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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the in-life phase, samples from new solutions removed from each treatment level, solvent control, and negative control were analyzed for test substance concentration on test days 0, 6, 14, and 20. Samples from aged solutions removed from each treatment level, solvent control, and negative control were analyzed for test substance concentration on test days 2, 8, 16, and 21. Solvent stocks were analyzed at each sampling interval.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of a primary stock solution in solvent by diluting appropriate amount of test substance to achieve concentration of 500 μg/mL; this was further diluted to produce test substance concentrations in solvent of 2.5, 1.3, 0.63, 0.31, 0.16 and 0.078 μg/mL.
- Controls: solvent control (0.3 mL, diluted to 3 L with dilution water)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): dimethylformamide
- Concentration of vehicle in test medium: 0.3 mL, diluted to 3 L with dilution water
- Test concentration separation factor: 2
- Evidence of undissolved material: all stock solutions were observed to be clear and colourless with no visible undissolved test substance

Test organisms (species):

Daphnia magna

Details on test organisms:

Freshwater invertebrates (Daphnia magna) were obtained from laboratory cultures maintained at the test laboratory.
Test organisms were <24 hours old at the initiation of exposure. The adult daphnids used to produce offspring did not contain ephippia, produced offspring prior to being 12 days old, produced on average at least three offspring per female per day over the seven days prior to exposure initiation, were not used in any portion of a previous test, and had no significant mortality (less than 5% of population died or showed signs of stress during the 48 hours prior to exposure initiation).
Regulated photoperiod of 16 hours light and 8 hours darkness, light intensity of 550 to 1100 lux at the surface of culture medium, provided by fluorescent bulbs.
Daphnids were cultured in 1-L glass vessels containing approximately 0.8 L of water. All vessels were maintained at 20 ± 2 ºC.
Daphnids were fed unicellular green algae (Ankistrodesmus falcatus) at a concentration of 4 x 10^7 cells/mL (250 to 350 μL, depending on age) and a suspension of YCT (50 μL, yeast, cereal leaves and flaked fish food).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

170 mg/L as CaCO3

Test temperature:

18 to 21 °C

pH:

7.8 to 8.9

Dissolved oxygen:

7.7 to 10 mg/L (84 to 110% of saturation)

Salinity:

Total alkalinity range of 84 to 90 mg/L

Conductivity:

750 to 830 µS/cm

Nominal and measured concentrations:

- Nominal test concentrations: 0.0078, 0.016, 0.031, 0.063, 0.13 and 0.25 μg/mL

Details on test conditions:

Animals were exposed individually in 100 mL clear glass beakers, each containing 80 mL of test solution. Exposure vessels were placed in a temperature controlled environmental chamber to maintain a temperature of 20 ± 2 °C. At exposure initiation and at each 48-hour interval thereafter, fresh exposure solutions were prepared, which were added to a second set of clean glass beakers. Daphnids were carefully transferred from the aged into the freshly prepared solutions.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.083 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.24 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.095 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Offspring per female per reproductive day

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 0.24 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

total body length

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.14 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

dry weight

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.064 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

total body length

Details on results:

All other endpoints investigated in the study (such as survival, production rate of first brood, offspring per female per reproduction day, total body length and dry weight) had EC10 values greater than that determined for the offspring per surviving female.

Reported statistics and error estimates:

The 95% confidence interval was 0.049 to 0.12 µg/L

Validity criteria fulfilled:

yes

Conclusions:

The substance demonstrated long-term toxicity to freshwater invertebrates (Daphnia magna), and the lowest 21-day EC10 value of 0.083 µg/L was observed for the number of offspring per surviving female daphnid.

Executive summary:

The effects of the substance on the survival, reproduction and growth of aquatic freshwater invertebrates (Daphnia magna) were determined under GLP to OECD TG 211 over a period of 21 days under semi-static conditions. Test substance was dissolved in dimethylformamide to produce stock solutions, which were then mixed with dilution water to give test solutions with nominal test concentrations of 0.0078, 0.016, 0.031, 0.063, 0.13 and 0.25 µg/L. These were tested alongside a dilution water and a solvent control. The test concentrations were confirmed analytically by LC-MS/MS and ranged from 92 to 120% of nominal concentrations. Mean measured concentrations were 0.0092, 0.015, 0.03, 0.064, 0.13 and 0.24 µg/L. The lowest 21-day EC10 value of 0.083 µg/L was calculated on the basis of mean measured concentration for the number of offspring per surviving female. The lowest NOEC of 0.064 µg/L was obtained for the total body length of adults.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr 2018 to 10 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Prior to exposure initiation, samples from one replicate of each treatment level and the control were collected and analyzed for the test substance concentration. In addition, samples from the saturator column effluent were analyzed. Results of these analyses were used to determine whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the definitive exposure.
- During the definitive exposure, samples were removed on days 0 (experimental initiation), 7, 14, 21, and 28 (F0 generation experimental termination) from one replicate of each concentration and the control for analysis of the test substance concentration. F1 generation observations were slightly extended to day 29 in four treatment replicates in order to ensure an equal observation period across all treatment levels and the control for this endpoint. However, the day 28 analytical interval, which encompassed all F0 mysid exposure and the majority of the F1 group exposures, adequately fulfilled the requirement of confirming analytical concentrations at test termination. Replicates were sequentially alternated between sampling intervals. All exposure samples were removed from the approximate midpoint of each aquarium using a pipette. Archive samples were also removed in the same manner on these exposure days and stored frozen as a precaution for potential future analysis. Samples of the syringe stock solution (i.e., solution from the gas-tight syringe) and the saturator column effluent (i.e., the continuous effluent to the drain) were also analyzed at each sampling interval during the exposure period.

