.05 kg silica sand) control treatments were set up, too. Acetone stock solutions of test substance were prepared for sediment dosing. A 10-mL aliquot of each stock solution was applied to 0.05 kg of fine silica sand and mixed with a spatula. The acetone was then allowed to evaporate for 30 minutes, prior to adding this pre-mixture to 2.0 kg of wet natural sediment filled in a jar. Proper mixing of sand and sediment was achieved by jar-rolling technique for 4 hours at 15 rpm. A volume of 100 mL (145 / 63.3 g wet/dry weight) of sediment was filled into a 300 mL glass vessel and overlaid with 175 mL of water. Eleven replicate vessels (8 used for biological response and 3 used for chemical analysis) each comprising 10 amphipods were maintained for each treatment and control group. Exposure concentrations of the test substance in sediment, pore water and overlying water were measured on days 0 and 10. The water quality during the experimental period was as follow: 22 - 24°C, pH 6.4 - 7.0, 4.1 - 7.1 mg/L of dissolved oxygen (41 - 84% of saturation) and, photoperiod of 16 hours light (460-530 lux) and 8 hours darkness.

Following 10 days of exposure, amphipod survival in the negative control and solvent control averaged 99 and 98%, respectively. During the same period, amphipod growth (dry weight) in the negative control and solvent control averaged 0.16 and 0.14 mg per amphipod, respectively, indicating a mean 8.6 and 7.6-fold increase in amphipod weight, respectively, based on the initial mean dry weight of 0.0185 mg per amphipod. At exposure termination (test day 10), statistical analysis (Steel’s Many-One Rank Sum Test) determined no significant difference in survival (91 – 99%) among amphipods exposed to any of the treatment levels tested compared to the negative control data (99%). At exposure termination (test day 10), statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in growth (0.13 – 0.16 mg mean dry weight) among amphipods exposed to any of the treatment levels tested compared to the negative control (0.16 mg). Based on mean measured sediment concentrations and amphipod survival and growth, the Lowest-Observed-Effect Concentration (LOEC) and No-Observed-Effect Concentration (NOEC) were determined to be >74 and 74 mg/kg sediment dry weight, respectively. The 10-day LC50/EC50 value was empirically estimated to be >74 mg/kg sediment dry weight.

Reference 2

Endpoint:

sediment toxicity: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jun 2015 to 6 Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1740 (Whole Sediment Acute Toxicity of Invertebrates, marine)

Deviations:

yes

Remarks:

See Deviations from the protocol in "Any other information on materials and methods incl. tables"

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- During the in-life phase of the definitive study, sediment, overlying water and pore water samples were removed and analyzed for the test substance concentration on test days 0 (exposure initiation) and 10 (exposure termination). On days 0 and 10, sediment, overlying water and pore water samples were removed and analyzed from replicate vessels F and H, respectively, for all treatment levels and the controls.
- Overlying water from each replicate vessel was decanted into separate graduated cylinders 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 a rate of approximately 10,000 g. The resulting pore water was removed from the centrifuge tube and its volume measured. Sediment samples were collected from each centrifuge tube with a stainless steel spatula, following centrifugation and removal of the pore water sample. After the removal of pore water, sediment samples were mixed well to ensure homogeneous subsamples.

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

STOCK SOLUTION PREPARATION
A 20 mg/mL primary stock solution was prepared by dissolving 1.0005 g of the test substance to a volume of 50 mL with acetone. The resulting stock solution was observed to be clear with a slight white hue and no visible undissolved test substance following preparation. Five individual dosing stock solutions were prepared in acetone for application of the test substance to the sediment. The dosing stock preparation scheme is provided in the Table 1 in "Any other information on materials and methods incl. tables". All dosing stock solutions were observed to be clear and colorless with no visible undissolved test substance following preparation

