3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Sediment toxicity

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 99.8%
Batch: DPX-JE874-313

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Test organisms (species):

Chironomus riparius

Details on test organisms:

Midges used in the test for the 28-day exposure were hatched from 12 egg masses collected from the cultures maintained at Wildlife International, Easton, Maryland, and midges used in the test for the 10-day exposure were hatched from 11 egg masses collected from the cultures. All midges were first instar (1-4 days post hatch) at the start of the test. Identification of the species was verified by the supplier of the original culture (Environmental Consulting and Testing of Superior, Wisconsin).

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

formulated sediment was composed of approximately 5% sphagnum peat moss, 20% silt and clay and 75% industrial quartz sand

Duration:

10 d

Exposure phase:

total exposure duration

Duration:

28 d

Exposure phase:

total exposure duration

Hardness:

92-144 as CaCO3 (negative control)
108-164 as CaCO3 (solvent control)
108-160 as CaCO3 (287 mg/kg group)

Test temperature:

20 ± 2ºC

pH:

8.1 to 9.1

Dissolved oxygen:

≥8.0 mg/L (≥89% of saturation) throughout the test.

Ammonia:

Conductivity:

262-373 µS/cm(negative control)
256-371 µS/cm(solvent control)
250-366 µS/cm(287 mg/kg group)

Nominal and measured concentrations:

Nominal: 10, 20, 40, 80, 160 and 320 mg famoxadone/kg

Details on test conditions:

Each test concentration had ten replicate test chambers. Four test chambers were maintained for biological observations with 20 organisms in each test chamber for a total of 80 organisms being observed for each test concentration. Five replicate chambers were maintained for analytical confirmation on Days 0, 7, 14, 21 and 28 of the test. The Day 7, 14, 21 and 28 analytical replicates contained 20 organisms placed in the test chambers at the same time as the biological replicates; however, the Day 0 replicates did not contain organisms. One additional replicate was maintained for each treatment group as an extra analytical replicate, if needed, and contained 20 organisms. In addition, four replicate test chambers were maintained for a 10 day period for evaluation of growth and survival. Each test chamber contained 20 organisms, for a total of 80 organisms in each control and treatment group for the 10 day evaluation and 80 organisms in each control and treatment group for the 28 day evaluation
Survival and growth (ash-free dry weights (AFDW)) were determined after the 10-day exposure period. Percent mortality observed in the treatment groups were used to calculate the 10-day LC50 value, and the AFDW data was used to calculate a 10-day EC50 value for growth. The total number of adults emerged at the end of the 28-day test period was recorded. Percent emergence and/or mortality in the treatment groups were used to calculate the 28-day LC50. The dose-response pattern and appropriate statistical analyses were used to define the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC).

Duration:

10 d

Dose descriptor:

LOEC

Effect conc.:

34 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

17 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

34 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

other: emergence ratios, development time and development rate

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

79 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

other: emergence ratios, development time and development rate

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

62 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

other: emergence and/or mortality of the midge

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

> 287 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

mortality

Duration:

10 d

Dose descriptor:

EC50

Effect conc.:

> 287 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Most adults that emerged appeared normal. There were occasional observations of larvae on the surface of the sediment or swimming in the water column and adults that emerged and died. However, these observations did not appear to be dose-responsive and were not considered to be treatment-related. Emergence was first noted on Day 12 of the test. The LC50 based on the number of emerged, surviving midges exposed to sediment incorporated 14C famoxadone was was 62 mg/kg, with a 95% confidence interval of 34 to 287 mg/kg..

 

Mean emergence ratios in the negative control, solvent control, 8.2, 17, 34, 79, 145 and 287 mg/kg mean measured treatment groups were 0.91, 0.85, 0.81, 0.71, 0.75, 0.40, 0.43 and 0.19, respectively. There were no statistically significant differences (p > 0.05) between the negative and solvent control group for emergence ratios; therefore, the treatment data was compared to the pooled control. Dunnett’s test indicated that there was a statistically significant difference in emergence in the 79, 145 and 287 mg/kg mean measured treatment groups in comparison to the pooled control (p ≤ 0.05). Therefore, the LOEC for emergence ratio was determined to be 79 mg/kg and the NOEC was 34 mg/kg.

 

Mean development times in the negative control, solvent control, 8.2, 17, 34, 79, 145 and 287 mg/kg mean measured treatment groups were 15.1, 15.0, 15.5, 16.0, 15.9, 16.7, 17.6 and 17.6 days, respectively. There were no statistically significant differences (p > 0.05) between the negative and solvent control group for development times; therefore, the treatment data was compared to the pooled control. Dunnett’s test indicated that there was a statistically significant difference in development times in the 79, 145 and 287 mg/kg mean measured treatment groups in comparison to the pooled control (p ≤ 0.05). Therefore, the LOEC for development times was determined to be 79 mg/kg and the NOEC was 34 mg/kg.

 

Mean development rates (defined as the portion of larval development which takes place per day) in the negative control, solvent control, 8.2, 17, 34, 79, 145 and 287 mg/kg mean measured treatment groups were 0.0693, 0.0700, 0.0679, 0.0656, 0.0659, 0.0626, 0.0590 and 0.0590, respectively. There were no statistically significant differences (p > 0.05) between the negative and solvent control group for development rate; therefore, the treatment data was compared to the pooled control. Dunnett’s test indicated that there was a statistically significant difference in development rate in the 79, 145 and 287 mg/kg mean measured treatment groups in comparison to the pooled control (p ≤ 0.05). Therefore, the LOEC for development times was determined to be 79 mg/kg and the NOEC was 34 mg/kg.

All organisms appeared normal during the test with some observations of organisms leaving the sediment or climbing the walls of the test compartments. The number of incidences of sediment avoidance on any one day was small and observations were made in all treatment groups and controls. Mean percent survival at the end of the 10-day exposure in the negative control, solvent control, 8.2, 17, 34, 79, 145 and 287 mg/kg mean measured treatment groups was 98, 91, 96, 100, 99, 98, 94 and 86%, respectively. There were no statistical differences (p > 0.05) between the negative and solvent control groups for survival on Day 10; therefore, the treatment data was compared to the pooled control. There was a statistically significant difference (p < 0.05) between the 287 mg/kg mean measured treatment group when compared to the pooled control for the day 10 survival data using a Bonferroni t-test. The NOEC for the day 10 survival was 145 mg/kg dry sediment. The LOEC for survival was 287 mg/kg and the 10-Day LC50 for survival was determined to be >287 mg/kg dry sediment, the highest concentration tested.

Prior to initiating the replicates used for the 10 day assessment of survival and growth, a group of 20 organisms from the culture was impartially selected and used for the determination of dry weight. It was determined that the average individual dry weight of those twenty organisms was 0.02 mg. The average individual ash-free dry weight per midge in the negative and solvent control groups was 1.08 and 1.10 mg, respectively. The average individual ash-free dry weight per midge in the 8.2, 17, 34, 79, 145 and 287 mg/kg mean measured treatment groups was 1.07, 1.00, 0.96, 0.92, 0.82 and 0.60 mg, respectively. There were no statistically significant differences (p > 0.05) between the negative and solvent control groups; therefore, the treatment groups were compared to the pooled control group. There was a statistically significant difference between the pooled control group and the 34, 79, 145 and 287 mg/kg mean measured treatment groups using a Bonferroni t-test (5). The NOEC for growth was 17 mg/kg and the LOEC for growth was determined to be 34 mg/kg. The 10-Day EC50 for growth was >287 mg/kg, the highest concentration tested.