Vehicle:

yes

Remarks:

methanol

Details on test solutions:

For this exposure, a glass wool saturator column was used to deliver the test substance to the exposure system. The glass column was packed with glass wool, and then coated with the test substance. The column was designed to provide a constant flow of saturated aqueous solution (1.0 mg/L) of the test substance to the diluter system without the use of a carrier solvent. The saturator column was constructed entirely of chemically inert materials (glass and Teflon). To construct the column, each 60 centimeter length × 4.8 centimeter diameter column was firmly packed with approximately 15% of the total column volume with glass wool. This provided ample surface area inside the column for the exposure substance to adhere once the column preparation was complete. After the column was packed, the end fittings were placed on the column. All fittings used to enclose the column and to attach the column to the water source were composed of Teflon.
To coat each column, approximately 8.0 grams of the test substance was diluted with 190 mL of methanol. The solution was mixed briefly with a glass rod and sonicated in an ultrasonic water bath for approximately 15 minutes until no undissolved test substance remained. This solution was then slowly poured into the glass column. After all of the solution was added, the column was attached to a vacuum pump. The vacuum pump was used to draw the solution evenly throughout the column to uniformly coat the glass wool with the test substance and draw off the remaining methanol. After it had visually appeared that all of the glass wool was coated and all the solvent was drawn off, the column was detached from the vacuum pump and attached to a pump which delivered a flow of 20‰ seawater through the column. During non-solvent dosing trials, the saturator column effluent was measured to be delivering a stable, consistent dose of approximately 1.0 mg/L for approximately one week at an effluent flow rate of 35 mL/min. This flow rate allowed an optimal retention time within the column. The saturator column was allowed to flush to a drain for approximately 48 hours prior to being used to dose the diluter system.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysids
- Age at study initiation: ≤ 24 hours old
- Source: Obtained from cultures maintained at the test facility (Had been maintained in the in-house culture for approximately two months prior to testing.

CULTIVATION
- Culture conditions: Mysids were cultured in six recirculating 80-L glass aquaria containing dilute, natural, filtered seawater.
- Aeration: Standard aquarium underwater gravel filters were used to provide aeration and a current conducive to feeding.
- Salinity: 21 to 23‰
- Dissolved oxygen: 87 to 94% of saturation
- pH: 7.8 to 7.9 (during the 14-days prior to exposure initiation)
- Temperatrue: 25 to 26 °C
- The brood stock and test organisms were cultured and tested in seawater from the same source.
- Photoperiod: 16 hours of light and 8 hours of darkness (Sudden transitions from light to dark, and vice versa, were avoided with a transition light period established by offsetting the culture lights from the general laboratory lighting.)
- Light intensity: 46 to 65 footcandles
- Health during cultivation: The culture organisms were observed to be in good health (e.g., no signs of sickness, disease, external parasites, physical damage, or widespread mortality) from the day of receipt to the time of female isolation and subsequent offspring collection. The juvenile mysids used for the test were determined to be in good health at the start of the exposure phase.

FEEDING DURING THE TETS
- Food type: Live brine shrimp (Artemia salina) nauplii, ≤ 48 hours old (post-hydration)
- Frequency: Twice daily (The second daily feeding was with brine shrimp nauplii enriched with a substance high in saturated fatty acids)
- Amount: Densities per mL of concentrated collections of nauplii from Artemia cultures created from daily hydrated cysts were determined by triplicate counts of aliquots pipetted onto filter paper. Based on these densities, various volumes of nauplii were fed to mysids per feeding in accordance with the following regime: rations for F0 retention chambers (containing 20 mysids each) were approximately 90 nauplii/mysid on test days 0 to 3, 135 nauplii/mysid on test days 4 to 6, 180 nauplii/mysid on test days 7 to 9, and 225 nauplii/mysid on test day 10 to pairing. From day of pairing until test termination, the F0 pairing chambers were fed approximately 450 nauplii/mysid and the retention chamberswere fed approximately 3600 nauplii/chamber. After F1 chambers were initiated, these chambers were fed a ration of approximately 90 nauplii/mysid. All treatments and the control received, as near as reasonably possible, the same ration of food. Excess brine shrimp and organic debris in the test chambers was removed prior to the addition of new nauplii.
- Food quality: Prior to use of a new batch of feed, a representative sample of the food source was analyzed for the presence of pesticides, PCBs, and toxic metals. None of these compounds were detected at concentrations that are considered toxic in any of the food samples analyzed in agreement with ASTM (2007) standard practice. Based on the analysis for pesticides and the performance of the control organisms, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline criteria.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