APPLICATION OF TEST SUBSTANCE TO SEDIMENT
A jar-rolling technique was used to apply the test substance to the sediment (Ditsworth et al., 1990). A 10-mL volume of each dosing stock solution was applied to 0.050 kg of fine silica sand which was then mixed with a metal spatula for approximately 2 minutes. The acetone solvent was allowed to completely evaporate from the sand for 30 minutes, leaving the material adhered to the sand. Since the solvent was allowed to completely evaporate to dryness, there was, in effect, no solvent introduced into the exposure system. Following evaporation, the entire sand/test substance mixture was added to 2.5 kg of wet sediment (1.008 kg total dry weight based on a percent solids value of 38.32% and including the 0.050 kg of sand) in a jar (e.g., 4-L glass jar). A scheme of applying the test substance to the sediment is provided in Table 2 in the "Any other information on materials and methods incl. tables".
Each jar was then rolled on a rolling mill for four hours at room temperature at approximately 15 rpm. Following four hours of rolling, the jars were stored upright in a dark refrigerator at 2 to 8ºC. Based on the physicochemical characteristics of the test material and recommendations of associated OCSPP draft guidelines (850.1740 and 850.1735, U.S. EPA, 1996a and U.S. EPA, 1996b, respectively), the spiked sediments were allowed to equilibrate for a 28-day period in a dark refrigerator (approximately one month). Once a week during the 28-day equilibration period and again prior to distribution of the sediments into the replicate test vessels, the jars were mixed on the rolling mill for an additional two hours at room temperature to ensure the sediment was homogeneous.
The negative control sediment group was prepared as described above using only untreated sediment (no test substance, solvent or added 0.050 kg of sand). A solvent control sample was prepared in the same manner as the treated sediment by adding 10 mL of acetone, containing no test substance, to 0.050 kg of fine silica sand and the solvent was allowed to evaporate. The sand was then added to 2.5 kg wet weight of sediment and processed in the same manner as the treated sediments. The control and solvent control sediments were maintained under the same conditions as the treatment sediments during the equilibration period.

Test organisms (species):

Leptocheirus plumulosus

Details on test organisms:

TEST ORGANISM
- Common name: Amphipod
- Source: Chesapeake Cultures, Hayes, Virginia
- Age and lenth: Approximately of the same size and age (immature amphipods ranging from 2.0 to 4.0 mm in length, correlating to those amphipods that pass through a 1.0-mm sieve and are retained on a 0.50-mm sieve, as described in OCSPP Draft Guideline 850.1740, U.S. EPA, 1996a).

ACCLIMATION
- Acclimation period: 48 hours prior to testing
- Acclimation conditions: Maintained in two 2-L glass beakers containing approximately 1.8 L of natural, filtered seawater that was diluted to a salinity of 20‰
- Organisms were acclimated in the same type of water that was used as overlying water during the definitive test.
- Dissolved oxygen range: 6.3 - 7.3 mg/L (85 to 97% air saturation)
- Salinity: 21‰
- Temperature: 24ºC
- Feeding: At a rate sufficient to maintain the population (approximately 100 mg of flaked fish food per beaker per day).
- No mortality was observed in the test population prior to exposure initiation.

FEEDING
- During holding and acclimation period: Amphipods in each holding vessel were fed a diet of flaked fish food suspended in laboratory well water
- During exposure: Amphipods were not fed.
- Representative samples of the food source used during organism holding 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:

static

Water media type:

saltwater

Type of sediment:

natural sediment

Limit test:

no

Duration:

10 d

Exposure phase:

total exposure duration

Test temperature:

25 ± 1 ºC

pH:

7.8 - 8.3

Dissolved oxygen:

5.9 - 7.8 mg O2/L (69 - 106 % of saturation)

Salinity:

- 20 - 22 ‰ (overlying water)
- Day 0: 24 - 25 ‰ (pore water)
- Day 10: 21 - 22 ‰ (pore water)

Ammonia:

- Day 0: 2.0 - 2.4 as nitrogen mg/L (overlying water)
- Day 10: 3.3 - 4.6 as nitrogen mg/L (overlying water)
- Day 0: 8.2 - 11 as nitrogen mg/L (pore water)
- Day 10: 4.3 - 6.8 as nitrogen mg/L (pore water)

Nominal and measured concentrations:

Nominal concentration: 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight
Mean measured sediment concentrations: 5.3, 11, 21, 43 and 90 mg/kg sediment dry weight, respectively. An complete overview of the measure concentrations in sediment, pore water and overlying water at different time intervals is presented in Tables 3, 4 and 5 in "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test container: 1000 mL glass beakers (chemically cleaned prior to use and rinsed several times using tap water)
- Sediment volume: 175 mL (approximately 2-cm layer)
- Weight of wet and dry sedimen: 223 g wet weight (85.6 g dry weight)
- Overlying water volume: 725 mL
- Total overlying water/sediment volume: Maintained at approximately 900 mL

