Dibenzylbenzene, ar-methyl derivative

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 18, 2021 to September 10, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 6, 14, 21, 28 and 32 (test termination) to determine concentrations of the test substance in the test chambers. Duplicate test water samples also were collected from each replicate test chamber in each treatment and control group at the beginning of the test and at Day 32 to serve as backups. All samples were collected at mid-depth in the test chambers and placed in glass vials and processed immediately for analysis or stored refrigerated until processed for analysis. The duplicate set of samples was stored under refrigeration as back-up samples for possible analysis.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

Stock solutions were prepared six times during the test. At each preparation, a primary stock solution was prepared in DMF at a nominal concentration of 1800 µg a.i./mL. The primary stock solutions were sonicated for ~10 minutes and then inverted at least twenty times to mix and appeared clear and colorless. Proportional dilutions of the primary stock were made in DMF to prepare additional secondary stock solutions at nominal concentrations of 17, 55, 175, and 550 µg a.i./mL. The secondary stock solutions were mixed by inversion and appeared clear and colorless. Stock solutions were stored under ambient conditions and fresh aliquots were placed in the syringe pumps daily during the test. The stock solutions were delivered to the diluter mixing chambers (at a target rate of 4.0 µL/minute) where they were mixed with dilution water (at a target rate of 200 mL/minute) to achieve the desired test concentrations of 0.34, 1.1, 3.5, 11 and 36 µg a.i./L. The solvent control was prepared by injecting HPLC-grade DMF into the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all dibenzyltoluene treatment groups was 20 µL/L.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnow embryos used in the test were obtained on spawning substrates from cultures maintained by the laboratory. Identification of the species was verified by the supplier of the original brood stock. The embryos were removed from the spawning substrates and examined under a dissecting microscope to select healthy, viable specimens at approximately the same stage of development (flat blastula to ¾ epiboly) (4,5). The brood fish cultured at the Easton site were acclimated for a minimum of 14 days prior to the collection of embryos for the test. The fish did not receive any treatment for diseases in the 14-day period prior to the test. There were <5% mortality of the population in the 7 days prior to the test. Embryos collected for use in the test were from 14 individual spawns and were <24 hours old when the test was initiated. To initiate the test, groups of 1 to 3 embryos were impartially distributed among incubation cups until each cup contained 20 embryos. One cup was placed in each treatment and control test chamber.
Newly hatched larvae were fed live brine shrimp nauplii (Artemia sp.) three times per day during the first seven days of post-hatch. Thereafter, they were fed live brine shrimp nauplii three times per day on weekdays and at least two times per day on weekends and holidays. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct, Ogden, Utah. The concentrations of selected pesticides and organic and inorganic constituents in the Artemia cysts are measured annually. Fish were not fed for approximately 24 hours prior to the termination of the test to allow for clearance of the digestive tracts before weight measurements were made. To ensure that the feeding rate per fish remained constant, rations were adjusted at least weekly to account for losses due to mortality.
Biomass loading at the end of the test, based on the mean wet weight of the negative control group, was 0.038 g of fish per liter of test solution that passed through the test chamber during a 24-hour period. Instantaneous loading (the total wet weight of fish per liter of water in the tank) at the end of the test was 0.39 g fish/L.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

The exposure period included a 4-day embryo hatching period, and a 28-day post-hatch juvenile growth period.

Hardness:

between 136 +/- 2.53 mg/L CaCO3 in the control
between 141 +/- 8.26 mg/L CaCO3 in the highest tested concentration

Test temperature:

25 +/- 1 °C

pH:

7.9 - 8.2

Dissolved oxygen:

Dissolved oxygen concentrations remained =61% of air saturation (5.0 mg/L).