28 d

Test temperature:

25 ± 2 °C

pH:

7.5 - 7.9

Dissolved oxygen:

- 5.26 - 7.05 mg O2/L
- 73 - 98% of saturation

Salinity:

20 - 22 ‰

Nominal and measured concentrations:

- Nominal concentration: 0 (nagative control), 1.0, 2.0, 4.0, 8.0, and 16 ng/L
- Measured concentration: < LOQ (nagative control), 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L, respectively. See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass test aquarium
- Size: 30 × 15 × 20 cm (L × W × H) with a 10 cm high side drain
- Filled volume: 4.5 L
- Type of flow-through: Proportional diluter
- Flow rate: Approximately 9.9 aquarium volume additions per day to provide a 90% test solution replacement rate of approximately 6 hours
- No. of vessels per concentration: 4
- No. of vessels per control: 4

BEFORE SEXUALLY MATURE
- Test chamber: Glass petri dishes
- Size: 10 cm in diameter, 2 cm deep, to which a 14 cm high Nitex screen collar (350-µm mesh size opening) was attached with silicone sealant.
- Filled volume: Approximately 785 mL
- Function of the chamber: Retain sexually immature mysids
- Retain period: First 11 days of the exposure
- No. of organisms per chamber: 20 mysids
- No. of chamber per test vessel: 1

AFTER SEXUALLY MATURE
- Test chamber: Petri dishes
- Size: 6 cm in diameter, to which a 14-cm high Nitex screen collar (350-µm mesh size opening) was attached with silicone sealant
- Filled volume: Approximately 250 mL
- Function of the chamber: Pairing sexually mature male and felame organisms
- Pairing period: From test day 12 to the end of the exposure (day 28)
- No. of organisms per chamber: 2 mysids (one mature male and one mature female)
- No. of chamber per test vessel: Maximum of 5 pairing chambers
- Unpaired mysids maintained in the initial retention chamber until they were paired or until test termination. Male mysids from this pool were used to replace dead males from the paired (male/female) groups. When three or fewer unpaired mysids remained in a retention chamber, they were transferred to a separate pairing chamber, to conserve space and reduce the overall surface area within the exposure vessel. Females that died in pairing chambers were replaced at the Study Director’s discretion. For example, if a female within a pairing chamber died prior to that replicate’s median first brood release and if that replicate’s retention chamber had not yet reproduced, the dead female was replaced with a female from the retention chamber, if available.

EXPOSURE SYSTEM
- Based on the very low test concentrations and the functional solubility of the test substance, a glass gas-tight syringe was used to deliver the test substance to the diluter system. The 10 mL syringe was filled directly with the effluent from the previously described saturator column, which flowed to a drain continuously (i.e., at 35 mL/min) throughout the exposure period. The syringe was filled daily. To avoid potential losses due to sorption, the syringe and plunger were conditioned for approximately 24 hours in a flow-through reservoir that received the saturator column effluent prior to use. A pump, in conjunction with the gas-tight syringe, was calibrated to continuously deliver 3.9 µL/min of the 1.0 mg/L saturator column effluent to the diluter system’s mixing chamber. The flow rate from the syringe, in combination with the calibrated dilution water cell, filled the mixing chamber at each cycle to a total volume of 1.940 L. The mixing chamber was positioned over a magnetic stir plate and the solution was continuously stirred with a Teflon-coated stir bar throughout the test. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration (16 ng/L) and was proportionally diluted by a constant factor of 2.0 to produce the remaining nominal test concentrations (8.0, 4.0, 2.0, and 1.0 ng/L).
- A set of control aquaria were also established which contained the same dilution water and were maintained under the same conditions as the treatment aquaria, but contained no test substance.
- The exposure system was in proper operation for 12 days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels. This extended equilibration time allowed for saturation of potential binding sites such as glassware and other substrates that typically exist in diluter systems.
- Flow-splitting cells were employed to equally distribute the solutions to the replicate vessels at a rate of 250 mL of control or test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within 10% of the nominal value. The diluter system was calibrated prior to exposure initiation and confirmed at exposure termination by measuring delivery volumes of toxicant solution and dilution water. The function of the diluter system (e.g., cycle rate and column effluent flow rate) was monitored daily and a visual check of the system’s operation was performed twice daily.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilute, filtered, natural seawater was used as dilution and control water during this study. Natural seawater was pumped from the Cape Cod Canal, Bourne, Massachusetts. The water was collected at about one to four meters offshore and a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 6080 L polyethylene holding tank. In the laboratory, the seawater was diluted to a salinity of 20 ± 3‰ with laboratory well water (a mixture of unadulterated water from an on-site 100 meter bedrock well and de-chlorinated Town of Wareham well water). The diluted seawater was then stored in a recirculating 30,280 L polyethylene tank, and then transferred
to one of two 22,680 L polyethylene tanks where it was continuously recirculated for at least 48 hours. The water was then filtered through 20 µm, 5 µm, and 1 µm polypropylene core filters, heated to the required test temperature, and then pumped under constant pressure through PVC pipes to the intermittent-flow proportional diluter system.
- TOC: Representative samples of the dilution water were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 1.6 and 0.81 mg/L for April and May 2018, respectively.
- Representative samples of the dilution 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 ASTM (2007) standard practices. Based on these analyses and the performance of the control organisms, the water source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline performance criteria.
- Intervals of water quality measurement: Temperature, dissolved oxygen concentration, pH, and salinity were measured in each replicate on day 0 and alternated between replicates daily thereafter throughout the exposure period, for each treatment level and the control. In addition, exposure solution temperature was continuously monitored in one control vessel.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours of light and 8 hours of darkness (Sudden transitions from light to dark, and vice versa, were avoided using a period of lower light intensity during a 30-minute transition period. )
- Light intensity and location: 28 to 57 footcandles (300 to 610 lux) above the test aquaria

