N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 December 1989 to 25 May 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Version / remarks:

No version noted

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

MGK-264 (Code #209-90)

Analytical monitoring:

yes

Details on sampling:

Fifty milliliters of aquatic test water were composited from the four replicate aquaria and placed in 70 ml culture tubes. Fortifications were prepared ranging from 0.147 to 7.33 mg/l. To each tube were added 10 ml of toluene with 0.02% polyethylene glycol (PEG) and the samples shaken vigorously for ~1 minute. The phases were allowed to separate and the toluene with 0.02% PEG was transferred to 15 ml culture tubes. Dilutions were then prepared using toluene with 0.02% PEG such that the concentration of the sample would fall within the standard curve range.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The MGK-264 diluter stock solution was allowed to flow through the diluter system prior to addition of daphnids in order to equilibrate the test levels. The diluter stock was prepared by weighing 5.3826 g of the MGK-264 technical material into a 100 ml volumetric flask. The volumetric flask was brought to volume with acetone. The final concentration of this diluter stock solution was 50,000 mg/l, corrected for compound purity. Since acetone was used to prepare the diluter stock solution, the solvent control received an aliquot of acetone (0.049 ml) which was equivalent to the amount received by the other treatment levels.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test specimens of Daphnia magna were obtained from an in-house daphnid culture which has been maintained by ABC since 1977. The primary culture was obtained from the Columbia National Fisheries Research Laboratory (CNFRL), Columbia, Missouri, in 1977. A trace of the daphnid strain indicated that CNFRL acquired their culture from the U.S. Fish and Wildlife Service Fish Control Laboratory, Lacrosse, Wisconsin, in 1960 and they obtained their culture from Pennsylvania State University in 1954 (3).

All daphnids were held in a temperature controlled area at 20 (±2)°C. The lighting was 50-70 footcandles on a 16-hour daylight photoperiod, with 30 minute dawn and dusk transition periods. During the holding period, the daphnids were fed a suspension of algae (Selenastrum capricornutum) supplemented with a trout chow/yeast suspension. Only first-instar daphnids (<24 hours old) were selected for testing. Detailed logs of the cultures used to obtain test daphnids were maintained and are included in the Raw Data. Test daphnids were not fed during the 48-hour study.

The definitive test was initiated on March 28, 1990 by random assignment of 10 first- instar Daphnia ~ (ABC Lot A-RC 1 ) to each of the four replicate test chambers, i.e. 40 daphnids were used per concentration. This represents a loading factor of ~1 daphnid per 100-ml of solution. All concentrations were observed at 24- and 48-hours for mortality and other abnormal effects such as surfacing, erratic movement and/or daphnids laying on the bottom.

Test type:

flow-through

Water media type:

other: The dilution water used in this study was reconstituted hard blended water. Reconstituted hard blended water is soft blended water (ABC well water/R.O. water mix) and four salts which are blended to achieve a final hardness of 160-180 mg/l as CaCO3

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

N/A

Hardness:

164-174 mg/l (as CaCO3) - From Table 1 - "Chemical Characteristics of Soft Blended Water"

Test temperature:

20°C (Measured throughout 48 hour study duration)

pH:

7.9-8.1 (Measured throughout 48 hour study duration)

Dissolved oxygen:

9.0 - 9.6 mg/l (Measured throughout 48 hour study duration)

Salinity:

Not Noted

Conductivity:

410-440 μMHOS/cm - From Table 1 - "Chemical Characteristics of Soft Blended Water"

Nominal and measured concentrations:

Nominal - 0.30, measured as 0.33 and 0.35 at 0 and 48 hours respectively. Mean concentration of 0.34 mg/l
Nominal - 0.60, measured as 0.59 and 0.69 at 0 and 48 hours respectively. Mean concentration of 0.64 mg/l
Nominal - 1.3, measured as 0.90 and 1.1 at 0 and 48 hours respectively. Mean concentration of 1.0 mg/l
Nominal - 2.5, measured as 1.7 and 2.1 at 0 and 48 hours respectively. Mean concentration of 1.9 mg/l
Nominal - 5.0, measured as 4.1 and 5.1 at 0 and 48 hours respectively. Mean concentration of 4.6 mg/l