EXPOSURE REGIME
- No. of organisms per container: 20
- No. of replicates per treatment group: 9 (5 replicates for evaluating the biological response and 4 additional replicates for chemical analysis of the test substance and pore water quality measurements)
- No. of replicates per control / vehicle control: 10
- No. of organisms per concentration for monitoring the biological responses: 100
- The additional replicates were maintained under the same conditions and contained test organisms, with the exception of replicates F and G which were sampled on day 0 prior to the addition of organisms. The additional replicates for analytical or pore water quality measurements were not used to evaluate the biological response of the test organisms.

OVERLYING WATER CHARACTERISTCS
- Type of water: Natural, filtered seawater
- Source and location of water: Pumped from the Cape Cod Canal, Bourne, Massachusetts from about 4 meters offshore at a depth of approximately 0.5 meters.
- The seawater was transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 3400 L fiberglass holding tank. The seawater was pumped under constant pressure through PVC pipes from the fiberglass holding tank to a laboratory holding reservoir. In the laboratory, the seawater was recirculated within an epoxy-coated concrete holding reservoir until use. Prior to use in this study, the seawater was diluted with laboratory well water.
- pH: 7.9 to 8.0 (original seawater source); 7.8 to 7.9 (after dilution with laboratory well water)
- Salinity: 30 to 31‰ (original seawater source); 19 to 21‰ (after dilution with laboratory well water)
- Representative samples of the 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 agreement with ASTM guidelines (2007). These analyses confirmed the acceptability of this overlying water for use during the conduct of the exposure.

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: The sediment used during this study was collected from Sequim Bay, Sequim, Washington.
- Prior to use and characterization, the sediment was wet pressed through a 0.25-mm sieve to remove large particles and any indigenous organisms. After sieving, the sediment was characterized.
- Mean percent organic carbon: 2.8%
- Particle size distribution: 29% sand, 50% silt and 21% clay
- pH: 7.8
- Moisture at 1/3 bar (water holding capacity): 68.1%
- Solids: 38.32%
- A sample of the sediment pore water was generated from the control sediment prior to exposure initiation and was found to have a measured ammonia concentration of 15 mg/L as nitrogen, which is below the guideline recommended limit of 60 mg/L.
- A representative sample of the sediment source was analyzed for the presence of pesticides, PCBs and toxic metals. None of these compounds were found detected at concentrations that would be considered to have an adverse impact on the results of the test.

ALLOCATION OF SEDIMENT TO TEST VESSELS
- One day prior to exposure initiation (day -1), the treated and control sediments and overlying water were allocated to each treatment and control vessel. Overlying water was gently added to each vessel to avoid suspension of the sediment layer. Each vessel was then placed in the water bath. Each test vessel was covered with a plastic plate and aeration was supplied with a constant trickle flow of bubbles from a 1 mL glass pipette.

EXPOSURE INITIATION
- At exposure initiation, amphipods were added impartially to each replicate, five at a time, until each replicate contained five amphipods. This procedure was repeated until each replicate contained twenty amphipods. The exposure was initiated when each respective replicate contained twenty amphipods.

OTHER TEST CONDITIONS
- Light source: Fluorescent bulbs
- Photoperiod: Continuously
- Light intensity: 560 to 790 lux

WATER PARAMETER
- At exposure initiation and termination, temperature, pH, dissolved oxygen concentration and salinity were measured in the overlying water of each replicate vessel of each treatment level and control used for biological monitoring (A through E) during the 10-day exposure. On test days 1 through 9, temperature, dissolved oxygen, pH and salinity were measured in one alternating replicate from each treatment level and control each day. 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.
- Total ammonia concentration (as nitrogen) of the overlying water was monitored at exposure initiation (day 0) and at exposure termination (day 10) in a sample of overlying water from each treatment and control group.
- At exposure initiation and again at termination, salinity, temperature, pH and total ammonia concentration (as nitrogen) was measured in a pore water sample from replicates G and I, respectively, of each treatment level and control.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- All vessels were examined at exposure initiation and daily thereafter, until test termination (day 10). Observations of mortality and abnormal behavior were made and the physical characteristics of the test samples were recorded. At exposure termination (day 10), the total number of surviving amphipods was determined in each test vessel by sieving the entire volume of sediment through a fine mesh net or sieve (≤ 0.50 mm opening) to remove all surviving amphipods. Missing animals or any animals observed failing to respond to gentle prodding (i.e., neuromuscular twitch of pleopods or antennae) were recorded as dead.