EFFECT PARAMETERS MEASURED
- Mysid counting and biological observations: In order to observe the mysids during the exposure period, each retention chamber was gently lifted from the aquaria daily. The chamber was then placed into a Pyrex dish containing dilution water at approximately 25 °C and the dish was placed on a light table. During this procedure, the water level in each chamber was reduced by allowing water to drain through the screen. The numbers of dead and living organisms were counted and any abnormal appearance or behavior was recorded. Due to the rapid movement of mysids in a single chamber containing up to 20 mysids, survival of the test organisms was estimated for the first 11 days of the test, i.e., prior to pairing of the mysids. After males and females had been paired (day 12) accurate counts of survival were made and the number of dead males and females, the number of offspring produced by each individual female and any abnormal appearance or behavior was recorded daily. Survival from day 12 to day 28 of exposure was denoted as post-pairing survival in the applicable data summaries. Dead parental mysids and offspring were recorded, removed, and discarded when observed during the test. Mortality was defined as lack of movement after gentle prodding with a glass pipet. Following the observation period, retention chambers were brushed clean, debris was removed, and then the chambers were returned to the respective aquaria.
- F1 generation (initialtion): During the reproductive phase, groups of 10 offspring per replicate, 40 per treatment were removed from mysid chambers in each replicate vessel, pooled, and transferred to a separate pairing chamber in that replicate. If 10 offspring could not be collected from a single replicate, offspring from multiple replicates in the same treatment level were pooled and collected. If 10 offspring could not be collected from multiple replicates of the same treatment level, a reduced number of young was collected. All groups of F1 offspring were
evaluated for a period of 96 hours post-release. Due to reduced reproductive output in several replicates, F1 generation collection began on test day 25 at the Study Director’s discretion in four treatment replicates (1.0 ng/L treatment level replicate D; 7.4 ng/L treatment level replicates A, B, and D). These groups were evaluated until test day 29 (after test termination) in order to ensure an equal observation period across all treatment levels and the control. Chambers were established based on the number of available juvenile F1 mysids; therefore, each chamber was not necessarily initiated on the same day. One F1 group was established and monitored for each replicate vessel.
- F1 generation (Biological observations and survival): At the time an F1 generation chamber was established and daily thereafter for 96 hours, observations of stress, abnormal behavior (including discoloration, immobilization, and inability to maintain position in the water column), and survival were made. Dead mysids (defined as those lacking mobility and failing to respond to gentle prodding) were recorded and removed from each replicate test vessel daily. Missing mysids were considered dead.
- Mean total body length and dry weight determinations: At test termination, all mysids were euthanized by immersion in ice-cold, deionized water. The mysids were carefully removed, blotted dry on absorbent paper, and separated into male and female groups for each replicate exposure level. A digital photograph was then taken of each mysid using a microscope equipped with a camera for individual body length measurements. Individual body length was measured to the nearest 0.01 mm using image analyzing software.
Following collection of digital photographs, male and female mysids were transferred to aluminum pans by group (i.e., paired males, unpaired males, paired females and unpaired females), dried in an oven at approximately 100 °C for 24 hours and then placed in a desiccator. Individual dry body weight to the nearest 0.01 mg was determined using a calibrated analytical balance. Individual lengths and weights of all surviving males and females were recorded separately for each replicate of each treatment level and the control.
- Reproductive success: Reproductive success was calculated for each replicate aquarium (treatments and the control) as the total number of offspring produced per female. The length of time for the appearance of the first brood for each replicate was also determined. In addition, the percentage of actively reproducing females in each replicate of each treatment and the control was determined.