Conductivity:

between 353 - 409 µS/cm

Nominal and measured concentrations:

nominal concentrations: 0.34, 1.1, 3.5, 11 and 36 µg a.i./L corresponding to the arithmetic mean measured test concentrations were calculated to be 0.22, 0.62, 1.9, 5.5 and 17 µg a.i./L, which represented 66, 56, 53, 50, and 48% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Details on test conditions:

TEST SYSTEM
A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC-grade dimethylformamide) control, and a negative (dilution water) control. Syringe pumps (Harvard Apparatus, Holliston, Massachusetts) were used to deliver the five test substance stock solutions and HPLC-grade dimethylformamide (DMF) for the solvent control into mixing chambers assigned to each treatment and the solvent control. The syringe pumps were calibrated prior to the test and verified at the end of the test. The stock solutions were diluted with well water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters, which were calibrated prior to test initiation and verified at approximately weekly intervals during the test. The flow of test water from each mixing chamber was split and allowed to flow into four replicate test chambers. The proportion of the test water that was split into each replicate was checked prior to the test and at approximately weekly intervals during the test to ensure that flow rates varied by no more than ± 10% of the mean for the four replicates. The diluter flow rate was adjusted to provide approximately 10 volume additions of test water in each test chamber per day. The general operation of the diluter was checked visually at least two times per day during the test and at least once at the end of the test. Periodically during the test, all organisms were transferred to clean test chambers to prevent the buildup of bacterial/fungal growth.
The test was conducted in a temperature-controlled environmental chamber designed to maintain the target test temperature throughout the test period. The test chambers were 9-L glass aquaria filled with approximately 7 L of test solution. The depth of the test water in a representative test chamber was approximately 15.4 cm. Test chambers were labeled with the study number, test concentration and replicate. Embryos were held in incubation cups constructed from glass cylinders approximately 50 mm in diameter with 425 µm nylon screen mesh attached to the bottom with silicone sealant. The cups were suspended in the water column of each test chamber and attached to a rocker arm. The reciprocating motion of the rocker arm (4 rpm) facilitated circulation of test water around the embryos during incubation.

TEST MEDIUM / WATER PARAMETERS
- The water used for organism holding and testing was freshwater obtained from a well approximately 40 meters deep located on the Eurofins-Easton site. The well water was passed through a sand filter to remove course particles, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.

OTHER TEST CONDITIONS
- Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at test initiation using a SPER Scientific Model 840006 light meter.
- The target test temperature during the test was 25 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, weekly during the test, and at the end of the test using a digital thermometer. Temperature also was monitored continuously in one negative control replicate using a validated environmental monitoring system (PointView Central Monitoring System), which was calibrated prior to exposure initiation and verified or calibrated approximately weekly during the test with a digital thermometer.
- Dissolved oxygen and pH were measured in alternating replicates of each treatment and control group at the beginning of the test, weekly during the test, and at the end of the test. Measurements of dissolved oxygen were made using a Thermo Orion Star A213 bench top RDO/DO meter and pH was measured using a Thermo Orion DUAL STAR pH/ISE meter.
- Hardness, alkalinity and specific conductance were measured in alternating replicates of the negative control (dilution water) and the highest concentration treatment group at the beginning of the test, weekly during the test and at the end of the test. Hardness and alkalinity were measured by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (6). Specific conductance was measured using Thermo Orion Star A122 portable conductivity meter.

RANGE-FINDING STUDY
In the range-finding study, fathead minnow embryos were exposed to a geometric series of five test concentrations, a negative control and a solvent control under flow-through conditions. The exposure period included a 4-day embryo hatching period, and a 14-day post-hatch juvenile growth period. The nominal test concentrations were 0.036, 0.36, 3.6, 18, and 36 µg a.i./L. Results for hatching success, post-hatch survival, overall survival, and wet weight are summarized in 'Biological detailed results'. Based on these results nominal test concentrations of 0.34, 1.1, 3.5, 11 and 36 µg a.i./L were chosen for the definitive test.

BIOLOGICAL OBSERVATIONS AND MEASUREMENTS:
- Observations of embryo mortality and the removal of dead embryos and/or embryos with fungus was performed daily until hatching was complete. Viable embryos started to hatch on Days 3 to 5 of the test and all viable embryos hatched or died by Day 5 of the test. When hatching reached >90% in the negative control group on Day 4 of the test, the larvae were released to their respective test chambers and the post-hatch period began. Any unhatched embryos were kept in the egg cups until they hatched and were released into the test chamber, or until death of the embryo occurred. During the 28-day post-hatch exposure period, the larvae were observed daily to evaluate the numbers of mortalities and the numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior. From these observations, time to hatch, hatching success, and post-hatch growth and survival were evaluated. Hatching success was calculated as the percentage of embryos that hatched successfully. Post-hatch survival was calculated as the number of larvae surviving to test termination divided by the total number of embryos that hatched successfully.
- Post-hatch growth of the fathead minnows was evaluated at the conclusion of the 28-day post-hatch exposure period. Total length for each surviving fish was measured to the nearest 1 mm using a metric ruler, and wet and dry weights were measured to the nearest 0.1 mg using an analytical balance. Fish were placed in an oven at approximately 60°C for approximately 45 hours to obtain dry weight data.