Validity criteria fulfilled:

yes

Conclusions:

The 10-day LC50 and EC50 value based on survival and growth of the midge, Chironomus riparius, exposed to sediment-incorporated 14C famoxadone was >287 mg/kg, the highest mean measured concentration tested. The 28-day LC50 value based on emergence and/or mortality of the midge, Chironomus riparius, exposed to sediment-incorporated 14C famoxadone was 62 mg/kg, with a 95% confidence interval of 34 – 287 mg/kg. There was a treatment-related effect observed on mean emergence ratios, development time and development rate in the 79, 145 and 287 mg/kg mean measured treatment groups. Therefore, the 28-day LOEC was 79 mg/kg and the 28-day NOEC was 34 mg/kg. There were also treatment related effects observed on day-10 growth measurements in the 34, 79, 145 and 287 mg/kg treatment groups when compared to the pooled control; therefore, the 10- day NOEC was 17 mg/kg and the 10-day LOEC was 34 mg/kg based on the 10-day growth measurements (the most sensitive endpoint).

Executive summary:

The midge, Chironomus riparius, was tested in a 28-day exposure period under static test conditions according to OECD guideline 218.

Each test concentration had ten replicate test chambers. Four test chambers were maintained for biological observations with 20 organisms in each test chamber for a total of 80 organisms being observed for each test concentration. Five replicate chambers were maintained for analytical confirmation on Days 0, 7, 14, 21 and 28 of the test. The Day 7, 14, 21 and 28 analytical replicates contained 20 organisms placed in the test chambers at the same time as the biological replicates; however, the Day 0 replicates did not contain organisms. One additional replicate was maintained for each treatment group as an extra analytical replicate, if needed, and contained 20 organisms. In addition, four replicate test chambers were maintained for a 10 day period for evaluation of growth and survival. Each test chamber contained 20 organisms, for a total of 80 organisms in each control and treatment group for the 10 day evaluation and 80 organisms in each control and treatment group for the 28 day evaluation

Survival and growth (ash-free dry weights (AFDW)) were determined after the 10-day exposure period. Percent mortality observed in the treatment groups were used to calculate the 10-day LC50 value, and the AFDW data was used to calculate a 10-day EC50 value for growth. The total number of adults emerged at the end of the 28-day test period was recorded. Percent emergence and/or mortality in the treatment groups were used to calculate the 28-day LC50. The dose-response pattern and appropriate statistical analyses were used to define the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC).

The 10-day LC50 and EC50 value based on survival and growth of the midge, Chironomus riparius, exposed to sediment-incorporated 14C famoxadone was >287 mg/kg, the highest mean measured concentration tested. The 28-day LC50 value based on emergence and/or mortality of the midge, Chironomus riparius, exposed to sediment-incorporated 14C famoxadone was 62 mg/kg, with a 95% confidence interval of 34 – 287 mg/kg. There was a treatment-related effect observed on mean emergence ratios, development time and development rate in the 79, 145 and 287 mg/kg mean measured treatment groups. Therefore, the 28-day LOEC was 79 mg/kg and the 28-day NOEC was 34 mg/kg. There were also treatment re-lated effects observed on day-10 growth measurements in the 34, 79, 145 and 287 mg/kg treatment groups when compared to the pooled control; therefore, the 10- day NOEC was 17 mg/kg and the 10-day LOEC was 34 mg/kg based on the 10-day growth measurements (the most sensitive endpoint).

Reference 2

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA 600/R-94/025: Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods

Version / remarks:

1994

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98.2%
Batch: DPX-JE874-496

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Test organisms (species):

Leptocheirus plumulosus

Details on test organisms:

Amphipods used in the test were 8 - 9 days old at test initiation and were obtained from cultures maintained at EAG Laboratories-Easton. The original culture was obtained from Aquatic Research Organisms, Hampton, New Hampshire. The supplier verified the identity of the species. The organisms were held in water from the same source as was used during testing. There was no sign of disease, abnormalities or sickness observed in the culture prior to the test. Known age organisms were obtained from the culture by isolating adults in the culture 9 days prior to the start of the test. At 7 days prior to the test, <48-hour old neonates were isolated from the culture tanks and held until the start of the test.

Study type:

laboratory study

Test type:

flow-through

Water media type:

saltwater

Type of sediment:

natural sediment

Remarks:

Natural marine sediment collected from the Wye River in Maryland, U.S.A.

Duration:

28 d

Exposure phase:

total exposure duration

Test temperature:

24.4 – 24.7°C

pH:

8.1-8.2

Dissolved oxygen:

7.0 – 7.1

Salinity:

20-21%

Ammonia:

Nominal and measured concentrations:

Nominal: 26, 64, 160, 400 and 1000 mg a.s./kg of sediment

Details on test conditions:

Groups of 8 - 9 day old amphipods were exposed to a geometric series of five test concentrations, a solvent control and a negative control in a flow-through system for 28 days. Five replicate test chambers were maintained in each treatment and control group for biological observations, with 20 amphipods in each test chamber, for a total of 100 amphipods per test concentration. Each test chamber contained a quantity of sediment and overlying water. Six additional replicates were included for each treatment group. Three replicates were used for water quality measurements and measurements of sediment pH and three replicates were used for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain amphipods, while amphipods were added at test initiation to the “analytical” and “water quality” replicates sampled on Days 14 and 28.
Five nominal test concentrations selected in consultation with the sponsor were 26, 64, 160, 400 and 1000 mg a.s./kg of sediment based on the dry weight of the sediment. Overlying water, pore water and sediment samples were collected and processed from the “analytical” replicates of each concentration on Days 0, 14 and 28. The results of the study are based on the mean measured concentrations of famoxadone in the sediment and pore water, as well as concentrations normalized for the organic carbon in the sediment.

Test compartments were positioned in a temperature controlled water bath 14 days prior to test initiation to condition the sediment prior to the introduction of organisms. Approximately 8 - 9 day old amphipods were sequentially and impartially added one or two at a time to transfer containers until each container held a complement of 20 individuals. The transfer containers were then indiscriminately assigned to exposure compartments. The amphipods in each transfer container were transferred to the exposure compartments below the air/water interface using a wide-bore pipette at test initiation. Observations of mortality and abnormal behavior were made daily during the test. Survival, reproduction and growth were measured at the end of the 28-day test period. The LC50 value based on survival and EC50 value based on growth and reproduction, the lowest-observed-effect-concentration (LOEC) and the no-observed-effect-concentration (NOEC) were determined (when possible) by the concentration-response pattern and statistical analysis of the survival, reproduction and growth data.

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

292 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 781 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

131 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

342 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 781 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

mortality

There were no significant differences between the solvent and negative control groups for any parameter; therefore, the treatment groups were compared to the pooled control group to determine if there were any significant reductions.