RANGE FINDING STUDY
- Study design: A preliminary 28-day flow-through exposure was conducted from 20 July to 17 August 2017 exposing juvenile mysids (80 mysids per treatment level; 20 per replicate) to the test substance. The procedures and conditions for this exposure were similar to those in the definitive exposure.
- Nominal concentrations: Control, 0.022, 0.076, 0.26, 0.88, and 3.0 ng/L.
- Results used to determine the conditions for the definitive study: During the post-pairing phase of the preliminary exposure, the majority of F0 and F1 generation mysids in the 0.076 ng/L treatment level replicates A and B; and the 0.88 ng/L treatment level replicate B were observed to be at the water surface displaying a partial lack of equilibrium, and within 2 days, those mysids that had shown sublethal effects had died. Based on the abruptness of the mortality in these few replicates and the lack of sublethal effects or overt mortality in all other replicates in the test, these three replicates were considered compromised, and the mortality was not considered a result of the test substance exposure. Therefore, after consultation with the Study Sponsor, these three replicates were excluded from the treatment means and all statistical analyses. No statistically significant effects were observed for any endpoint evaluated. Based on the results of preliminary testing and consultation with the Study Sponsor, the nominal concentrations selected for the definitive exposure were 1.0, 2.0, 4.0, 8.0, and 16 ng/L.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

F0 generation

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

4.2 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Male Survival, Female Survival and Offspring Per Female

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

7.4 ng/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: F1 generation Survival, Male Total Length, Female Total Length, Male Dry Weight and Female Dry Weight

Details on results:

An overview of the results in provided in Table 3 to Table 7 in 'Any other information on results incl. tables'

SURVIVAL OF F0 GENERATION
The calculation of male and female post-pairing survival began following test day 12; any deaths which occurred prior to pairing are captured in the 28-day survival endpoint. On days 13 through 21, the majority of the F0 and F1 generation mysids in the 7.4 ng/L treatment level were observed to be lethargic.
At test termination, mean survival among male mysids in the control was 95%. Mean survival among male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 91, 89, 94, 86, and 71%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in male survival among organisms exposed to the 7.4 ng/L treatment level tested compared to the control. Based on male survival, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively. At test termination, mean survival among female mysids in the control was 98%. Mean survival among female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 100, 100, 93, 91, and 66%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in female survival among organisms exposed to the 4.2 and 7.4 ng/L treatment levels tested compared to the control. Based on the individual replicate distribution of survival in the 4.2 ng/L treatment level (i.e., three of the four replicates were ≥ 88% survival), the significant response at 4.2 ng/L was determined to have been an artifact of a single replicate (replicate B, 78% survival), and was not related to exposure from the test substance. The mean female survival at the 4.2 ng/L treatment level (91%) was also within expectations of the survival of female control mysids. Therefore, based on female survival, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively. Following 28 days of exposure, overall mean survival among mysids in the control was 96%. Mean survival among mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 91, 95, 82, 81, and 54%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in overall survival among organisms exposed to the 2.3, 4.2, and 7.4 ng/L treatment levels tested compared to the control. Based on 28-day survival, the NOEC and LOEC were determined to be 1.0 and 2.3 ng/L, respectively.

NUMBER OF OFFSPRING PER FEMALE
At test termination, the mean number of offspring per female in the control was 10. The mean number of offspring per female among mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 11, 5.1, 6.9, 11, and 6.1, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in offspring per female among organisms exposed to the 1.0 and 7.4 ng/L treatment levels tested compared to the control. Based on the lack of a statistically significant reduction at the next two highest treatment levels (2.3 and 4.2 ng/L), the effect at 1.0 ng/L was not considered to be toxicant-related, nor biologically relevant. Therefore, based on the mean number of offspring per female, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively.

GROWTH
The mean total body length of male mysids in the control was 7.77 mm. The mean total body length of male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 7.95, 7.73, 7.83, 7.76, 7.53 mm, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body length of male mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on male total body length, the NOEC and LOEC were determined to be 7.4 and >7.4 ng/L, respectively. The mean total body length of female mysids in the control was 7.92 mm. The mean total body length of female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 8.17, 8.05, 8.14, 8.10, and 7.92 mm, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body length of female mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on female total body length, the NOEC and LOEC were determined to be 7.4 and >7.4 ng/L, respectively. The mean dry body weight of male mysids in the control was 0.93 mg. The mean dry body weight of male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 1.01, 0.97, 1.01, 0.97, and 0.92 mg, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body weight of male mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on male dry body weight, the NOEC and LOEC were determined to be 7.4 and >7.4 ng/L, respectively. The mean dry body weight of female mysids in the control was 1.28 mg. The mean dry body weight of female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 1.40, 1.38, 1.42, 1.42, and 1.34 mg, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body weight of female mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on female dry body weight, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively.

