Bis(dibutyldithiocarbamato-S,S')copper

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:

2019

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)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical Sampling
Water samples were collected from one test chamber of each treatment and control group four, three, two and one day prior to test initiation to confirm concentrations after conditioning the diluter system for three, four, five and six days, respectively. Test solution samples were collected from one replicate test chamber in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. Additional samples were collected on Days 14, 15, 21, 22, 28, 29 and 32 at the Study Director’s request. Sampling alternated between the replicate test chambers in each group at each sampling interval. Test solution samples (10.0 mL) were collected from mid-depth of the test solutions, were placed in glass scintillation vials and were processed immediately for LSC analysis or were held refrigerated for possible future analysis.
Additional samples were collected from the 0.13 and 2.0 µg/L treatment groups at the beginning of the test, on Day 14 of the test and at the end of the test to measure concentrations of the non-radiolabelled test substance. Sampling alternated between the replicate test chambers in the 0.13 and 2.0 µg/L treatment groups at each sampling interval. Test solution samples (10.0 mL) were collected from mid-depth of the test solutions, were placed in glass scintillation vials containing 10.0 mL of methanol and were processed immediately for LC/MS/MS analysis.

Vehicle:

yes

Remarks:

Methanol

Details on test solutions:

* Preparation of Test Concentrations
Stock solutions of 14C-CDBC were prepared in methanol at a nominal concentration of 50.0 mg/L weekly during the test. Before measurement, the radiolabeled test substance was removed from frozen storage and allowed to equilibrate to room temperature. A 100-mL primary stock solution was prepared by weighing out 0.00500 g of the radiolabeled test substance. The radiolabeled test substance was then transferred to a 100 mL glass volumetric flask and brought to volume with methanol. The primary stock solutions were sonicated and then mixed by inversion and ranged in appearance from clear and brown to clear and brownish-yellow.

Stock solutions of 12C-CDBC were also prepared weekly during the study by dissolving non radiolabelled CDBC in methanol at a nominal concentration of 1.0 mg/mL. A 20-mL primary stock solution was prepared by weighing out 0.0200 g of the non-radiolabelled test substance. The non-radiolabeled test substance was then rinsed into a 20 mL glass volumetric flask and brought to volume with methanol. The primary stock solutions were sonicated for approximately 15 minutes and then mixed by inversion. The primary stock solutions appeared clear and black.

Dispensing stock solutions at nominal concentrations of 0.0013, 0.0025, 0.0050, 0.010 and 0.020 mg/mL were prepared in methanol using calculated volumes of the radiolabelled and non-radiolabelled stock solutions to achieve nominal radioactivities of 1.92 x 105, 1.00 x 105, 5.00 x 104, 2.50 x 104 and 1.25 x 104 dpm/µg, respectively in the stock solutions. The dispensing stock solutions were prepared on Day -7, -1, 6, 13, 20 and 27. The five dispensing stock solutions were injected into the diluter mixing chambers (at a rate of 20.0 µL/minute) where they were mixed with well water (at a rate of 200 mL/minute) to achieve the targeted test concentrations. The solvent control was prepared by injecting methanol into the mixing chamber, using the same target rates as mentioned above, for the solvent control. The concentration of methanol in the solvent control and all 14C-CDBC treatment groups was 0.1 mL/L.

The concentrations of the test were selected in consultation with the Sponsor and were based on the solubility of the test substance in water. The highest concentration selected, 2 µg/L, was selected based on preliminary solubility work conducted at Eurofins-Easton in dilution water.

* Dilution Water
The water used for 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 particles greater than approximately 25 ¿m 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 ¿m to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) of the well water during the approximate four-week period immediately preceding the test were measured.

Test organisms (species):

Pimephales promelas

Details on test organisms:

* Test Organism
The fathead minnow, Pimephales promelas, was selected as the test species for this study. Fathead minnows are one of the preferred fish species to test the toxicity of test substances during the early life-stages of fish. This species was selected for use in the test based upon past use and ease of handling in the laboratory. Fathead minnow embryos used in the test were obtained from cultures maintained by Eurofins Easton. Identification of the species was verified by the supplier of the original culture. The brood fish cultured at Eurofins were acclimated for at least 14 days prior to the collection of embryos for the test. During the 7 days immediately preceding the test, the brood fish used for the test showed no signs of disease or stress and there was no mortality.