 

The mean percent survival of Leptocheirus in the negative control, solvent control, pooled control, 16, 47, 131, 342 and 781 mg a.s./kg treatment groups were 91, 84, 88, 92, 93, 83, 75 and 93%, respectively, at test termination. A Bonferroni t-test indicated that there was no significant reduction in survival in any treatment group (p > 0.05) when compared to the pooled control. Consequently, the LOEC for survival was >781 mg a.s./kg dry weight of sediment, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and >0.22 mg a.s./L) and the NOEC for survival was 781 mg a.s./kg (corresponding to 325417 mg a.s./kg-OC and 0.22 mg a.s./L). The 28-day LC50 based on survival was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L).

 

The mean male growth rate of Leptocheirus in the negative control, solvent control, pooled control, 16, 47, 131, 342 and 781 mg a.s./kg treatment groups were 0.0907, 0.1033, 0.0970, 0.0908, 0.0968, 0.1033, 0.0811 and 0.0722 mg/day, respectively, at test termination. The mean female growth rate of Leptocheirus in the negative control, solvent control, pooled control, 16, 47, 131, 342 and 781 mg a.s./kg treatment groups were 0.0774, 0.0686, 0.0730, 0.0608, 0.0702, 0.0664, 0.0574 and 0.0560 mg/day, respectively, at test termination. A Bonferroni t-test indicated there was a significant difference in male growth rate in the 781 mg a.s./kg treatment group and in female growth rate in the 342 and 781 mg a.s./kg treatment groups when compared to the pooled control. Consequently, the LOEC for growth was 342 mg a.s./kg dry weight of sediment (corresponding to 142500 mg a.s./kg-OC and 0.14 mg a.s./L) and the NOEC for growth was 131 mg a.s./kg (corresponding to 54444 mg a.s./kg-OC and 0.11 mg a.s./L). The 28-day EC50 based on growth was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L).

The number of young per surviving female in the negative control, solvent control, pooled control, 16, 47, 131, 342 and 781 mg a.s./kg treatment groups were 12.7, 16.6, 14.7, 17.8, 14.0, 10.9, 6.90 and 2.56 young/adult, respectively, at test termination. A Bonferroni t-test indicated there was a significant difference in number of young per surviving female in the 342 and 781 mg a.s./kg treatment groups. Consequently, the LOEC for reproduction was 342 mg a.s./kg dry weight of sediment (corresponding to 142500 mg a.s./kg-OC and 0.14 mg a.s./L) and the NOEC for reproduction was 131 mg a.s./kg (corresponding to 54444 mg a.s./kg-OC and 0.11 mg a.s./L). The 28-day EC50 based on reproduction was 292 mg a.s./kg (corresponding to 121440 mg a.s./kg-OC and 0.13 mg a.s./L).

Validity criteria fulfilled:

yes

Conclusions:

Groups of amphipods were exposed to sediment spiked with famoxadone at nominal concentrations of 26, 64, 160, 400 and 1000 mg a.s./kg of sediment based on the dry weight of the sediment for 28-days under flow-through conditions. The mean measured concentrations of famoxadone in the sediment, based on the average sediment concentration determined from analyses conducted on Days 0, 14 and 28 of the exposure were 16, 47, 131, 342 and 781 mg a.s./kg, respectively. The results of the test are based on the mean measured concentrations of famoxadone in the sediment and are also reported based on the mean measured concentrations in the sediment adjusted for a percent organic carbon content of 0.24%: 6514, 19389, 54444, 142500 and 325417 mg a.s./kg-OC, respectively and the mean measured concentrations in the pore water: 0.059, 0.085, 0.11, 0.14 and 0.22 mg a.s./L, respectively. The 28-day LC50 based on survival was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L). The 28-day EC50 based on growth rate was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L). The 28-day EC50 based on reproduction was 292 mg a.s./kg (corresponding to 121440 mg a.s./kg-OC and 0.13 mg a.s./L). The LOEC was 342 mg a.s./kg dry weight of sediment (corresponding to 142500 mg a.s./kg-OC and 0.14 mg a.s./L) and the NOEC was 131 mg a.s./kg (corresponding to 54444 mg a.s./kg-OC and 0.11 mg a.s./L).

Executive summary:

The objective of this study was to determine the effects of sediment-incorporated famoxadone on the amphipod, Leptocheirus plumulosus, during a 28-day exposure period in a flow-through system providing intermittent renewal of overlying water. The measured endpoints of the test were survival, growth rate and reproduction. The study was conducted based on procedures outlined in the U.S. Environmental Protection Agency Guidance Document, EPA 600/R-01/020: Method for Assessing the Chronic Toxicity of Marine and Estuarine Sediment-Associated Contaminants with the Amphipod Leptocheirus plumulosus; and EPA 600/R-94/025: Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods.

Groups of 8 - 9 day old amphipods were exposed to a geometric series of five test concentrations, a solvent control and a negative control in a flow-through system for 28 days. Five replicate test chambers were maintained in each treatment and control group for biological observations, with 20 amphipods in each test chamber, for a total of 100 amphipods per test concentration. Each test chamber contained a quantity of sediment and overlying water. Six additional replicates were included for each treatment group. Three replicates were used for water quality measurements and measurements of sediment pH and three replicates were used for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain amphipods, while amphipods were added at test initiation to the “analytical” and “water quality” replicates sampled on Days 14 and 28.

Five nominal test concentrations selected in consultation with the sponsor were 26, 64, 160, 400 and 1000 mg a.s./kg of sediment based on the dry weight of the sediment. Overlying water, pore water and sediment samples were collected and processed from the “analytical” replicates of each concentration on Days 0, 14 and 28. The results of the study are based on the mean measured concentrations of famoxadone in the sediment and pore water, as well as concentrations normalized for the organic carbon in the sediment.

 

Test compartments were positioned in a temperature controlled water bath 14 days prior to test initiation to condition the sediment prior to the introduction of organisms. Approximately 8 - 9 day old amphipods were sequentially and impartially added one or two at a time to transfer containers until each container held a complement of 20 individuals. The transfer containers were then indiscriminately assigned to exposure compartments. The amphipods in each transfer container were transferred to the exposure compartments below the air/water interface using a wide-bore pipette at test initiation. Observations of mortality and abnormal behavior were made daily during the test. Survival, reproduction and growth were measured at the end of the 28-day test period. The LC50 value based on survival and EC50 value based on growth and reproduction, the lowest-observed-effect-concentration (LOEC) and the no-observed-effect-concentration (NOEC) were determined (when possible) by the concentration-response pattern and statistical analysis of the survival, reproduction and growth data.

Groups of amphipods were exposed to sediment spiked with famoxadone at nominal concentrations of 26, 64, 160, 400 and 1000 mg a.s./kg of sediment based on the dry weight of the sediment for 28-days under flow-through conditions. The mean measured concentrations of famoxadone in the sediment, based on the average sediment concentration determined from analyses conducted on Days 0, 14 and 28 of the exposure were 16, 47, 131, 342 and 781 mg a.s./kg, respectively. The results of the test are based on the mean measured concentrations of famoxadone in the sediment and are also reported based on the mean measured concentrations in the sediment adjusted for a percent organic carbon content of 0.24%: 6514, 19389, 54444, 142500 and 325417 mg a.s./kg-OC, respectively and the mean measured concentrations in the pore water: 0.059, 0.085, 0.11, 0.14 and 0.22 mg a.s./L, respectively. The 28-day LC50 based on survival was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L). The 28-day EC50 based on growth rate was >781 mg a.s./kg, the highest concentration tested (corresponding to >325417 mg a.s./kg-OC and 0.22 mg a.s./L). The 28-day EC50 based on reproduction was 292 mg a.s./kg (corresponding to 121440 mg a.s./kg-OC and 0.13 mg a.s./L). The LOEC was 342 mg a.s./kg dry weight of sediment (corresponding to 142500 mg a.s./kg-OC and 0.14 mg a.s./L) and the NOEC was 131 mg a.s./kg (corresponding to 54444 mg a.s./kg-OC and 0.11 mg a.s./L).