SURVIVAL OF F1 GENERATION
Following the 96-hour observation period, mean percent survival among F1 mysids in the control was 98%. Mean percent survival among F1 mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 98, 100, 100, 98, and 92%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in F1 mysid survival among organisms exposed to any of the treatment levels tested compared to the control. Based on F1 96-hour post-release survival, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively

Reported statistics and error estimates:

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

Table 3. First Generation (F0) Survival

Mean Measured Concentration (ng/L)	Replicate	Male Survival^a (%)	Female Survival^a (%)	Post-Pairing Survival (%)	28-Day Survival^b (%)
Control	A	100	100	100	100
	B	100	100	100	100
	C	90	90	90	90
	D	90	100	95	95
	Mean (SD)^c	95 (6)	98 (5)	96 (5)	96 (5)
0.52	A	88	100	94	89
	B	100	100	100	100
	C	100	100	100	89
	D	75	100	92	86
	Mean (SD)	91 (12)	100 (0)	97 (4)	91 (6)
1.0	A	100	100	100	100
	B	67	100	83	83
	C	91	100	95	95
	D	100	100	100	100
	Mean (SD)	89 (16)	100 (0)	95 (8)	95 (8)
2.3	A	100	86	92	75
	B	89	88	88	83
	C	88	100	94	89
	D	100	100	100	82
	Mean (SD)	94 (7)	93 (8)	94 (5)	82^d(6)
4.2	A	70	88	78	78
	B	100	78	89	85
	C	75	100	86	67
	D	100	100	100	95
	Mean (SD)	86 (16)	91^d(11)	88 (9)	81^d(12)
7.4	A	71	40	58	37
	B	78	63	71	67
	C	67	100	83	63
	D	70	60	67	50
	Mean (SD)	71^d(5)	66^d(25)	70 (10)	54^d(13)

a Evaluations of male and female survival began after pairing and encompasses days 12 to 28, once sexual maturity was reached. Post-pairing survival includes the total percentage of males and females from day 12 to day 28 (test termination). The sex ratio may not be 1:1 in all replicates due to non-toxicant-related mortalities and differences in the ratios of F0 generation males and females.

b 28-Day survival encompasses all mortalities from day 0 through termination, regardless of pairing status (i.e., overall mysid survival from exposure initiation to termination). Mortality prior to pairing is considered in this calculation; therefore, male and female survival is not synonymous with 28-day survival.

c Mean values are presented with standard deviations (SD) in parentheses.

d Significantly reduced compared to the control, based on Cochran-Armitage’s Trend Step-Down Test. Based on the individual replicate distribution of survival in the 4.2 ng/L treatment level (i.e., three of the four replicates were ≥ 88% survival), the significant response at 4.2 ng/L was determined to have been an artifact of a single replicate (replicate B, 78% survival), and was not related to exposure from the test substance. The mean female survival at the 4.2 ng/L treatment level (91%) was also within expectations of the survival of female control mysids. Therefore, based on female survival, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively.

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values

Table 4. First Generation (F0) Reproductive Success (Offspring per Female)

Mean measured concentratio(ng/L)	Replicate	Percent of females producing young	Number of offspring Per Female
Control	A	100	8.4
	B	100	8.0
	C	100	12.0
	D	100	13.0
	Mean (SD)^a	100 (0)	10 (2.5)
0.52	A	100	11.4
	B	100	9.8
	C	100	9.0
	D	100	13.0
	Mean (SD)	100 (0)	11 (1.8)
1.0	A	80	5.8
	B	100	9.2
	C	60	2.8
	D	100	2.6
	Mean (SD)	85 (19)	5.1^b(3.1)
2.3	A	100	5.8
	B	80	6.4
	C	100	8.8
	D	80	6.6
	Mean (SD)	90 (12)	6.9 (1.3)
4.2	A	80	7.6
	B	80	9.8
	C	100	11.8
	D	100	13.6
	Mean (SD)	90 (12)	11 (2.6)
7.4	A	40	5.6
	B	80	4.4
	C	80	9.8
	D	60	4.6
	Mean (SD)	65 (19)	6.1^c(2.5)

a Mean values are presented with standard deviations (SD) in parentheses.

b Significantly reduced compared with the control, based on Dunnett’s Multiple Comparison Test. Based on the lack of a statistically significant reduction at the next two highest treatment levels (2.3 and 4.2 ng/L), the effect at 1.0 ng/L was not considered to be toxicant-related, nor biologically relevant.

c Significantly reduced compared with the control, based on Dunnett’s Multiple Comparison Test.