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks on result:

other: Dry weight - compared to solvent control

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

weight

Remarks on result:

other: Wet weight

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

length

Remarks on result:

other: Compared to solvent control

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

larval development

Remarks on result:

other: Larval survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

larval development

Remarks on result:

other: post hatch larval survival

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

time to hatch

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

17 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: hatching success

Remarks on result:

other: compared to solvent control

Details on results:

TIME TO HATCH :
Time to hatch of the fathead minnow embryos exposed to dibenzyltoluene is summarized in Table 5, and individual replicate data are presented in attached document 'Biological detailed results'. Percent inhibition in time to hatch as compared to the pooled control is presented in Table 8. Fathead minnow embryos began hatching on Day 3 of the test. The majority of fathead minnow embryos in the control and treatment replicates hatched on Day 4 of the test. Hatching reached >90% in the negative control groups on Day 4 of the test, at which time the larvae were released to their respective test chambers.
The mean time to hatch in the negative and solvent control groups was 4.0 days. Since there was not a statistically significant difference in mean time to hatch between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons among the treatment groups. The mean time to hatch in the pooled control and the 0.22, 0.62, 1.9, 5.5, and 17 µg a.i./L treatment groups was 4.0, 4.0, 4.1, 4.0, 4.2, and 4.0 days, respectively. According to Dunnett’s one-tailed test, there were no statistically significant differences in any treatment group in comparison to the pooled control group (p > 0.05). Consequently, the NOEC and LOEC for time to hatch were determined to be 17 µg a.i./L and >17 µg a.i./L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the IC10 and IC20 values for mean time to hatch were empirically estimated to be greater than the highest concentration tested.

HATCHING SUCCESS :
Hatching success of fathead minnow embryos exposed to dibenzyltoluene is summarized in Table 5, and individual replicate data are presented in attached document 'Biological detailed results'. Percent inhibition in hatching success as compared to the solvent control group is presented in Table 8.
Hatching success in the negative and solvent control groups was 98.8 and 90.0%, respectively. The negative control and solvent control were found to be statistically significantly different (p = 0.05); therefore, the treatment group data were compared to the solvent control. Hatching success in the 0.22, 0.62, 1.9, 5.5 and 17 µg a.i./L treatment groups was 91.3, 65.0, 98.8, 93.8 and 100%, respectively. Fisher’s Exact test indicated that there was a statistically significant decrease in hatching success in the 0.62 µg a.i./L treatment group in comparison to the solvent control (p = 0.05). However, this was not considered to be treatment-related, since the decrease did not follow a dose-responsive pattern.
Consequently, the NOEC for hatching success was 17 µg a.i./L and the LOEC was >17 µg a.i./L. The LC10 and LC20 values for hatching success could not be estimated, since the calculated LCx values were extrapolated beyond the data range used in the calculation and/or the 95% confidence intervals were overly wide.

POST-HATCH AND OVERALL SURVIVAL :
Survival of the fathead minnow larvae exposed to dibenzyltoluene through Day 28 post-hatch is summarized in Table 5, and individual replicate data are presented in attached document 'Biological detailed results'. Percent inhibition in survival as compared to the pooled control groups is presented in Table 8.
At test termination, post-hatch larval survival in the negative and solvent control groups was 88.6 and 87.5%, respectively, while overall survival in the negative and solvent control groups was 87.5 and 78.8%, respectively. Since there were no statistically significant differences between the negative and solvent control groups for either survival endpoint (p > 0.05), the control data were pooled for comparisons with the treatment groups. Post-hatch larval survival in the pooled control and the 0.22, 0.62, 1.9, 5.5, and 17 µg a.i./L treatment groups was 88.1, 94.5, 82.7, 89.9, 85.3, and 93.7%, respectively, while overall survival at test termination in the pooled control and the 0.22, 0.62, 1.9, 5.5, and 17 µg a.i./L treatment groups was 83.1, 86.3, 53.8, 88.8, 80.0, and 93.7%, respectively. According to Fisher’s Exact test, there were no statistically significant effects in any of the treatment groups in comparison to the pooled control for post-hatch survival (p > 0.05). However, Fisher’s Exact test indicated that there was a statistically significant decrease in overall survival in the 0.62 µg a.i./L treatment group in comparison to the pooled control (p = 0.05), but this was not considered to be treatment-related, since the decrease did not follow a dose-responsive pattern.
Consequently, the NOEC and LOEC for both post-hatch survival and overall survival were determined to be 17 µg a.i./L and >17 µg a.i./L, respectively. Since there was less than 10% inhibition in any treatment group in comparison to the pooled control, the LC10 and LC20 values for post-hatch larval survival were empirically estimated to be greater than the highest concentration tested. The LC10 and LC20 values for overall survival were not reportable, since the estimated LCx values were extrapolated beyond the range of the test concentrations and/or the 95% confidence intervals were overly wide.