Reference 3

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ASTM E1706 (Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrate

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS 850.1770 (draft)

Version / remarks:

2009

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates

Version / remarks:

2000

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98.2%
Batch: DPX-JE874-496

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Test organisms (species):

Hyalella azteca

Details on test organisms:

Amphipods used in the test were 7 - 8 days old at test initiation and were obtained from cultures maintained at EAG Laboratories-Easton. The original culture was obtained from Aquatic Bio Systems, Fort Collins, Colorado. The supplier verified the identity of the species. The organisms were held in water from the same source as was used during testing. There was no sign of disease, abnormalities or sickness observed in the culture prior to the test. Known age organisms were obtained from the culture by isolating adults in the culture 8 days prior to the start of the test. At 7 days prior to the test, <24-hour old neonates were isolated from the culture tanks containing the isolated adults and held until the start of the test.

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

natural sediment

Remarks:

natural sediment collected at West Bearskin Lake, Minnesota

Duration:

42 d

Exposure phase:

total exposure duration

Hardness:

137-149 as CaCO3

Test temperature:

22.7 – 22.8°C

pH:

8.1 – 8.2

Dissolved oxygen:

7.9 – 8.0 mg/L

Ammonia:

Conductivity:

393 - 401

Nominal and measured concentrations:

Nominal: 13, 32, 80, 200 and 500 mg a.s./kg of sediment based on the dry weight of the sediment.

Details on test conditions:

Groups of 7 – 8 day old amphipods were exposed to a geometric series of five test concentrations of sediment spiked with famoxadone, a solvent control and a negative control for 42 days under flow-through test conditions. Test concentrations were chosen in consultation with the sponsor and were based on a 10-day range-finding study. For the definitive test, twelve replicate test compartments were maintained in each treatment and control group, with 10 amphipods in each test compartment, for a total of 120 amphipods per test concentration. Each test compartment contained a quantity of sediment and overlying water. One additional replicate was added to each treatment and control group for water quality measurements and measurements of sediment pH. Three additional replicates were added in each treatment and control group for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain amphipods, while amphipods were added at test initiation to the “analytical” replicates sampled on Days 14 and 28.
Overlying water, pore water and sediment samples were collected from the “analytical” replicates of each concentration on Days 0, 14 and 28 and were processed. The results of the study are based on the mean measured concentrations of famoxadone in the sediment and pore water as well as concentrations normalized for the organic carbon in the sediment.
Test compartments were positioned in the test system 14 days prior to test initiation to condition the sediment prior to the introduction of organisms. Approximately 7-8 day old amphipods were sequentially and impartially added one and two at a time to transfer containers until each container held a complement of 10 individuals. The transfer containers were then indiscriminately assigned to exposure compartments. The amphipods in each transfer container were transferred to the exposure compartments below the air/water interface using a wide-bore pipette at test initiation. Observations of mortality and abnormal behavior were made daily during the test. On Day 28, surviving amphipods were removed from the sediment by sieving. Four of the 12 replicates in each treatment group were evaluated for survival and growth (length and weight). The surviving adult organisms from the remaining eight replicates were transferred to corresponding clean, water-only test compartments and monitored for survival, reproduction and growth. Reproduction was determined on Days 35 and 42 of the test. Any young observed on Day 28 of the test were also counted and discarded. Adult amphipods surviving to Day 42 were collected for growth measurements and the numbers of males and females were determined, the number of females surviving to Day 42 was used to determine the number of young per female per replicate.

Duration:

42 d

Dose descriptor:

NOEC

Effect conc.:

24 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Duration:

42 d

Dose descriptor:

LOEC

Effect conc.:

60 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

growth rate

Duration:

42 d

Dose descriptor:

LC50

Effect conc.:

> 378 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

mortality

Details on results:

Day 28 Survival
The Day 28 mean survival of H. azteca in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups (based on 12 replicates) were 97, 98, 98, 98, 94, 96 and 97%, respectively. There were no statistically significant reductions (p > 0.05) in survival of any treatment group when compared to the pooled control using a Kruskal-Wallis test. Consequently, the LOEC and NOEC for Day 28 survival was empirically estimated to be >378 and 378 mg a.s./kg dry weight of sediment, respectively (>9450 and 9450 mg a.s./kg-OC; >0.97 and 0.97 mg a.s./L mean measured in pore water, respectively). The LC50 was >378 mg a.s./kg, (>9450 mg a.s./kg-OC; >0.97 mg a.s./L, mean measured in pore water) the highest concentration tested, since the percent mortality was less than 50% in all treatment groups and controls.

Day 28 Growth
The average length of Hyalella on Day 28 in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups was 5.18, 5.10, 4.58, 4.92, 4.54, 4.30 and 4.89 mm, respectively. The Day 28 average individual dry weight of Hyalella in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups was 0.803, 0.725, 0.505, 0.630, 0.518, 0.395 and 0.635 mg, respectively. A Bonferroni t-test indicated that there were statistical differences in Day 28 length measurements between the pooled control and the 10, 60 and 151 mg a.s./kg treatment groups. The same statistical differences were observed using a Dunnett’s test on the Day 28 weight measurements. However, since the statistical differences were not dose responsive, they were not considered to be biologically meaningful. Therefore, the Day 28 LOEC was >378 mg a.s./kg and the Day 28 NOEC was 378 mg a.s./kg, the highest concentration tested.

Day 42 Survival
On Day 42, the mean adult survival in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups was 86, 96, 99, 99, 88, 95 and 88%, respectively. A Bonferroni t-test indicated that there were no statistically significant differences in the Day 42 percent survival. The 42-Day LC50 for survival was >378 mg a.s./kg, the highest concentration tested. (>9450 mg a.s./kg-OC, >0.97 mg a.s./L pore water). The LOEC and NOEC for Day 42 survival were >378 mg a.s./kg and 378 mg a.s./kg, respectively (>9450 and 9450 mg a.s./kg-OC; >0.97 and 0.97 mg a.s./L pore water, respectively).

Day 42 Growth
The average individual lengths of adult male Hyalella on Day 42 in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups were 6.03, 6.19, 5.83, 5.78, 5.32, 5.47 and 5.65 mm, respectively. The average individual lengths of adult female Hyalella on Day 42 in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups were 5.61, 5.57, 5.20, 5.38, 4.92, 5.02 and 5.06 mm, respectively. The average dry weight values on Day 42 in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg were 1.05, 1.10, 0.929, 1.03, 0.731, 0.855 and 0.904 mg, respectively. A Dunnett’s test indicated that there was a statistical difference in Day 42 average male body length between the pooled control and all treatment groups and in the Day 42 average female body length between the pooled control and the 10, 60, 151 and 378 mg a.s./kg treatment groups. Since the statistical difference in the 10 mg a.s./kg treatment group was not dose responsive and the difference in the 24 mg a.s./kg treatment group was not observed in the female body lengths, the difference observed in the 10 and 24 mg a.s./kg treatment groups were not considered to be biologically meaningful. A Dunnett’s test indicated that there were statistical differences in the 10, 60, 151 and 378 mg a.s./kg treatment groups for dry weight when compared to the pooled control group. Since the statistical difference in the 10 mg a.s./kg treatment group was not dose responsive; it was not considered to be biologically meaningful. Therefore, the Day 42 LOEC for growth was determined to be 60 mg a.s./kg and the Day 42 NOEC for growth was 24 mg a.s./kg.