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 1.9 - < 4.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 1.9 - < 4.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The 24-hour and 48-hour LC 50 values (Table 4) for MGK-264 based on mortality were 2.7 and 2.3 mg/l, respectively. At 24 and 48 hours the 95% confidence limits were 1.9 and 4.6 mg/l using the binominal method. The 48-hour dose response slope was calculated to be 5.4. All results were based on the mean measured concentrations of: 0.34, 0.64, 1.0, 1.9 and 4.6 mg/l. The no-effect concentration based on the lack of mortality or other abnormal effects was 1. 0 mg/l after 48-hours. The adverse effects of morality, daphnids on the bottom of the test chambers, quiescence and erratic movement were observed in the 2. 5 and 5.0 mg/1 test concentrations (Table 5). One mortality was observed in the solvent control during the conduct of the study. The 0.6 mg/1 test concentration (Level 2) had 3 daphnids out of 40 show abnormal behaviour (1 mortality, 2 on bottom). These effects were not considered to be compound related since the surrounding levels (0.3 and 1.3 mg/l) had no adverse effects.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

24 Hour LC50 = 2.7 (95% confidence interval 1.9 - 4.6) Calculated uring the Binominal Method
48 Hour LC50 = 2.3 (95% confidence interval 1.9 - 4.6) Calculated uring the Binominal Method
The 48-hour dose response slope was calculated to be 5.4.
The 48-hour no-effect concentration was 1.0 mg/l which is based on the lack of mortality and/or abnormal effects.

Validity criteria fulfilled:

not specified

Conclusions:

48-Hour LC 50 = 2.3 mg/l
48-Hour No-Effect Concentration = 1.0 mg/l
48-Hour Dose-Response Slope = 5.4

Executive summary:

The definitive flow-through acute toxicity of MGK-264 to Daphnia magna was conducted from March 28, 1990 to March 30, 1990. The study was conducted at the following nominal concentrations of levels 1 to 5: 0.3, 0.6, 1.3, 2.5 and 5.0 mg/l. Forty first-instar daphnids were exposed to each of the test concentrations and controls. The mean measured concentrations calculated from analytical sampling at 0 and 48 hours are as follows: 0.34, 0.64, 1.0, 1.9 and 4.6 mg/l. The toxicity of MGK-264 based on mean measured test concentrations was as follows:

48-Hour LC 50 =2.3 mg/l

48-Hour No-Effect Concentration = 1.0 mg/l

48-HourDose-Response Slope = 5.4

 

Water quality parameters of temperature, dissolved oxygen and pH were measured in the control, solvent control and all test concentrations at 0- and 48-hours and were determined to be within acceptable limits. The overall ranges for these parameters were: daily diluter temperature observations = 20°C, pH = 7. 9-8 .1 and dissolved oxygen = 9.0-9.6 mg/1.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 May 2011 to 26 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1025 (Bivalve Acute Toxicity (shell deposition test))

Version / remarks:

Draft Version

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

One replicate solution of each treatment level and the dilution water control was sampled prior to the start of the definitive exposure and analyzed for MGK-264 concentration. Results of these pretest analyses were used to judge whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the oyster shell deposition exposure.

During the definitive study, water samples were removed from one replicate solution of each treatment level and control at each sampling interval. At exposure initiation, replicate A was sampled and at test termination, replicate B was sampled and analyzed for MGK-264 concentration.

Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations of MGK-264 approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.

All exposure solutions and QC samples were analyzed for MGK-264 using high performance liquid chromatography with ultraviolet detection (HPLC/UV) based on methodology validated at Smithers Viscient. The method validation study was conducted prior to the initiation of the definitive test and established an average recovery of 102% ± 1.67% from filtered seawater (Appendix 2). Defined limits for acceptance of QC sample recovery were set at 80.0% to 120%. The conditions and procedures used during this study were the same as those used in the method validation study.

Vehicle:

yes

Details on test solutions:

A 50 mg a.i./mL diluter stock solution was prepared prior to exposure initiation by adding approximately 18.34 g (17.50 g as active ingredient) of test substance to 350 mL of acetone (CAS No. 67-64-1). The stock solution was observed to be clear and colorless following preparation.
In addition, a 0.50 mL/mL solvent stock solution was prepared by bringing 50 mL of acetone to a final volume of 100 mL with reagent grade water.