BIOLOGICAL OBSERVATIONS :
Biological observations of sublethal effects during the 28-day post-hatch period were performed daily. Observations made during on days in which organisms were transferred to clean test chambers are presented in attached document 'Biological detailed results'. In general, the majority of the fish in the control groups and in all treatment groups appeared normal throughout the test. Observations of unusual behavior or appearance included appearing small, weak, morphologically deformed, lying on the bottom of the test chamber with little motion other than minor gill movement, having their jaw locked in open position, having a swollen abdomen, having their head stuck in eggshell, missing their bottom half of caudal fin, and having a left eye being smaller than the right. However, these observations were not present in the majority of the population and did not follow a dose-responsive pattern, and therefore, were not considered treatment-related.

GROWTH :
Growth measurements at the end of the 28-day post-hatch period are summarized in Table 6, and the individual replicate data for total length, wet weight and dry weight are presented in attached document 'detailed results'. Percent inhibition in growth as compared to the appropriate control group is presented in Table 8.
- Total Length
Mean total length at test termination in the negative and solvent control groups was 25.1 and 24.7 mm, respectively. The negative control and solvent control were found to be statistically significantly different (p = 0.05); therefore, the treatment group data were compared to the solvent control. Mean total length at test termination in the 0.22, 0.62, 1.9, 5.5, and 17 µg a.i./L treatment groups was 24.7, 25.2, 24.6, 24.3. and 24.0 mm, respectively. A statistically significant concentration-related decreasing trend in the 17 µg a.i./L treatment group was indicated according to the one-sided Jonckheere-Terpstra step-down trend test when the solvent control data was included in the calculation (p = 0.05).
Consequently, the NOEC and LOEC for total length were determined to be 5.5 µg a.i./L and 17 µg a.i./L, respectively. Since there was a less than a 10% inhibition for total length between the treatment group means and the solvent control mean, the IC10 and IC20 values were empirically estimated to be greater than the highest treatment group.
- Wet Weight
Mean wet weight at test termination in the negative and solvent control groups was 139 and 136 mg, respectively. There were no statistically significant differences in mean wet weight between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Mean wet weight at test termination in the pooled control and the 0.22, 0.62, 1.9, 5.5, and 17 µg a.i./L treatment groups was 137, 137, 147, 128, 136, and 117 mg, respectively. According to the one-sided Jonckheere-Terpstra step-down trend test, a statistically significant decreasing trend was evident in the wet weight data for the 17 µg a.i./L treatment group when the pooled control was included in the calculation (p < 0.05).
Consequently, the NOEC and LOEC for wet weight were determined to be 5.5 µg a.i./L and 17 µg a.i./L, respectively. The IC10 for wet weight was calculated to be 11 µg a.i./L, with a 95% confidence interval of 6.7 to 20 µg a.i./L. The IC20 value for wet weight was could not be estimated, since the calculated IC20 value was extrapolated beyond the data range used in the calculation and/or the 95% confidence intervals were overly wide.
- Dry Weight
Mean dry weight at test termination in the negative and solvent control groups was 32.2 and 30.0 mg, respectively. The negative control and solvent control were found to be statistically significantly different (p = 0.05); therefore, the treatment group data were compared to the solvent control. Mean dry weight at test termination in the 0.22, 0.62, 19, 5.5, and 17 µg a.i./L treatment groups was 30.0, 30.1, 26.9, 29.4, and 25.6 mg, respectively. According to Dunnett’s one-tailed test, there was a statistically significant decrease in the 1.9 and 17 µg a.i./L treatment groups in comparison to the solvent control group (p = 0.05). However, the decrease in the 1.9 µg a.i./L treatment group was not dose-responsive and was not considered to be treatment-related.
Consequently, the NOEC and LOEC for dry weight were determined to be 5.5 µg a.i./L and 17 µg a.i./L, respectively. The IC10 and IC20 values for dry weight could not be estimated, since the calculated ICx values were extrapolated beyond the data range used in the calculation and/or the 95% confidence intervals were overly wide.