Reproduction
The number of young per surviving female in the negative control, solvent control, 10, 24, 60, 151 and 378 mg a.s./kg treatment groups was 12.2, 8.9, 6.9, 11.0, 7.9, 8.4 and 11.3 young/female, respectively, at test termination. There was no statistically significant difference in comparison of the number of young per surviving female in any treatment group when compared to the pooled control group (Dunnett’s test). Therefore, the Day 42 LOEC for reproduction was determined to be >378 mg a.s./kg and the Day 42 NOEC for reproduction was 378 mg a.s./kg, the highest concentration tested.

Validity criteria fulfilled:

yes

Conclusions:

The 42-Day LC50 value for Hyalella azteca exposed to famoxadone in sediment was >378 mg a.s./kg, the highest concentration tested, based on mortality data (>9450 mg a.s./kg-OC and >0.97 mg a.s./L pore water). The Day 42 LOEC was 60 mg a.s./kg (1497 mg a.s./kg-OC, 0.21 mg a.s./L pore water) and the Day 42 NOEC was 24 mg a.s./kg (594 mg a.s./kg-OC, 0.098 mg a.s./L pore water).

Executive summary:

The objective of this study was to determine the effects of sediment-incorporated Famoxadone on the amphipod, Hyalella azteca, during a 42-day test period in a flow-through system providing intermittent renewal of overlying water. The measured endpoints of the test were survival, growth and reproduction. The study was conducted on based on procedures in the U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates; U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines (draft), OPPTS Number 850.1770: Whole Sediment Life Cycle Toxicity Test with Hyalella azteca; and ASTM Standard E 1706-05: Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates.

Groups of 7 – 8 day old amphipods were exposed to a geometric series of five test concentrations of sediment spiked with famoxadone, a solvent control and a negative control for 42 days under flow-through test conditions. Test concentrations were chosen in consultation with the sponsor and were based on a 10-day range-finding study. For the definitive test, twelve replicate test compartments were maintained in each treatment and control group, with 10 amphipods in each test compartment, for a total of 120 amphipods per test concentration. Each test compartment contained a quantity of sediment and overlying water. One additional replicate was added to each treatment and control group for water quality measurements and measurements of sediment pH. Three additional replicates were added in each treatment and control group for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain amphipods, while amphipods were added at test initiation to the “analytical” replicates sampled on Days 14 and 28.

Overlying water, pore water and sediment samples were collected from the “analytical” replicates of each concentration on Days 0, 14 and 28 and were processed. The results of the study are based on the mean measured concentrations of famoxadone in the sediment and pore water as well as concentrations normalized for the organic carbon in the sediment.

Test compartments were positioned in the test system 14 days prior to test initiation to condition the sediment prior to the introduction of organisms. Approximately 7-8 day old amphipods were sequentially and impartially added one and two at a time to transfer containers until each container held a complement of 10 individuals. The transfer containers were then indiscriminately assigned to exposure compartments. The amphipods in each transfer container were transferred to the exposure compartments below the air/water interface using a wide-bore pipette at test initiation. Observations of mortality and abnormal behavior were made daily during the test. On Day 28, surviving amphipods were removed from the sediment by sieving. Four of the 12 replicates in each treatment group were evaluated for survival and growth (length and weight). The surviving adult organisms from the remaining eight replicates were transferred to corresponding clean, water-only test compartments and monitored for survival, reproduction and growth. Reproduction was determined on Days 35 and 42 of the test. Any young observed on Day 28 of the test were also counted and discarded. Adult amphipods surviving to Day 42 were collected for growth measurements and the numbers of males and females were determined, the number of females surviving to Day 42 was used to determine the number of young per female per replicate.

 

The mean measured concentrations of famoxadone in the sediment, based on the average sediment concentration determined from analyses conducted on Days 0, 14 and 28 of the exposure were 10, 24, 60, 151 and 378 mg a.s./kg, respectively. The results of the test are based on the mean measured concentrations of famoxadone in the sediment as well as the mean measured concentrations in the sediment adjusted for a percent organic carbon content of 4% and the mean measured concentration in pore water. The 42-Day LC50 value for Hyalella azteca exposed to famoxadone in sediment was >378 mg a.s./kg, the highest concentration tested, based on mortality data (>9450 mg a.s./kg-OC and >0.97 mg a.s./L pore water). The Day 42 LOEC was 60 mg a.s./kg (1497 mg a.s./kg-OC, 0.21 mg a.s./L pore water) and the Day 42 NOEC was 24 mg a.s./kg (594 mg a.s./kg-OC, 0.098 mg a.s./L pore water).

Reference 4

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: BBA recommended 'Long term test with Chironomus riparius (1995)’

Version / remarks:

1995

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 96.9%
Batch: DPX-JE874-221

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetonitrile

Test organisms (species):

Chironomus riparius

Details on test organisms:

strain used originated from a culture supplied by CEFAS，Bumham-on-Crouch. The study was initiated with first instar Chironomus riparius, cultured in house and less than 24 hours old.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

Artificial sediment was prepared according to OECD guideline 207

Duration:

28 d

Exposure phase:

total exposure duration

Test temperature:

19-20°C

pH:

6.2 – 7.4

Dissolved oxygen:

5.2 – 8.9

Nominal and measured concentrations:

Nominal test concentrations: 0.01, 0.032, 0.1, 0.32, and 1.0 mg Famoxadone/L.
Actual applied concentrations: 0.01, 0.032, 0.1. 0.32, and 1.0 mg Famoxadone/L.

Details on test conditions:

Five test concentrations plus a dilution water control were prepared each consisting of three replicates together with a solvent control (100 µL auxiliary solvent per litre) consisting of six replicates (20 animals per replicate vessel). Four additional test vessels were prepared for use as 'destructive' samples for the analysis of water, sediment and pore water concentrations on Days 1 and 7: two at the highest test concentration. 1 mg/L and two at the lowest test concentration, 0.01 mg Famoxadone/L. Chironomids were exposed to the test or control conditions for a 28 day period without renewal of the test medium. the vessels were monitored daily for signs of growth and development of the chironomids. From Day 14, when the first flies emerged, adults were sexed and removed from the vessels. The 28 day percentage emergence success was calculated for each treatment.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

emergence rate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.01 mg/L

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

development rate

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

emergence rate

Data from the analyses of the overlying water showed that test substance quickly dispersed, in most vessels, in the overlying water. After 6 hours, test concentrations ranged from 54% at the highest test concentration to 100% at the lowest concentrations. In one replicate (replicate two) at test concentration 0.032 mg/L Famoxadone. high concentrations were found at time zero and 6 hours after application - approximately five times nominal concentration. The disparity between replicates at this concentration was not apparent at subsequent analyses. By Day 7, overlying water samples found to contain between 36 and 53% of applied [14C] Famoxadone and by Day 28 overlying water concentrations had declined to a range from 10 to 26% applied [14C] Famoxadone.