RANGE FINDING STUDY
- Prior to initiating the definitive study, a preliminary range-finding exposure of the test substance was conducted at the test facility at nominal concentrations of 0.39, 1.6, 6.3, 25 and 100 mg/kg sediment dry weight, a control and a solvent (acetone) control. Sediments were prepared following similar procedures as the definitive test and were equilibrated for approximately 1 month prior to the preliminary exposure. Three replicate vessels, each containing 20 amphipods, were established for each treatment level and the controls.
- Results: Following 10 days of exposure, amphipod survival in both the control and solvent control averaged 98 and 100%, respectively. At exposure termination (test day 10), survival observed among amphipods in the 0.39, 1.6, 6,3, 25 and 100 mg/kg sediment dry weight treatment levels was 97, 97, 98, 90 and 0%, respectively. Based on these results and consultation with the Study Sponsor, the definitive exposure was conducted at with the following nominal the test substance concentrations: 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight.

Reference substance (positive control):

no

Key result

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

58 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

95% C.I.: 55 - 60 mg/kg sediment dw

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

43 mg/kg sediment dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

90 mg/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 Tables 6 and 7 in “Any other information on results incl. tables”.
Following 10 days of exposure, amphipod survival in both the control and solvent control averaged 99 and 100%, respectively. Amphipod survival observed in the control and solvent control during this period met the minimum standard criteria established by the EPA test method (i.e., ≥ 90% mean survival).
At exposure termination (test day 10), survival observed among amphipods in the 5.3, 11, 21, 43 and 90 mg/kg sediment dry weight treatment levels was 99, 98, 99, 90 and 0%, respectively. Statistical analysis (Steel Many-One Rank Sum Test) determined no significant difference in survival among amphipods exposed to the 5.3, 11, 21 and 43 mg/kg treatment level compared to the negative control (99%). The 90 mg/kg sediment dry weight treatment level was censored from statistical analyses due to 100% mortality in that treatment level. No sublethal or behavioral effects were noted among the amphipods in any of the treatment replicates.

Reported statistics and error estimates:

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

Table 6. Amphipod survival at exposure termination of the 10-day study exposing amphipods (Leptocheirus plumulosus) to the test substance

Mean Measured Sediment Concentration (mg/kg sediment dry weight)	Test Day 10
	Replicate	Number Surviving	Mean Percent Survival (SD^a)
Control	A	20
	B	20
	C	19	99 (2)
	D	20
	E	20
Solvent Control	A	20
	B	20
	C	20	100 (0)
	D	20
	E	20
5.3	A	20
	B	20
	C	20	99 (2)
	D	19
	E	20
11	A	20
	B	20
	C	20	98 (3)
	D	19
	E	19
21	A	20
	B	20
	C	20	99 (2)
	D	20
	E	19
43	A	20
	B	18
	C	16	90 (8)
	D	17
	E	19
90	A	0
	B	0
	C	0	0 (0)
	D	0
	E	0

a SD = Standard Deviation.

Table 7. Established endpoints for the exposure of amphipods (Leptocheirus plumulosus) to the test substance applied to sediment

	Amphipod Survival
Endpoint	Mean Measured Sediment Concentration (mg/kg)
Lowest-Observed-Effect Concentration (LOEC)	90
No-Observed-Effect Concentration (NOEC)	43
10-Day LC50 (95% confidence intervals)	58 (55 - 60)

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the 10-d NOEC of amphipod survival was determined to be 43 mg/kg sediment dry weight, and the LC50 was determined to be 58 mg/kg sediment dry weight (corresponding 95 % confidence intervals 55 - 60 mg/kg sediment dry weight).