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

Table 5. First Generation (F0) Male and Female Total Body Length

Mean measured concentration (ng/L)	Replicate	Mean total body length (mm)
		Males	Females
Control	A	7.62	7.86
	B	7.67	7.75
	C	7.90	8.11
	D	7.91	7.98
	Mean (SD)^ab	7.77 (0.15)	7.92 (0.15)
0.52	A	7.75	8.23
	B	7.92	8.14
	C	8.13	8.18
	D	8.00	8.12
	Mean (SD)	7.95 (0.16)	8.17 (0.05)
1.0	A	7.65	7.97
	B	7.58	8.14
	C	8.00	8.06
	D	7.70	8.04
	Mean (SD)	7.73 (0.19)	8.05 (0.07)
2.3	A	8.02	8.20
	B	7.67	8.20
	C	7.92	8.09
	D	7.71	8.07
	Mean (SD)	7.83 (0.17)	8.14 (0.07)
4.2	A	7.76	8.07
	B	7.75	8.18
	C	7.87	8.28
	D	7.67	7.88
	Mean (SD)	7.76 (0.08)	8.10 (0.17)
7.4	A	7.51	7.94
	B	7.78	8.03
	C	7.28	7.88
	D	7.54	7.86
	Mean (SD)	7.53 (0.21)	7.92 (0.08)

a Mean values are presented with standard deviations (SD) in parentheses.

b Mean values include paired and non-paired F0 generation mysids.

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

Table 6. First Generation (F0) Male and Female Dry Body Weight

Mean measured concentration (ng/L)	Replicate	Dry body weight (mg)
		Males	Females
Control	A	0.83	1.29
	B	0.90	1.19
	C	1.01	1.37
	D	0.97	1.27
	Mean (SD)^ab	0.93 (0.08)	1.28 (0.07)
0.52	A	1.01	1.49
	B	1.01	1.47
	C	1.03	1.41
	D	0.99	1.23
	Mean (SD)	1.01 (0.02)	1.40 (0.12)
1.0	A	0.95	1.32
	B	0.90	1.44
	C	1.07	1.38
	D	0.97	1.40
	Mean (SD)	0.97 (0.07)	1.38 (0.05)
2.3	A	1.06	1.44
	B	0.96	1.35
	C	1.06	1.42
	D	0.97	1.46
	Mean (SD)	1.01 (0.06)	1.42 (0.05)
4.2	A	0.92	1.43
	B	0.92	1.43
	C	1.04	1.46
	D	0.99	1.38
	Mean (SD)	0.97 (0.06)	1.42 (0.03)
7.4	A	0.89	1.40
	B	0.96	1.51
	C	0.88	1.21
	D	0.96	1.26
	Mean (SD)	0.92 (0.04)	1.34 (0.13)

 

a Mean values are presented with standard deviations (SD) in parentheses.

b Mean values include paired and non-paired F0 generation mysids.

Table 7. Second Generation (F1) Survival at 96-Hours Post-Release

Mean Measured Concentration (ng/L)	Replicate	96-Hour Survival (%)
Control	A	90
	B	100
	C	100
	D	100
	Mean (SD)^a	98 (5)
0.52	A	100
	B	100
	C	100
	D	90
	Mean (SD)	98 (5)
1.0	A	100
	B	100
	C	100
	D	100
	Mean (SD)	100 (0)
2.3	A	100
	B	100
	C	100
	D	100
	Mean (SD)	100 (0)
4.2	A	100
	B	100
	C	90
	D	100
	Mean (SD)	98 (5)
7.4	A	100
	B	80
	C	89
	D	100
	Mean (SD)	92 (10)

a Mean values are presented with standard deviations (SD) in parentheses.

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the NOEC for F0 generation survival (the most sensitive indicator of toxicity) was determined to be 1.0 ng/L.

Executive summary:

This 28-day study was performed to determine the potential chronic (full life-cycle) toxicity of the test substance to the mysid, Americamysis bahia, under flow-through conditions, following OCSPP Guideline 850.1350. The study was in compliance with GLP criteria. Based on preliminary testing and in consultation with the Study Sponsor, the following nominal concentrations were chosen for the definitive exposure: 1.0, 2.0, 4.0, 8.0, and 16 ng/L (0.52, 1.0, 2.3, 4.2, and 7.4 ng/L mean measured). The test was conducted using an exposure system consisting of a glass wool saturator column, an intermittent-flow proportional diluter, a temperature-controlled water bath, and a set of 24 exposure aquaria. The test area was illuminated at an intensity range of 28 to 57 footcandles (300 to 610 lux), measured during the in-life exposure, using a photoperiod of 16 hours light and 8 hours darkness with a 30-minute transition period. The study was conducted in a water bath designed to maintain the test solution at a target temperature 25 ± 2 °C. Exposure solution concentrations were analytically confirmed on exposure days 0 (experimental initiation), 7, 14, 21, and 28 (experimental termination).

The calculation of male and female post-pairing survival began following test day 12; any deaths which occurred prior to pairing are captured in the 28-day survival endpoint. On days 13 through 21, the majority of the F0 and F1 generation mysids in the 7.4 ng/L treatment level were observed to be lethargic. At test termination, mean survival among male mysids in the control was 95%. Mean survival among male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 91, 89, 94, 86, and 71%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in male survival among organisms exposed to the 7.4 ng/L treatment level tested compared to the control. Based on male survival, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively.