 

Pore water samples from the Day1 and 7 destructive samples contained very low concentrations of [14C] Famoxadone at both 0.01 and 1.0 mg/L concentrations (,0.0001 and 0.0009 rng/L respectively). Pore water samples from Day 28 samples showed concentrations had increased in the highest and lowest concentrations compared with Day 1 and 7 destructive sampling analyses and, in all concentrations, measured concentrations were higher than the nominal applied concentration.

 

Sediment analyses from Day 1 and 7 destructive sampling vessels showed an increase in concentration with time. Concentrations for the 1.0 mg/L concentration increased from 1.43 mg/kg on Day 1 to 7.5 mg/kg on Day 1 and for 0.01 mg/L concentration increased from 0.038 on Day 1 to 0.004 mg/kg on Day 7. By Day 28 0.01 gs/L concentration sediments were found to contain 0.07 mg/kg [14C] Famoxadone and 1.0 rng/L concentration sediments contained 7.48 mg/kg [14C] Famoxadone. These data suggest that

[UC] Famoxadone partitioned by more than 50% from overlying water to pore water and sediment during the study.

Validity criteria fulfilled:

yes

Conclusions:

The test substance [14C] Famoxadone affected the development rate and emergence success of the test species Chironomus ripaius under the conditions of this test. The EC50 for emergence was determined to be 0.41 mg [14C] Famoxadone/L. The no-observed effect concentration for emergence was 0.1 mg [14C] Famoxadone/L. The no observed effect concentration for development rate was 0.010 mg [14C] Famoxadone/L.

Executive summary:

The midge, Chironomus riparius, was tested in a 28-day exposure period under static test conditions according to BBA recommended 'Long term test with Chironomus riparius (1995)

Nominal test concentrations in the test were 0.01, 0.032, 0.1, 0.32, and 1.0 mg Famoxadone/L and the actual applied concentrations were 0.01, 0.032, 0.1. 0.32, and 1.0 mg Famoxadone/L.

 

Five test concentrations plus a dilution water control were prepared each consisting of three replicates together with a solvent control (100 µL auxiliary solvent per litre) consisting of six replicates (20 animals per replicate vessel). Four additional test vessels were prepared for use as 'destructive' samples for the analysis of water, sediment and pore water concentrations on Days 1 and 7: two at the highest test concentration. 1 mg/L and two at the lowest test concentration, 0.01 mg Famoxadone/L. Chironomids were exposed to the test or control conditions for a 28 day period without renewal of the test medium. the vessels were monitored daily for signs of growth and development of the chironomids. From Day 14, when the first flies emerged, adults were sexed and removed from the vessels. The 28 day percentage emergence success was calculated for each treatment.

 

 

The test substance [14C] Famoxadone affected the development rate and emergence success of the test species Chironomus ripaius under the conditions of this test. The EC50 for emergence was determined to be 0.41 mg [14C] Famoxadone/L. The no-observed effect concentration for emergence was 0.1 mg [14C] Famoxadone/L. The no observed effect concentration for development rate was 0.010 mg [14C] Famoxadone/L.

Reference 5

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ASTM E1706 (Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates)

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates

Version / remarks:

2000

GLP compliance:

yes

Specific details on test material used for the study:

Purity: 98.2%
Batch: DPX-JE874-496

Analytical monitoring:

yes

Vehicle:

yes

Remarks:

acetone

Test organisms (species):

Chironomus dilutus (previous name: Chironomus tentans)

Details on test organisms:

The organisms were obtained in two collections from cultures maintained at EAG Laboratories-Easton. The identity of the species was verified by the supplier of the original culture (Aquatic BioSystems, Inc., Fort Collins, Colorado). The organisms were collected as egg masses and held in glass beakers containing water from the same source as the water used during the test.

Study type:

laboratory study

Test type:

flow-through

Water media type:

freshwater

Type of sediment:

natural sediment

Remarks:

natural sediment collected at West Bearskin Lake, Minnesota

Duration:

47 d

Exposure phase:

total exposure duration

Hardness:

132- 148 as CaCO3

Test temperature:

23.0 – 23.1 ºC

pH:

7.9 – 8.0

Dissolved oxygen:

7.7 – 7.9 mg/L

Ammonia:

Conductivity:

321 -363 µS/cm

Nominal and measured concentrations:

Nominal: 9.0, 22, 56, 140 and 350 mg a.s./kg sediment, based on the dry weight of the sediment.

Details on test conditions:

Groups of first instar larvae were exposed to a geometric series of five test concentrations of sediment-incorporated famoxadone, a solvent control and a negative control for 47 days in a flow-through system providing intermittent renewal of overlying water. The test was initiated with 12 replicate test compartments, with an additional four replicates (for use as auxiliary male replicates) initiated on Day 6, for a total of 16 replicate test compartments in each treatment and control group. Each test compartment was initiated with 12 midge larvae for a total of 192 individuals per treatment group and control. Each test compartment contained a quantity of sediment and overlying water. Three additional replicates (defined as “water quality” replicates) were added to each treatment and control group to be used for measurements of sediment pH and pore water ammonia. Three additional replicates (defined as “analytical” replicates) were added in each treatment and control group to be used for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain midge larvae, while midge larvae were added at test initiation to the “analytical” and “water quality” replicates to be sampled on Days 16 and 47.

Test compartments were positioned in the test system 14 days prior to test initiation to condition the sediment prior to the introduction of organisms (the time required for sediment conditioning was determined during an equilibration trial). On Day 0 of the test, first instar midge larvae (up to 4 days post hatch) were sequentially and impartially added one and/or two at a time to test compartments (replicates A – L; O, P, Q and R) until each test compartment contained a total of 12 larvae. Replicates M and N were not initiated with organisms since they were removed from the test system on Day 0 for analytical and water quality measurements. Because male midges typically emerge sooner than female midges, additional replicates were set up six days after the original replicates to be able to obtain males to mate with the females during their emergence period and in order to be able to obtain viable egg masses. On Day 6 of the test, first instar midge larvae from a second collection of organisms (up to 4 days post hatch) were sequentially and impartially added one and/or two at a time to auxiliary male test compartments (replicates S, T, U and V) until each test compartment contained a total of 12 larvae. All transfers to the test compartments were made below the air/water interface using a wide-bore pipette. Observations of mortality and abnormal behavior (leaving the sediment, climbing the walls of the test chamber, etc.) were made daily during the test. On Day 16 of the test, approximately 20-day old surviving larvae were isolated from the sediment using 0.425 μm sieves and shallow sorting pans in four replicates (replicates I, J, K and L) for each treatment group and control for the assessment of survival and growth. Emergence traps were placed on replicates A – L and O - R, on Day 15 of the test and stayed on for the remainder of the test. Emergence traps were placed on the four auxiliary male replicates (replicates S, T, U and V) on Day 14 of their exposure (Day 20 of the test, corresponding with the first sign of pupation). The total number of adults emerged at the end of the test period was recorded. During the period of emergence, males and females were paired with other adults from the same treatment group and monitored for production of egg masses. Primary egg masses were collected and the number of eggs per egg mass was determined. The egg masses were held for six days to determine hatchability, with one exception. There was one egg mass that was inadvertently counted after five days. Mortality of the first generation organisms observed in the treatment groups over the course of the test was used to calculate the LC50 value, when possible. The lowest-observed-effect-concentration (LOEC) and the no-observed-effect-concentration (NOEC) were determined by the concentration-response pattern and statistical analysis of the survival, emergence, reproduction and growth data.