Prior to test initiation, two 100-mL Glenco® gas tight syringes in conjunction with a Harvard Apparatus syringe pump were calibrated to deliver 0.03 mL/minute of the 50 mg a.i./mL diluter stock solution into the diluter's chemical mixing chamber which also received 0.300 L/minute of dilution water. The contents of the mixing chamber were continuously stirred, using a magnetic stirrer and Teflon®-coated stir bar and water-driven magnetic stir plate partially submerged in an ultrasonic water bath, to aid in the solubilization of the test substance. The concentration of MGK-264 in the solution contained within the mixing chamber was equivalent to that of the highest nominal test concentration (5.0 mg a.i./L), which was serially diluted (50%) to produce the remaining nominal test concentrations (0.31, 0.63, 1.3 and 2.5 mg a.i./L).

The same system was used to deliver 0.03 mL/minute of the 0.50 mL/mL solvent control stock solution into the diluter's solvent control mixing chamber which received 0.150 L/minute dilution water. The resulting solution in the mixing chamber contained the same amount of solvent present in the highest treatment level tested (0.10 mL/L). The dilution water control contained no solvent or test substance.
The operation of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check of the systems’ function was performed twice daily. In addition, analysis of the exposure solutions for test substance concentration was used to evaluate the proper operation of the diluter system. The exposure system was in proper operation for three days prior to test initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels.

Test organisms (species):

other aquatic mollusc: Eastern Oyster (Crassostrea virginica)

Details on test organisms:

Eastern oysters (Crassostrea virginica) were selected because of the availability of the species. The test organisms were obtained from Circle C Oysters, Ridge, Maryland. Oysters were transported to Smithers Viscient in an insulated foam container. Upon arrival, the oysters were carefully examined and assigned SMV Lot No. 11A65B. If any oyster appeared unsuitable for testing due to the presence of boring sponges and/or mudworms, it was discarded. In addition, several oysters were opened and carefully examined to confirm that no parasites were present, then discarded. Oysters were determined to be reproductively immature by pressing the tissue in the area where gametes are stored to confirm that none were present. The oysters were of similar age and had a mean valve height of 39 ± 3.6 mm (N = 30). Oysters were held in a polypropylene acclimation tray through which seawater (salinity 19 to 20%) flowed continuously at Smithers Viscient for 14 days prior to testing. During this acclimation period, the seawater in which the oysters were held had a temperature range of 19 to 23 ºC, a pH range of 7.2 to 8.2 and a dissolved oxygen concentration range of 85 to 100% of saturation. At their source, the oysters were collected from water which had a salinity of approximately 6‰. During the acclimation period at Smithers Viscient, the salinity was gradually increased from 10 to 20‰. During the culture period, oysters were fed a supplementary algal diet of Tetraselmus maculata prepared in seawater from a commercially available concentrate. No mortalities were observed among the oyster population during the seven days prior to test initiation and, therefore, the population was deemed acceptable for the test.
Prior to testing, 3 to 5 mm of the new peripheral shell growth of each oyster was removed by grinding the shell to a blunt edge using a fine-grit grinding wheel. Approximately ten oysters, in addition to the number required for testing, were prepared; any oysters which appeared less than optimal were discarded.

During the exposure, the oysters received supplemental feedings of algae (Tetraselmus maculata). Concentrated volumes of algal suspension (approximately 7.5 x 102 cells/mL) were added to each test aquarium three times daily to maintain an average concentration of approximately 105 cells/mL in the test solutions during the exposure.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

N/A

Hardness:

Not Measured

Test temperature:

20 degrees C

pH:

7.1 to 7.8

Dissolved oxygen:

5.1 to 7.9 mg/L

Salinity:

19 to 20%

Conductivity:

Not Measured

Nominal and measured concentrations:

Nominal Concentration (mg a.i./L) 0.31 mg/L; Measured mean concentration 0.077 mg/L
Nominal Concentration (mg a.i./L) 0.63 mg/L; Measured mean concentration 0.16 mg/L
Nominal Concentration (mg a.i./L) 1.3 mg/L; Measured mean concentration 0.29 mg/L
Nominal Concentration (mg a.i./L) 2.5 mg/L; Measured mean concentration 0.73 mg/L
Nominal Concentration (mg a.i./L) 5 mg/L; Measured mean concentration 1.3 mg/L