At test termination, mean survival among female mysids in the control was 98%. Mean survival among female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 100, 100, 93, 91, and 66%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in female survival among organisms exposed to the 4.2 and 7.4 ng/L treatment levels tested compared to the control. Based on the individual replicate distribution of survival in the 4.2 ng/L treatment level (i.e., three of the four replicates were ≥88% survival), the significant response at 4.2 ng/L was determined to have been an artifact of a single replicate (replicate B, 78% survival), and was not related to exposure from the test substance. The mean female survival at the 4.2 ng/L treatment level (91%) was also within expectations of the survival of female control mysids. Therefore, based on female survival, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively. Following 28 days of exposure, overall mean survival among mysids in the control was 96%. Mean survival among mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 91, 95, 82, 81, and 54%, respectively. Cochran-Armitage’s Trend Step-Down Test determined a significant reduction in overall survival among organisms exposed to the 2.3, 4.2, and 7.4 ng/L treatment levels tested compared to the control. Based on 28-day survival, the NOEC and LOEC were determined to be 1.0 and 2.3 ng/L, respectively.

At test termination, the mean number of offspring per female in the control was 10. The mean number of offspring per female among mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 11, 5.1, 6.9, 11, and 6.1, respectively. Dunnett’s Multiple Comparison Test determined a significant reduction in offspring per female among organisms exposed to the 1.0 and 7.4 ng/L treatment levels tested compared to the control. Based on the lack of a statistically significant reduction at the next two highest treatment levels (2.3 and 4.2 ng/L), the effect at 1.0 ng/L was not considered to be toxicant-related, nor biologically relevant. Therefore, based on the mean number of offspring per female, the NOEC and LOEC were determined to be 4.2 and 7.4 ng/L, respectively.

The mean total body length of male mysids in the control was 7.77 mm. The mean total body length of male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 7.95, 7.73, 7.83, 7.76, 7.53 mm, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body length of male mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on male total body length, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively. The mean total body length of female mysids in the control was 7.92 mm. The mean total body length of female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 8.17, 8.05, 8.14, 8.10, and 7.92 mm, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body length of female mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on female total body length, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively.

The mean dry body weight of male mysids in the control was 0.93 mg. The mean dry body weight of male mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 1.01, 0.97, 1.01, 0.97, and 0.92 mg, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body weight of male mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on male dry body weight, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively.

The mean dry body weight of female mysids in the control was 1.28 mg. The mean dry body weight of female mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 1.40, 1.38, 1.42, 1.42, and 1.34 mg, respectively. Dunnett’s Multiple Comparison Test determined no significant difference in total body weight of female mysids among organisms exposed to any of the treatment levels tested compared to the control. Based on female dry body weight, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively.

Following the 96-hour observation period, mean percent survival among F1 mysids in the control was 98%. Mean percent survival among F1 mysids exposed to the 0.52, 1.0, 2.3, 4.2, and 7.4 ng/L treatment levels was 98, 100, 100, 98, and 92%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant reduction in F1 mysid survival among organisms exposed to any of the treatment levels tested compared to the control. Based on F1 96-hour post-release survival, the NOEC and LOEC were determined to be 7.4 and > 7.4 ng/L, respectively.

Based on mean measured concentrations and 28-day survival (the most sensitive indicator of toxicity), the NOEC was determined to be 1.0 ng/L, and the LOEC was determined to be 2.3 ng/L for mysids exposed the test substance. Since no concentration tested resulted in ≥ 50% mortality, LC50 values were empirically estimated to be greater than the highest mean concentration tested (i.e., > 7.4 ng/L).

Description of key information

Freshwater, 21-d EC10 = 0.083 µg/L, Daphnia magna, offspring per female per reproductive day, OECD TG 211, Conway 2020 

Marine water, 28-d NOEC = 1 ng/L, Americamysis bahia, F0 generation survival, EPA TG 850.1350, Marini 2018

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.083 µg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

NOEC

Effect concentration:

1 ng/L

Additional information

Freshwater

The chronic toxicity of the substance to freshwater invertebrates was studied under GLP to OECD TG 211 in two reliable and valid reproduction toxicity studies with Daphnia magna con. The substance had an effect on the growth and reproductive performance of daphnids. The lowest EC10 value of 0.083 µg/L was observed for the number of offspring per surviving female over a period of 21 days (Conway 2020). This value is used in the characterisation of aquatic hazards and environmental risk.

Marine water

The chronic, full life-cycle toxicity of the test substance to a marine mysid, Americamysis bahia, under flow-through conditions was studied in a valid and fully reliable test under GLP over a period of 28 days following OCSPP Guideline 850.1350 (Marini 2018). The most sensitive indicator of toxicity in this study was the overall 28-day survival of exposed adult organisms, and a significant reduction in the survival of male and female organisms was seen in the course of the test. Based on 28-day survival, the NOEC and LOEC were determined to be 1.0 and 2.3 ng/L, respectively. No treatment-related adverse effects on the number of offspring per female, survival of F1 mysids, the total body length of mysids, or the total dry body weight of mysids were observed.