Duration:

47 d

Dose descriptor:

NOEC

Effect conc.:

5.5 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

other: male development rate

Duration:

47 d

Dose descriptor:

LOEC

Effect conc.:

14 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

other: male development rate

Duration:

47 d

Dose descriptor:

EC50

Effect conc.:

> 196 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Basis for effect:

mortality

Day 16 Results

 

Survival

On Day 16 of the test, four of the original 12 replicates were terminated for the determination of survival and growth. The Day 16 mean survival of C. dilutus in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups were 87.5, 87.5, 87.5, 93.8, 81.3, 100, 66.7 and 68.8%, respectively. There was a statistically significant reduction (p < 0.05) in survival of the 89 mg a.s./kg treatment group when compared to the pooled control using a Bonferroni t-test; however, the difference was not dose responsive and not considered to be evidence of a toxic effect. Consequently, the LOEC and NOEC for Day 16 survival was empirically estimated to be >196 and 196 mg a.s./kg dry weight of sediment, respectively (>4900 and 4900 mg a.s./kg-OC; >0.43 and 0.43 mg a.s./L mean measured in pore water, respectively). The LC50 was >196 mg a.s./kg, (>4900 mg a.s./kg-OC; >0.43 mg a.s./L, mean measured in pore water) the highest concentration tested, since the percent mortality was less than 50% in all treatment groups and controls.

 

Growth

The Day 16 average individual ash-free dry weight of C. dilutus in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups were 2.45, 2.34, 2.40, 1.90, 2.60, 1.82, 1.59 and 1.01 mg, respectively. There were statistically significant reductions (p ≤ 0.05) in the ash-free dry weight of the 5.5, 35, 89 and 196 mg a.s./kg treatment groups in comparison with the pooled control using a Bonferroni t-test. Since there was no significant reduction in the 14 mg a.s./kg treatment group, the difference observed in the 5.5 mg a.s./kg treatment group is not considered to be biologically meaningful. Consequently, the LOEC and NOEC for Day 16 growth was 35 and 14 mg a.s./kg dry weight of sediment, respectively (884 and 350 mg a.s./kg-OC; 0.10 and 0.035 mg a.s./L mean measured in pore water, respectively).

 

Day 47, Test Termination Results

There were a total of 33, 35, 40, 42, 42, 30 and 20 mated females in the negative control, solvent control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups, respectively. There were a total of 29, 32, 40, 40, 38, 30 and 18 primary egg masses produced in the negative control, solvent control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups, respectively. The average number of eggs per primary egg mass was 1456, 1393, 1424, 1445, 1498, 1393, 1353 and 1274, respectively in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups. The average percent hatch of eggs in the primary egg masses produced was 98, 88, 93, 76, 91, 77, 90 and 92, respectively. There were 0.89, 0.94, 0.91, 1.00, 0.96, 0.89, 1.00 and 0.91 primary egg masses per mated female and 1309, 1222, 1266, 1445, 1388, 1235, 1286 and 1169 average number of eggs per mated female in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups, respectively. The mean time to oviposition was 1.00, 1.03, 1.02, 1.03, 1.13, 1.00, 1.09 and 1.00 days, respectively. A Bonferroni t-test indicated that there were statistical differences in percent hatchability between the pooled control and the 5.5 and 35 mg a.s./kg treatment groups; however, the differences were not dose responsive and not considered to be treatment related. There were no statistical differences noted for any of the other reproductive endpoints using a Kruskal-Wallis or Bonferroni t-test (as appropriate). Consequently, the LOEC and NOEC for the reproductive endpoints was >196 and 196 mg a.s./kg, respectively (>4900 and 4900 mg a.s./kg-OC; >0.43 and 0.43 mg a.s./L mean measured in pore water, respectively).

 

Emergence was first noted in the original eight replicates on Day 16 of the test. Most adults that emerged appeared normal. There were a few observations of larvae leaving the surface of the sediment or swimming in the water column. There also were observations of dead pupae or larvae, partial emergence and adults that emerged and died. The frequency of these observations was comparable among control and most treatment groups and is considered normal in this type of test system.

Mean emergence ratios in the negative control, solvent control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups were 0.92, 0.79, 0.85, 0.85, 0.81, 0.68 and 0.50, respectively. There were statistically significant differences (p < 0.05) between the negative control group and the 89 and 196 mg a.s./kg treatment groups using a Dunnett’s test. Therefore, the NOEC for emergence ratios was 35 mg a.s./kg and the LOEC for emergence ratios was 89 mg a.s./kg (884 and 2229 mg a.s./kg-OC; 0.10 and 0.18 mg a.s./L mean measured in pore water, respectively).

Mean development times were 22.6, 21.7, 22.2, 22.6, 23.1, 24.1, 25.0 and 29.9 days, respectively in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups. Mean male development rates (defined as the portion of larval development which takes place per day and calculated as the inverse of the development time) in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups were 0.0530, 0.0523, 0.0527, 0.0515, 0.0495, 0.0479, 0.0444 and 0.0352, respectively. Mean female development rates in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups were 0.0420, 0.0435, 0.0427, 0.0414, 0.0406, 0.0396, 0.0391 and 0.0332, respectively. There were statistically significant differences for development time and female development rate (p < 0.05) between the pooled control group and the 35, 89 and 196 mg a.s./kg treatment groups using a Dunnett’s test. There were statistically significant differences for male development rate (p < 0.05) between the pooled control group and the 14, 35, 89 and 196 mg a.s./kg treatment groups using a Dunnett’s test. Therefore, (based on the male development rate, the most sensitive endpoint) the NOEC for development was 5.5 mg a.s./kg and the LOEC was 14 mg a.s./kg (139 and 350 mg a.s./kg-OC; 0.025 and 0.035 mg a.s./L mean measured in pore water, respectively).

 

The mean time to death of midges after complete emergence was also examined. The number of days it took for an adult to die after complete emergence was recorded for each emerged adult (excluding the organisms from the auxiliary male replicates). A mean was calculated for each replicate and for each treatment group. The male mean time to death in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups was 4.32, 4.54, 4.43, 4.92, 4.97, 4.65, 4.54 and 5.02 days, respectively. The female mean time to death in the negative control, solvent control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups was 5.21, 4.19, 4.38, 4.30, 4.09, 4.13 and 3.74 days, respectively. The mean time to death (males and females pooled) in the negative control, solvent control, pooled control, 5.5, 14, 35, 89 and 196 mg a.s./kg treatment groups was 4.83, 4.36, 4.59, 4.66, 4.75, 4.28, 4.38 and 4.06 days, respectively. There were statistically significant differences (p ≤ 0.05) between female mean time to death between the negative control and the 35, 89 and 196 mg a.s./kg treatment groups using a Dunnett’s test. The NOEC for mean female time to death, the most sensitive gender, was 14 mg a.s./kg and the LOEC was 35 mg a.s./kg (350 and 884 mg a.s./kg-OC; 0.035 and 0.10 mg a.s./L pore water).