Executive summary:

The purpose of this study was to determine the effect of the test substance on the survival of an estuarine, sediment-dwelling amphipod (Leptocheirus plumulosus) for 10 days following EPA 850.1740 guideline and in compliance with GLP. Amphipods were exposed to nominal sediment concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight (5.3, 11, 21, 43 and 90 mg/kg sediment dry weight mean measured concentration by LC/MS/MS), a control and a solvent (10 mL acetone to 0.05 kg silica sand) control. Acetone stock solutions of test substance were prepared for sediment dosing. A 10-mL aliquot of each stock solution was applied to 0.05 kg of fine silica sand and mixed with a spatula. The acetone was then allowed to evaporate for 30 minutes, prior to adding this pre-mixture to 2.0 kg of wet natural sediment filled in a jar. Proper mixing of sand and sediment was achieved by jar-rolling technique for 4 hours at 15 rpm. A volume of 175 mL (223 / 85.6 g wet/dry weight) of sediment was filled into a 1 L glass beaker and overlaid with approximately 900 mL of filtered seawater. Nine replicate beakers (5 used for biological response and 4 used for chemical analysis) each comprising 10 amphipods were maintained for each treatment and control group. Exposure concentrations of the test substance in sediment, overlying water and pore water were measured on days 0 (exposure initiation) and 10 (exposure termination). The test condition during the experimental period was as follow: 24 - 26 °C, pH 7.8 – 8.3, dissolved oxygen 5.2 – 7.8 mg/L of (69 - 106% of saturation) and, continuous illumination at an intensity in the range of 560 to 790 lux. The salinity of overlying water was 20 - 22‰ and the salinity of pore water was 24 - 25‰ on day 0 and 21 - 22‰ on day 10.

Following 10 days of exposure, amphipod survival in both the control and solvent control averaged 99 and 100%, respectively. At exposure termination (test day 10), statistical analysis (Steel Many-One Rank Sum Test) determined no significant difference in survival (90 – 99%) among amphipods exposed to the 5.3, 11, 21 and 43 mg/kg treatment level compared to the negative control (99%). The 90 mg/kg sediment dry weight treatment level was censored from statistical analyses due to 100% mortality in that treatment level. No sublethal or behavioural effects were noted among the amphipods in surviving animals at any of the treatment concentrations.Based on mean measured sediment concentrations and amphipod survival, the NOEC and LOEC were determined to be 43 and 90 mg/kg sediment dry weight, respectively. The 10-day LC50 value, as determined by untrimmed Spearman-Kärber method, was estimated to be 58 mg/kg sediment dry weight with 95% confidence intervals of 55 to 60 mg/kg.

Description of key information

Freshwater, 10-d EC50 > 74 mg/kg sediment dry weight, Hyalella azteca, survivial and growth, EPA 850.1740, Bradley 2015a

Marine water, 10-d LC50 = 58 mg/kg sediment dry weight (corresponding 95% confidence intervals 55 - 60 mg/kg sediment dry weight), Leptocheirus plumulosus, mortality, EPA 850.1740, Bradley 2015b

Key value for chemical safety assessment

EC50 or LC50 for marine water sediment:

58 mg/kg sediment dw

Additional information

Fresh water

The purpose of this study was to determine the effect of the test substance, applied to sediment, on the survival and growth (final dry weight) of the 8 days old benthic freshwater amphipod, Hyalella azteca. The test was conducted according to EPA 850.1735 guideline (draft) and in compliance with GLP. The definitive exposure was performed under intermittent, static-renewal conditions of overlying water for a period of 10 days, at nominal test concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight (5.1, 9.9, 20, 41 and 74 mg/kg sediment dry weight, respectively, mean measured concentration by LC/MS/MS). In addition, blank and solvent (10 mL acetone to 0.05 kg silica sand) control treatments were set up, too. Acetone stock solutions of test substance were prepared for sediment dosing. A 10-mL aliquot of each stock solution was applied to 0.05 kg of fine silica sand and mixed with a spatula. The acetone was then allowed to evaporate for 30 minutes, prior to adding this pre-mixture to 2.0 kg of wet natural sediment filled in a jar. Proper mixing of sand and sediment was achieved by jar-rolling technique for 4 hours at 15 rpm. A volume of 100 mL (145 / 63.3 g wet/dry weight) of sediment was filled into a 300 mL glass vessel and overlaid with 175 mL of water. Eleven replicate vessels (8 used for biological response and 3 used for chemical analysis) each comprising 10 amphipods were maintained for each treatment and control group. Exposure concentrations of the test substance in sediment, pore water and overlying water were measured on days 0 and 10. The water quality during the experimental period was as follow: 22 - 24°C, pH 6.4 - 7.0, 4.1 - 7.1 mg/L of dissolved oxygen (41 - 84% of saturation) and, photoperiod of 16 hours light (460-530 lux) and 8 hours darkness.