MEASUREMENT OF TEST CONCENTRATIONS
Nominal concentrations selected for use in the study were 0.34, 1.1, 3.5, 11 and 36 µg a.i./L. During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
Since the highest nominal test concentration was above the reported solubility of the test substance in water, selected water samples collected during the pretest intervals were analyzed as centrifuged and non-centrifuged for comparison (Table 1). Measured dibenzyltoluene concentrations in centrifuged samples were lower than non-centrifuged samples. Although no precipitate was observed at any point in mixing or test chambers throughout the equilibration phase or during the test, all samples during the test were centrifuged prior to analysis as a conservative precaution.
The measured concentrations of samples collected to verify the diluter system prior to the test ranged from
PHYSICAL AND CHEMICAL MEASUREMENTS OF WATER
Measurements of temperature, dissolved oxygen and pH in the test chambers are presented in attached document 'physico-chemicals parameters'. Water temperatures were within the 25 ± 1°C range established for the test. Dissolved oxygen concentrations remained =61% of air saturation (5.0 mg/L). Measurements of pH ranged from 7.9 to 8.2 during the test. Weekly measurements of specific conductance, hardness and alkalinity in the negative control water and in the highest test concentration are presented in attached document 'physico-chemicals parameters'. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by dibenzyltoluene concentration. Light intensity at test initiation was 695 lux at the surface of the water of one representative test chamber.

VALIDITY CRITERIA OF THE STUDY :
The following criteria were used to judge the validity of the test and were met:
1) The dissolved oxygen concentration should be > 60% of the air saturation value throughout the test. The dissolved oxygen concentration remained = 61% (5.0 mg/L) of the air saturation value throughout the test.
2) The water temperature measurements will not differ by more than ±1.5ºC between test chambers or between successive days at any time during the test, and should be within the 25 ± 1°C range specified for the test species. The water temperature did not differ by more than ±1.5ºC between test chambers or between successive days at any time during the test, and temperature measured in the test chambers throughout the test ranged from 24.8 to 26.2°C.
3) Evidence will be available to demonstrate that the concentrations of the test substance in solution have been satisfactorily maintained within ±20% of the mean measured value. The coefficients of variation for all treatment groups ranged from 5.1 to 12.7% of the mean measured concentrations.
4) The percent hatching success of fertilized eggs in the control group will be >70%, and the minimum percent post-hatch larval survival will be 75%. Percent hatching success in the negative and solvent control groups were 98.8 and 90.0%, respectively. At test termination, post-hatch larval survival in the negative and solvent control groups was 88.6 and 87.5%, respectively.

Table 5 Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed to Dibenzyltoluene

Mean Measured Concentration (μg a.i./L)	Number Exposed	Total Number Hatched	Hatching Success (%)	Mean Time to Hatch ± Std. Dev. (Days)^1	Number Surviving to Termination	Post-Hatch Survival (%)^1	Overall Survival (%)
Negative Control	80	79	98.8	4.0 ± 0.05	70	88.6	87.5
Solvent Control	80	72	90.0	4.0 ± 0.03	63	87.5	78.8
Pooled Control	160	151	--^3	4.0 ± 0.05	133	88.1	83.1
0.22	80	73	91.3	4.0 ± 0.03	69	94.5	86.3
0.62	80	52	65.0^♦2	4.1 ± 0.13	43	82.7	53.8 ^*2
1.9	80	79	98.8	4.0 ± 0.04	71	89.9	88.8
5.5	80	75	93.8	4.2 ± 0.20	64	85.3	80.0
17	80	80	100.0	4.0 ± 0.12	74^4	93.7	93.7