On the morning of study initiation, 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. Embryos collected for use in the test were from 22 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. Brine shrimp nauplii were obtained by hatching cysts purchased from Brine Shrimp Direct of Ogden, Utah. The concentrations of selected pesticides and organic and inorganic constituents in the Artemia cysts are measured annually and the results from the most recent analysis are presented in Appendix 3. Fish were not fed for at least 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 0.0238 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 0.245 g fish/L.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Remarks on exposure duration:

32 Days (4-Day Hatch and 28-Day Post-Hatch)

Hardness:

136 - 144 mg/L as CaCO3

Test temperature:

24.7 - 25.0 °C

pH:

7.9 - 8.1

Dissolved oxygen:

7.5 - 8.2 mg/L

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

nominal concentrations: 0.13 µg/L, 0.25 µg/L, 0.50 µg/L, 1.0 µg/L, 2.0 µg/L (The highest concentration was selected based on preliminary solubility work n in dilution water)
measured concentrations: 0.14 µg/L, 0.23 µg/L, 0.44 µg/L, 1.0 µg/L, 1.6 µg/L

Details on test conditions:

* Test Apparatus
A continuous-flow diluter was used to deliver each concentration of the test substance, a solvent (HPLC grade methanol) 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 methanol 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 16 cm. Test chambers were labeled with the study number, test concentration and replicate designation. 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.
* Environmental 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. Water temperature also was monitored continuously during the test in one negative control test chamber using a validated environmental monitoring system (Pointview Real-time Data Reporting System). The system measurements were calibrated prior to exposure initiation and verified or recalibrated 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 Scientific Orion Star A213 Benchtop RDO/DO meter and pH was measured using a Thermo Scientific 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. Specific conductance was measured using a Thermo Scientific Orion Star A122 Portable Conductivity meter.

Reference substance (positive control):

no

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

time to hatch

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

weight

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

1.6 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

length

Details on results:

* RESULTS
Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 0.13, 0.25, 0.50, 1.0 and 2.0 µg/L. The concentrations were selected in consultation with the Sponsor and were based on the solubility of the test substance in water. The highest concentration selected, 2 µg/L, was selected based on preliminary solubility work in dilution water.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 test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution. The test solutions in the 0.13, 0.25 and 0.50 µg/L mixing chambers appeared clear and colorless during the test, with no evidence of precipitation observed. At test initiation, the test solutions in the 1.0 and 2.0 µg/L mixing chambers were clear and colorless. However, the toxicant delivery lines in the 1.0 and 2.0 µg/L mixing chambers had brown material on them and the sides of the mixing chambers were brown where the delivery lines were touching. At test termination, the test solutions in the 1.0 and 2.0 µg/L treatment groups mixing chambers were clear and colorless but the ends of the toxicant delivery lines were stained brown.

The measured concentrations by LSC of samples collected to verify the diluter system prior to the test ranged from 54.0 to 122% of nominal concentrations. Samples of the test solution samples collected during the test had measured concentrations by LSC that ranged from 63.2 to 161% of nominal concentrations. Additional samples were collected from various treatment groups on Days 14, 15, 21, 22, 28, 29 and 32 when unusually high or low recoveries were measured. The mean from all analyzed samples from the respective treatment groups on those days were calculated for each interval and used to calculate the mean measured concentrations. When the measured concentrations by LSC of test solution samples collected during the test were averaged, the mean measured test concentrations were 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L, which represented 104, 93, 87, 100 and 82% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations by LSC.

Samples of the test solution samples from the 0.14 and 1.6 µg/L treatment groups collected during the test had measured concentrations by LC/MS/MS that ranged from
Physical and Chemical Measurements of Water
Water temperatures were within the 25 ± 1°C range established for the test. Dissolved oxygen concentrations remained = 91% of air saturation (7.5 mg/L). Measurements of pH ranged from 7.9 to 8.2 during the test. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by CDBC concentration. Light intensity at test initiation was 602 lux at the surface of the water of one representative test chamber.