Details on test conditions:

The toxicity test was conducted using an exposure system consisting of a constant-flow diluter (Benoit et. al, 1982) with a dilution factor of 50% between concentrations, a temperature-controlled water bath and a set of 14 exposure aquaria. The test system was designed to provide five test concentrations of MGK-264, a dilution water control and a solvent control. All treatment levels and the controls were maintained in duplicate. The test aquaria were impartially positioned in a temperature-controlled water bath which was designed to maintain a test solution temperature of 20 ± 2 oC. The exposure vessels were maintained in an area illuminated with Sylvania Oktron fluorescent bulbs at a light intensity range of 31 to 62 footcandles (330 to 670 lux). Light intensity was measured with a VWR Traceable light meter. The photoperiod was the same as that of the culture area. Sudden transitions from light to dark and vice versa were avoided.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

>= 0.15 - <= 0.63 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Shell growth

Remarks on result:

other: The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L.

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

< 0.077 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Lack of sub-lethal response

Remarks on result:

other: The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Details on results:

No mortality or adverse effects were observed among oysters at any of the treatment levels tested or the controls. Individual shell growth measurements are presented in Table 3. Table 4 presents the mean shell growth of oysters at each treatment level. Following 96 hours of exposure, 26, 10, 33, 86 and 98% reduction in shell growth was observed among oysters exposed to the 0.077, 0.16, 0.29, 0.73 and 1.3 mg a.i./L treatment levels, respectively. Williams’ Multiple Comparison Test determined a statistically significant difference in shell growth among oysters exposed to all of the treatment levels tested (0.077, 0.16, 0.29, 0.73 and 1.3 mg a.i./L) compared to the dilution water control.

The concentration-response curve data is presented in Figure 2. The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

The concentration-response curve data is presented in Figure 2. The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Validity criteria fulfilled:

yes

Conclusions:

The concentration-response curve data is presented in Figure 2 of the attached report. The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Executive summary:

Growth among dilution water control and solvent control oysters at test termination both averaged 2.3 mm, respectively (pooled control = 2.3 mm, Table 3). The Standard Evaluation Procedures (U.S. EPA, 1985, Emended August 1990) for this study states that a minimum mean of 2.0 mm of new shell (based on the longest finger of new growth) must be deposited by the end of 96 hours. Based on these data, the amount of shell deposition observed during this study is considered representative for this species and acceptable for establishing the relative toxicity of MGK-264 to Eastern oysters.

Statistical analysis (t-test) determined no significant difference between control and solvent control growth data. The treatment data was compared to that of the dilution water control.

No mortality or adverse effects were observed among oysters at any of the treatment levels tested or the controls. Individual shell growth measurements are presented in Table 3. Table 4 presents the mean shell growth of oysters at each treatment level. Following 96 hours of exposure, 26, 10, 33, 86 and 98% reduction in shell growth was observed among oysters exposed to the 0.077, 0.16, 0.29, 0.73 and 1.3 mg a.i./L treatment levels, respectively. Williams’ Multiple Comparison Test determined a statistically significant difference in shell growth among oysters exposed to all of the treatment levels tested (0.077, 0.16, 0.29, 0.73 and 1.3 mg a.i./L) compared to the dilution water control.

The concentration-response curve data is presented in Figure 2. The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Since the primary intent of this testing is to establish an EC50 value, in consultation with the Study Sponsor, further testing to define a NOEC value was not performed.

Description of key information

Daphnia:

48-Hour LC 50 = 2.3 mg/l

48-Hour No-Effect Concentration = 1.0 mg/l

48-Hour Dose-Response Slope = 5.4

Oyster:

The 96-hour EC50 value was determined by linear interpolation to be 0.41 mg a.i./L, with 95% confidence intervals of 0.15 to 0.63 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be < 0.077 mg a.i./L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.3 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.41 mg/L

Additional information

Note: The EC50 of 0.41 mg/L for marine water invertebrates is not used in the environmental risk assessments as there will be no exposure of MKG 264 to the marine environment.