^* Statistically significant decrease in comparison to the pooled control, according to Fisher’s Exact test (p ≤ 0.05). ^♦ Statistically significant decrease in comparison to the solvent control, according to Fisher’s Exact test (p ≤ 0.05). ^1 There were no statistically significant decreases evident in any treatment group in comparison to the pooled control, according to Dunnett’s one-tailed test (p > 0.05). ^2 Not considered to be treatment-related, since the decrease did not follow a dose-responsive pattern. ^3 The negative control and solvent control were found to be statistically different (p ≤ 0.05); therefore, the treatment group data were compared to the solvent control. ^4 One fish in the 17 μg a.i./L treatment group was inadvertently killed during transfer to clean test chamber; this fish will be excluded from survival calculations.

 

Table 6 Summary of Larval Growth of Fathead Minnows Exposed to Dibenzyltoluene

Mean Measured Concentration (μg a.i./L)	Mean Total Length ± Std. Dev. (mm)	Mean Wet Weight ± Std. Dev. (mg)	Mean Dry Weight ± Std. Dev.^1 (mg)
Negative Control	25.1 ± 0.16	139 ± 6.7	32.2 ± 1.40
Solvent Control	24.7 ± 0.25	136 ± 3.0	30.0 ± 0.56
Pooled Control	--^1	137 ± 5.0	--^1
0.22	24.7± 0.12	137 ± 3.6	30.0 ± 0.67
0.62	25.2 ± 0.43	147 ± 6.0	30.1 ± 1.36
1.9	24.6 ± 0.60	128 ± 6.6	26.9 ± 2.21 ^¿2
5.5	24.3 ± 0.89	136 ± 4.7	29.4 ± 0.58
17	24.0± 0.09 ^¦	117 ± 9.3 ^+	25.6 ± 1.52^¿
^¦ A statistically significant concentration-related decreasing trend was evident in the data according to the one-sided Jonckheere-Terpstra step-down trend test (p ≤ 0.05) when the solvent control data was included in the calculation. ^+ A statistically significant concentration-related decreasing trend was evident in the data according to the one-sided Jonckheere-Terpstra step-down trend test (p ≤ 0.05) when the pooled control data was included in the calculation. ^¿ Statistically significant decrease in comparison to the solvent control , according to Dunnett’s one-tailed test (p ≤ 0.05). ^1 The negative control and solvent control were found to be statistically different (p ≤ 0.05); therefore, the treatment group data were compared to the solvent control only. ^2 Not considered to be treatment-related, since the decrease did not follow a dose-responsive pattern.

 

Table 7 10% and 20% Inhibition Concentrations and 95% Confidence Intervals

Endpoint	LC/IC10 (95% CI) (μg a.i./L)	LC/IC20 (95% CI) (μg a.i./L)	NOEC (μg a.i./L)		LOEC (μg a.i./L)
Hatching Success (compared to solvent control)	NC	NC	17		>17
Time to Hatch	>17^1	>17^1	17		>17
Post-Hatch Larval Survival	>17^1	>17^1	17		>17
Overall Larval Survival	NC	NC	17		>17
Total Length (compared to solvent control)	>17^2	>17^2	5.5		17
Wet Weight	11 (6.7 – 20)	NC	5.5		17
Dry Weight (compared to solvent control)	NC	NC	5.5		17
No-Observed-Effect Concentration:				5.5 μg a.i./L
Lowest-Observed-Effect-Concentration:				17 μg a.i./L

^1 Empirically estimated to be greater than the highest test concentration, since there was less than 10% inhibition in any treatment group in comparison to the pooled control. ^2 Empirically estimated to be greater than the highest test concentration, since there was less than 10% inhibition in any treatment group in comparison to the solvent control. CI Confidence Interval NC Not calculable, since the LC/ICx value was extrapolated beyond the range of the test concentrations and/or the 95% confidence interval was overly wide.