Time to Hatch
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 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 3.96 and 4.07 days, respectively. There were no statistically significant differences in mean time to hatch between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. The mean time to hatch in the pooled control and the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was study day 4.01, 4.01, 4.01, 4.08, 4.07 and 4.16, respectively. According to Dunnett’s test, there was a statistically significant increase in the 1.6 µg/L treatment groups in comparison to the pooled control group (p = 0.05). However, this increase was very slight (3.7%) in comparison to the pooled control and was not considered to be biologically meaningful. Therefore, the NOEC and LOEC for time to hatch were determined to be 1.6 µg/L and > 1.6 µg/L, respectively.

Hatching Success
Hatching success in the negative and solvent control groups was 97.5 and 96.3%, respectively. There were no statistically significant differences in hatching success between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Hatching success in the pooled control and the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 96.9, 98.8, 93.8, 96.3, 96.3 and 96.3, respectively. According to the Jonckheere Terpstra step-down trend test, a statistically significant decreasing trend was not evident in the hatching success data (p > 0.05). Consequently, the NOEC and LOEC for hatching success were determined to be 1.6 µg/L and > 1.6 µg/L, respectively. Since there was a less than a 10% reduction in hatching success between the treatment group means and the pooled control mean, the LC10 and LC20 values were empirically estimated to be greater than the highest treatment group.

Post-Hatch and Overall Survival
At test termination, post-hatch larval survival in the negative and solvent control groups was 92.4 and 97.4%, respectively. There were no statistically significant differences in post-hatch larval survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Post-hatch larval survival in the pooled control and the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 94.9, 92.3, 98.7, 97.4, 94.4 and 89.6%, respectively. Overall survival in the negative and solvent control groups was 90.0 and 93.8%, respectively. There were no statistically significant differences in overall survival between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Overall survival at test termination in the pooled control and the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 91.9, 91.3, 92.5, 93.8, 91.3 and 86.3%, respectively. According to the Jonckheere Terpstra step-down trend test, a statistically significant decreasing trend was not evident in either the post-hatch survival data or overall survival data (p > 0.05). Consequently, the NOEC and LOEC for both post-hatch survival and overall survival were determined to be 1.6 µg/L and > 1.6 µg/L, respectively. Since there was a less than a 10% reduction in post-hatch and overall survival between the treatment group means and the pooled control mean, the LC10 and LC20 values were empirically estimated to be greater than the highest treatment group.

Biological Observations
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, discolored (pale), morphologically deformed (e.g. crooked spine, curled, protrusion on side of abdomen, and swollen abdomen) and lying on the bottom of the tank with little motion other than minor gill movement. However, these observations were infrequent, did not follow a dose-responsive pattern, and were comparable in the controls.

Total Length
Mean total length at test termination in the negative and solvent control groups was 22.6 and 22.4 mm, respectively. There were no statistically significant differences in mean total length between the negative and solvent control groups (p > 0.05). Therefore, the control data were pooled for comparisons with the treatment groups. Mean total length at test termination in the pooled control and the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 22.5, 22.1, 21.8, 22.0, 21.8 and 22.3 mm, respectively. According to Dunnett’s test, there were statistically significant decreases in the 0.14, 0.23, 0.44 and 1.0 µg/L treatment groups in comparison to the pooled control group (p = 0.05). However, the decreases in the 0.14, 0.23, 0.44 and 1.0 µg/L treatment groups were not considered to be treatment-related since the decrease was not dose responsive. Consequently, the NOEC and LOEC for total length were determined to be 1.6 µg/L and > 1.6 µg/L, respectively. Since there was a less than a 10% inhibition for total length between the treatment group means and the pooled 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 85.8 and 79.9 mg, respectively. There was a statistically significant difference between the negative and solvent control groups (p = 0.05), therefore, the treatment groups were compared to the solvent control group for analysis. Mean wet weight at test termination in the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 79.0, 77.2, 77.1, 72.2 and 79.9 mg, respectively. According to Dunnett’s test, there was a statistically significant decrease in the 1.0 µg/L treatment group in comparison to the solvent control group (p = 0.05). However, the decrease was not considered to be treatment-related since the decrease was not dose-responsive. Consequently, the NOEC and LOEC for wet weight were determined to be 1.6 µg/L and > 1.6 µg/L, respectively. Since there was a less than a 10% inhibition for wet weight 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.