Following 10 days of exposure, amphipod survival in the negative control and solvent control averaged 99 and 98%, respectively. During the same period, amphipod growth (dry weight) in the negative control and solvent control averaged 0.16 and 0.14 mg per amphipod, respectively, indicating a mean 8.6 and 7.6-fold increase in amphipod weight, respectively, based on the initial mean dry weight of 0.0185 mg per amphipod. At exposure termination (test day 10), statistical analysis (Steel’s Many-One Rank Sum Test) determined no significant difference in survival (91 – 99%) among amphipods exposed to any of the treatment levels tested compared to the negative control data (99%). At exposure termination (test day 10), statistical analysis (Dunnett’s Multiple Comparison Test) determined no significant difference in growth (0.13 – 0.16 mg mean dry weight) among amphipods exposed to any of the treatment levels tested compared to the negative control (0.16 mg). Based on mean measured sediment concentrations and amphipod survival and growth, the Lowest-Observed-Effect Concentration (LOEC) and No-Observed-Effect Concentration (NOEC) were determined to be >74 and 74 mg/kg sediment dry weight, respectively. The 10-day LC50/EC50 value was empirically estimated to be >74 mg/kg sediment dry weight.

 

Marine water

The purpose of this study was to determine the effect of the test substance on the survival of an estuarine, sediment-dwelling amphipod (Leptocheirus plumulosus) for 10 days following EPA 850.1740 guideline and in compliance with GLP. Amphipods were exposed to nominal sediment concentrations of 6.3, 13, 25, 50 and 100 mg/kg sediment dry weight (5.3, 11, 21, 43 and 90 mg/kg sediment dry weight mean measured concentration by LC/MS/MS), a control and a solvent (10 mL acetone to 0.05 kg silica sand) control. Acetone stock solutions of test substance were prepared for sediment dosing. A 10-mL aliquot of each stock solution was applied to 0.05 kg of fine silica sand and mixed with a spatula. The acetone was then allowed to evaporate for 30 minutes, prior to adding this pre-mixture to 2.0 kg of wet natural sediment filled in a jar. Proper mixing of sand and sediment was achieved by jar-rolling technique for 4 hours at 15 rpm. A volume of 175 mL (223 / 85.6 g wet/dry weight) of sediment was filled into a 1 L glass beaker and overlaid with approximately 900 mL of filtered seawater. Nine replicate beakers (5 used for biological response and 4 used for chemical analysis) each comprising 10 amphipods were maintained for each treatment and control group. Exposure concentrations of the test substance in sediment, overlying water and pore water were measured on days 0 (exposure initiation) and 10 (exposure termination). The test condition during the experimental period was as follow: 24 - 26 °C, pH 7.8 – 8.3, dissolved oxygen 5.2 – 7.8 mg/L of (69 - 106% of saturation) and, continuous illumination at an intensity in the range of 560 to 790 lux. The salinity of overlying water was 20 - 22‰ and the salinity of pore water was 24 - 25‰ on day 0 and 21 - 22‰ on day 10.

 

Following 10 days of exposure, amphipod survival in both the control and solvent control averaged 99 and 100%, respectively. At exposure termination (test day 10), statistical analysis (Steel Many-One Rank Sum Test) determined no significant difference in survival (90 – 99%) among amphipods exposed to the 5.3, 11, 21 and 43 mg/kg treatment level compared to the negative control (99%). The 90 mg/kg sediment dry weight treatment level was censored from statistical analyses due to 100% mortality in that treatment level. No sublethal or behavioural effects were noted among the amphipods in surviving animals at any of the treatment concentrations. Based on mean measured sediment concentrations and amphipod survival, the NOEC and LOEC were determined to be 43 and 90 mg/kg sediment dry weight, respectively. The 10-day LC50 value, as determined by untrimmed Spearman-Kärber method, was estimated to be 58 mg/kg sediment dry weight with 95% confidence intervals of 55 to 60 mg/kg.