 

Table 8 Percent Inhibition from the Solvent Control or Pooled Control

Percent Inhibition (%) Compared to the Control Group^1
Mean measured Concentration (µg a.i./L)	Hatching Success	Mean Time to Hatch	Post-Hatch Larval Survival	Overall Survival	Mean Total Length		Mean Wet Weight	Mean Dry Weight
	SC	PC	PC	PC	SC		PC	SC
0.22	-1.4	-0.3	-7.3	-3.8		0.0	0.0	0.0
0.62	27.8	-1.2	6.1	35.3		-2.0	-7.3	-0.4
1.9	-9.7	0.3	-2.0	-6.8		0.4	6.6	10.3
5.5	-4.2	-3.5	3.1	3.8		1.6	0.7	2.2
17	-11.1	0.9	-5.0	-11.3		2.8	14.6	14.6

¹ Stimulation or a greater response in the test substance treatment than the control is reported as a negative percent inhibition. Calculated using Excel 2016; manual calculations may differ slightly.

PC = compared to pooled control; SC = compared to solvent control.

Validity criteria fulfilled:

yes

Conclusions:

Fathead minnows (Pimephales promelas) were exposed to dibenzyltoluene at mean measured concentrations of 0.22, 0.62, 1.9, 5.5 and 17 µg a.i./L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period). There were no treatment-related effects on hatching success, mean time to hatch, overall survival, or post-hatch survival at concentrations =17 µg a.i./L. There were statistically significant effects on hatching success and overall survival in the 0.62 µg a.i./L treatment group, but these were not considered to be treatment-related. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to dibenzyltoluene at a concentration of 17 µg a.i./L had statistically significant reductions in total length, wet weight and dry weight in comparison to the appropriate controls. Consequently, the overall NOEC and LOEC for the study were determined to be 5.5 and 17 µg a.i./L.

Executive summary:

The objective of this study was to determine the effects of dibenzyltoluene on the time to hatch, hatching success, survival, and growth of fathead minnows, Pimephales promelas, under flow-through conditions for 32 days during early life-stage development (4-Day Hatch and 28-Day Post-Hatch).

 

Test concentrations:

Nominal	Mean Measured
Negative Control Solvent Control  0.34 μg a.i./L  1.1 μg a.i./L  3.5 μg a.i./L  11 μg a.i./L  36 μg a.i./L	<LOQ <LOQ 0.22 μg a.i./L 0.62 μg a.i./L 1.9 μg a.i./L 5.5 μg a.i./L 17 μg a.i./L

 

Summary of the LC10 - LC20 - NOEC and LOEC values:

Endpoint	LC/IC10 (95% CI) (μg a.i./L)	LC/IC20 (95% CI) (μg a.i./L)	NOEC (μg a.i./L)		LOEC (μg a.i./L)
Hatching Success (compared to solvent control)	NC	NC	17		>17
Time to Hatch	>17^1	>17^1	17		>17
Post-Hatch Larval Survival	>17^1	>17^1	17		>17
Overall Larval Survival	NC	NC	17		>17
Total Length (compared to solvent control)	>17^2	>17^2	5.5		17
Wet Weight	11 (6.7 – 20)	NC	5.5		17
Dry Weight (compared to solvent control)	NC	NC	5.5		17
No-Observed-Effect Concentration:				5.5 μg a.i./L
Lowest-Observed-Effect-Concentration:				17 μg a.i./L

^1 Empirically estimated to be greater than the highest test concentration, since there was less than 10% inhibition in any treatment group in comparison to the pooled control. ^2 Empirically estimated to be greater than the highest test concentration, since there was less than 10% inhibition in any treatment group in comparison to the solvent control. CI Confidence Interval NC Not calculable, since the LC/ICx value was extrapolated beyond the range of the test concentrations and/or the 95% confidence interval was overly wide.

Description of key information

Fathead minnows (Pimephales promelas) were exposed to dibenzyltoluene at mean measured concentrations of 0.22, 0.62, 1.9, 5.5 and 17 µg a.i./L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period). There were no treatment-related effects on hatching success, mean time to hatch, overall survival, or post-hatch survival at concentrations =17 µg a.i./L. There were statistically significant effects on hatching success and overall survival in the 0.62 µg a.i./L treatment group, but these were not considered to be treatment-related. Growth, measured as total length, wet and dry weight, was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to dibenzyltoluene at a concentration of 17 µg a.i./L had statistically significant reductions in total length, wet weight and dry weight in comparison to the appropriate controls. Consequently, the overall NOEC and LOEC for the study were determined to be 5.5 and 17 µg a.i./L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

5.5 µg/L