Dry Weight
Mean dry weight at test termination in the negative and solvent control groups was 17.6 and 16.5 mg, respectively. There was a statistically significant difference between the negative and solvent control groups (p = 0.05), therefore, the treatment groups were compared to the solvent control group for analysis. Mean dry weight at test termination in the 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L treatment groups was 16.0, 16.2, 15.8, 15.0 and 16.1 mg, respectively. According to Dunnett’s test, there was a statistically significant decrease in the 1.0 µg/L treatment group in comparison to the solvent control group (p = 0.05). However, the decrease in the 1.0 µg/L treatment group was not considered to be treatment-related since the decrease was not dose responsive. Consequently, the NOEC and LOEC for dry weight were determined to be 1.6 µg/L and > 1.6 µg/L, respectively. Since there was a less than a 10% inhibition for dry weight 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.

Results with reference substance (positive control):

-

Reported statistics and error estimates:

Test endpoints analyzed statistically included time to hatch, hatching success, post-hatch larval survival, overall survival and growth (total length, wet weight and dry weight). Data from the negative and solvent control groups for each parameter were compared using a t-test. Since no differences were detected between the two control groups (p > 0.05) for time to hatch, hatching success, post-hatch survival, overall survival and total length, the control data for these parameters were pooled for comparison among the treatment groups. There were statistically significant differences between the two control groups (p = 0.05) for wet weight and dry weight. Therefore, the treatment group data for dry weight were compared to the solvent control data.

The statistical tests were performed using a personal computer with SAS software.

The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and LC/ICx values. However, scientific judgement was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. The LOEC was defined as the lowest tested concentration at which the test substance is observed to have had a statistically significant adverse effect (at p = 0.05) on time to hatch, hatching success, survival or growth when compared to the controls. However, all test concentrations above the LOEC should have a harmful effect equal to or greater than those observed at the LOEC. The NOEC was defined as the test concentration immediately below the LOEC, which when compared with the control groups, had no statistically significant adverse effect (at p = 0.05).

Measured Concentrations of¹⁴C-CDBC in the Primary Stock Solutions

 

Sample Type	Sample Number (15175-)	Nominal ^14C-CDBC Concentration (mg/L)	Specific Activity (dpm/mg)	Total [^14C] Found (dpm)	^14C-CDBC Equivalents Found^1 (mg/L)	Percent of Nominal
Stock	090419-1-A	50.0	250072	586397	46.9	93.8
Stock	090419-1-B	50.0	250072	575431	46.0	92.0
Stock	090419-1-C	50.0	250072	580306	46.4	92.8
				Mean	46.4	92.9
Stock	091119-2-A	50.0	250072	617894	49.4	98.8
Stock	091119-2-B	50.0	250072	639329	51.1	102
Stock	091119-2-C	50.0	250072	623518	49.9	99.7
				Mean	50.1	100
Stock	091819-3-A	50.0	250072	572157	45.8	91.5
Stock	091819-3-B	50.0	250072	520621	41.6	83.3
Stock	091819-3-C	50.0	250072	589406	47.1	94.3
				Mean	44.8	89.7
Stock	092619-5-A	50.0	250072	585952	46.9	93.7
Stock	092619-5-B	50.0	250072	578658	46.3	92.6
Stock	092619-5-C	50.0	250072	581590	46.5	93.0
				Mean	46.6	93.1
Stock	100219-6-A	50.0	250072	579998	46.4	92.8
Stock	100219-6-B	50.0	250072	580930	46.5	92.9
Stock	100219-6-C	50.0	250072	588185	47.0	94.1
				Mean	46.6	93.3
Stock	100919-7-A	50.0	250072	673641	53.9	108
Stock	100919-7-B	50.0	250072	670814	53.6	107
Stock	100919-7-C	50.0	250072	673152	53.8	108
				Mean	53.8	108
^1  14C-CDBC Equivalents = (Total [^14C] found / Specific Activity/ Sample Volume (mL)) x Dilution Factor  Sample Volume = 0.0500 mL; Dilution Factor = 1.00