Validity criteria fulfilled:

yes

Conclusions:

There were statistically and/or biologically significant decreases in growth and development. The NOEC for male development rate (the most sensitive endpoint) was 5.5 mg a.s./kg dry weight of sediment (139 mg a.s./kg-OC, 0.025 mg a.s./L pore water). The LOEC was 14 mg a.s./kg dry weight of sediment (350 mg a.s./kg-OC, 0.035 mg a.s./L pore water). The 47-Day LC50 for survival was >196 mg a.s/kg dry weight of sediment, the highest concentration tested (>4900 mg a.s./kg-OC, >0.43 mg a.s./L pore water).

Executive summary:

Midge larvae (Chironomus dilutus) were exposed to famoxadone in sediment at mean measured concentrations of 5.5 to 196 mg a.s./kg sediment dry weight, and then observed for 47 days. The test was conducted according to the procedures outlined in the protocol, “Famoxadone (DPX-JE874) Technical: A Life Cycle Toxicity Test with the Midge (Chironomus dilutus) Using Spiked Sediment”. The protocol was based on procedures in the U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates; and ASTM Standard E 1706-05: Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates.

 

Groups of first instar larvae were exposed to a geometric series of five test concentrations of sediment-incorporated famoxadone, a solvent control and a negative control for 47 days in a flow-through system providing intermittent renewal of overlying water. The test was initiated with 12 replicate test compartments, with an additional four replicates (for use as auxiliary male replicates) initiated on Day 6, for a total of 16 replicate test compartments in each treatment and control group. Each test compartment was initiated with 12 midge larvae for a total of 192 individuals per treatment group and control. Each test compartment contained a quantity of sediment and overlying water. Three additional replicates (defined as “water quality” replicates) were added to each treatment and control group to be used for measurements of sediment pH and pore water ammonia. Three additional replicates (defined as “analytical” replicates) were added in each treatment and control group to be used for analytical sampling of water and sediment. The “analytical” and “water quality” replicates sampled on Day 0 did not contain midge larvae, while midge larvae were added at test initiation to the “analytical” and “water quality” replicates to be sampled on Days 16 and 47.

 

Test compartments were positioned in the test system 14 days prior to test initiation to condition the sediment prior to the introduction of organisms (the time required for sediment conditioning was determined during an equilibration trial). On Day 0 of the test, first instar midge larvae (up to 4 days post hatch) were sequentially and impartially added one and/or two at a time to test compartments (replicates A – L; O, P, Q and R) until each test compartment contained a total of 12 larvae. Replicates M and N were not initiated with organisms since they were removed from the test system on Day 0 for analytical and water quality measurements. Because male midges typically emerge sooner than female midges, additional replicates were set up six days after the original replicates to be able to obtain males to mate with the females during their emergence period and in order to be able to obtain viable egg masses. On Day 6 of the test, first instar midge larvae from a second collection of organisms (up to 4 days post hatch) were sequentially and impartially added one and/or two at a time to auxiliary male test compartments (replicates S, T, U and V) until each test compartment contained a total of 12 larvae. All transfers to the test compartments were made below the air/water interface using a wide-bore pipette. Observations of mortality and abnormal behavior (leaving the sediment, climbing the walls of the test chamber, etc.) were made daily during the test. On Day 16 of the test, approximately 20-day old surviving larvae were isolated from the sediment using 0.425 μm sieves and shallow sorting pans in four replicates (replicates I, J, K and L) for each treatment group and control for the assessment of survival and growth. Emergence traps were placed on replicates A – L and O - R, on Day 15 of the test and stayed on for the remainder of the test. Emergence traps were placed on the four auxiliary male replicates (replicates S, T, U and V) on Day 14 of their exposure (Day 20 of the test, corresponding with the first sign of pupation). The total number of adults emerged at the end of the test period was recorded. During the period of emergence, males and females were paired with other adults from the same treatment group and monitored for production of egg masses. Primary egg masses were collected and the number of eggs per egg mass was determined. The egg masses were held for six days to determine hatchability, with one exception. There was one egg mass that was inadvertently counted after five days. Mortality of the first generation organisms observed in the treatment groups over the course of the test was used to calculate the LC50 value, when possible. The lowest-observed-effect-concentration (LOEC) and the no-observed-effect-concentration (NOEC) were determined by the concentration-response pattern and statistical analysis of the survival, emergence, reproduction and growth data

 

There were statistically and/or biologically significant decreases in growth and development. The NOEC for male development rate (the most sensitive endpoint) was 5.5 mg a.s./kg dry weight of sediment (139 mg a.s./kg-OC, 0.025 mg a.s./L pore water). The LOEC was 14 mg a.s./kg dry weight of sediment (350 mg a.s./kg-OC, 0.035 mg a.s./L pore water). The 47-Day LC50 for survival was >196 mg a.s/kg dry weight of sediment, the highest concentration tested (>4900 mg a.s./kg-OC, >0.43 mg a.s./L pore water).

Description of key information

Freshwater

28-day LC50 (midge, Chironomus riparius) = 62 mg/kg, 28-day NOEC = 34 mg/kg, OECD 218, Reliability = 1

28-day EC50 (midge, Chironomus riparius) = 0.41 mg/L; 28-day NOEC = 0.01 mg/L, BBA recommended 'Long term test with Chironomus riparius’ (1995), Reliability = 1

47-day LC50 (midge, Chironomus dilutus) > 196 mg/kg, 47-day NOEC = 5.5 mg/kg, ASTM E1706 and U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, Reliability = 1

42-day LC50 (amphipod, Hyalella azteca) > 378 mg/kg, 42-day NOEC = 24 mg/kg, ASTM E1706, OPPTS 850.1770 and U.S. Environmental Protection Agency Guidance Document, EPA 600/R-99/064: Methods for Measuring the Toxicity and Bioaccumulation of Sediment-associated Contaminants with Freshwater Invertebrates, Reliability = 1

Saltwater

28-day EC50 (amphipod, Leptocheirus plumulosus) = 292 mg/kg, 28-day NOEC = 131 mg/kg, EPA 600/R-94/025: Methods for Assessing the Toxicity of Sediment-Associated Contaminants with Estuarine and Marine Amphipods and EPA 600/R-01/020: Method for Assessing the Chronic Toxicity of Marine and Estuarine Sediment-Associated Contaminants with the Amphipod Leptocheirus plumulosus, Reliability = 1

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

62 mg/kg sediment dw

EC50 or LC50 for marine water sediment:

292 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

5.5 mg/kg sediment dw

EC10, LC10 or NOEC for marine water sediment:

131 mg/kg sediment dw

Additional information

Famoxadone was tested in 4 freshwater sediments (3 midge species and in Hyalella Azteca).  The EC/LC50s ranged from 62 - >196 mg/kg dw sediment and the NOEC ranged from 5.5 – 34 mg/kg dw sediment.  In the one saltwater sediment tested, the EC50 was 292 mg/kg dw sediment and the NOEC was 131 mg/kg dw sediment.