Measured Concentrations of¹⁴C-CDBC in Dispensing Stock Solutions

 

Sample Type	Sampling Interval (Day)	Sample Number (524A-138-)	Nominal CDBC Concentration (mg/mL)	Specific Activity (dpm/mg)	Total [^14C] Found (dpm)	^14C-CDBCEquivalents[AG1] [YB2]  Found^1 (mg/mL)	Percent Recovery
Background	-1	BKG	--	--	0	--	--
Dosing Solution	-1	S-1	0.0013	192350	49666	0.00129	99.3
Dosing Solution	-1	S-2	0.0025	100011	50648	0.00253	101
Dosing Solution	-1	S-3	0.0050	50003	50546	0.00505	101
Dosing Solution	-1	S-4	0.010	25001	50955	0.0102	102
Dosing Solution	-1	S-5	0.020	12500	51110	0.0204	102
^1 ^14C-CDBC Equivalents = (Total [^14C] found / Specific Activity / Sample Volume (mL)) x Dilution Factor   Sample Volume = 0.200 mL; Dilution Factor = 1.00

Measured Concentrations of¹²C-CDBC in Dispensing Stock Solutions

 

Sample Number (524A-138-)	Sampling Interval (Day)	CDBC Nominal Concentration (mg/mL)	^12C-CDBC Nominal Concentration (mg/mL)^1	^12C-CDBC Measured Concentration (mg/mL)^2	^12C-CDBC Percent of Nominal^1,3
S-1	-1	0.0013	0.00030	0.000471	157
S-2	-1	0.0025	0.0015	0.00147	98.0
S-3	-1	0.0050	0.0040	0.00410	103
S-4	-1	0.010	0.0090	0.00931	103
S-5	-1	0.020	0.019	0.0194	102
^1The stock solutions for the test system were prepared as a mixture of radiolabeled and non-labeled material. Nominal concentrations of CDBC in the stock solutions were corrected for the ratio of^12C-CDBC in each test solution. The corrected nominal values were calculated as follows [Cold Material (mg)/(Hot Material (mg) + Cold Material (mg))] x CDBC Nominal Concentration. ^2Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly. ^3   Calculated by Excel 2010, based upon measured values of^12C-CDBC/^12C-CDBC nominal concentration.

Measured Concentrations of¹⁴C-CDBC in Pretest Verification Samples

 

	Nominal CDBC Concentration (µg/L)	Sample Number (524A-138-)	Sampling Interval (Day)	Specific Activity (dpm/µg)	Total [^14C] Found (dpm)^1	^14C-CDBC Equivalents Found^1, 2 (µg/L)	Percent of Nominal
	Negative Control	PT-1-RE^3	-4	NA	< LOQ	< LOQ	--
	(0.0)	PT-11	-3	NA	< LOQ	< LOQ	--
		PT-21	-2	NA	< LOQ	< LOQ	--
		PT-31	-1	NA	< LOQ	< LOQ	--
	Solvent Control	PT-2-RE^3	-4	NA	< LOQ	< LOQ	--
	(0.0)	PT-12	-3	NA	< LOQ	< LOQ	--
		PT-22	-2	NA	< LOQ	< LOQ	--
		PT-32	-1	NA	< LOQ	< LOQ	--
	0.13	PT-3-RE^3	-4	192350	305	0.159	122
		PT-13	-3	192350	221	0.115	88.4
		PT-23	-2	192350	199	0.103	79.6
		PT-33	-1	192350	226	0.117	90.4
	0.25	PT-4-RE^3	-4	100011	225	0.225	90.0
		PT-14	-3	100011	210	0.210	84.0
		PT-24	-2	100011	203	0.203	81.2
		PT-34	-1	100011	225	0.225	90.0
	0.50	PT-5-RE^3	-4	50003	165	0.330	66.0
		PT-15	-3	50003	210	0.420	84.0
		PT-25	-2	50003	165	0.330	66.0
		PT-35	-1	50003	209	0.418	83.6
	1.0	PT-6-RE^3	-4	25001	167	0.668	66.8
		PT-16	-3	25001	176	0.704	70.4
		PT-26	-2	25001	176	0.704	70.4
		PT-36	-1	25001	201	0.804	80.4
	2.0	PT-7-RE^3	-4	12500	257	2.06	103
		PT-17	-3	12500	202	1.62	80.8
		PT-27	-2	12500	135	1.08	54.0
		PT-37	-1	12500	201	1.61	80.4
^1 The limit of quantification (LOQ) for^14C-CDBC in water is 50 dpm, based upon the LOQ of the liquid scintillation counter (LSC). ^2 ^14C-CDBC Equivalents = [(Total dpm found/Sample volume)/Specific activity]    Sample volume = 0.0100 L ^3 Sample was reanalyzed due to instrument contamination. The sample was analyzed on a different instrument and the result is presented here.

 

Measured Concentrations of¹⁴C-CDBC in Test Solution Samples

 

	Nominal CDBC Concentration (µg/L)	Sample Number (524A-138-)	Sampling Interval (Day)	Specific Activity (dpm/µg)	Total [^14C] Found (dpm)^1	^14C-CDBC Equivalents Found^1, 2, 3 (µg/L)	Percent of Nominal	Mean^14C-CDBC Equivalents Found (µg/L)/Standard Deviation/ %CV^1,2,3	Mean Percent of Nominal^1
	Negative	4	0	NA	< LOQ	< LOQ	--	--	--
	Control	16	7	NA	< LOQ	< LOQ	--
		26	14	NA	< LOQ	< LOQ	--
		41	21	NA	< LOQ	< LOQ	--
		55	28	NA	< LOQ	< LOQ	--
		67	32	NA	< LOQ	< LOQ	--
	Solvent	5	0	NA	< LOQ	< LOQ	--	--	--
	Control	17	7	NA	< LOQ	< LOQ	--
		27	14	NA	< LOQ	< LOQ	--
		42	21	NA	< LOQ	< LOQ	--
		56	28	NA	< LOQ	< LOQ	--
		68	32	NA	< LOQ	< LOQ	--
	0.13	6	0	192350	266	0.138	106	0.14	104
		18	7	192350	277	0.144	111	SD = 0.0117
		28	14	192350	248	0.129	99.2	%CV = 8.66
		43	21	192350	257	0.134	103
		57	28	192350	223	0.116	89.2
		69	32	192350	286	0.149	114
Measured Concentrations of^14C-CDBC in Test Solution Samples   Nominal CDBC Concentration (µg/L)   Sample Number (524A-138-)   Sampling Interval (Day)   Specific Activity (dpm/µg) Total [^14C] Found (dpm) ^14C-CDBC Equivalents Found^1, 2, 3 (µg/L)   Percent of Nominal Mean^14C-CDBC Equivalents Found (µg/L)/Standard Deviation/ %CV^1, 2, 3 Mean Percent of Nominal^1 0.25 7 0 100011 229 0.229 91.6 0.23 92.7   19 7 100011 210 0.210 84.0 SD = 0.0378     29^4 14 100011 163 0.163 65.2 %CV = 16.3     35^4 14 100011 214 0.214 85.6         14 Average^4     0.189^5 75.6       44 21 100011 212 0.212 84.8       58 28 100011 258 0.258 103       70 32 100011 293 0.293 117                       0.50 8 0 50003 209 0.418 83.6 0.44 87.3   20 7 50003 196 0.392 78.4 SD = 0.0699     30^6 14 50003 164 0.328 65.6 %CV = 16.0     36^6 14 50003 170 0.340 68.0       37^6 15 50003 242 0.484 96.8         14 Average^6     0.384^5 76.8       45^6 21 50003 175 0.350 70.0       48^6 21 50003 175 0.350 70.0       50^6 22 50003 261 0.522 104         21 Average^6     0.407^5 81.4       59 28 50003 223 0.446 89.2       71 32 50003 304 0.608 122       BU-71^7 32 50003 268 0.536 107         32 Average^7     0.572^5 114     Measured Concentrations of^14C-CDBC in Test Solution Samples     Nominal CDBC Concentration (µg/L)   Sample Number (524A-138-)   Sampling Interval (Day)   Specific Activity (dpm/µg) Total [^14C] Found (dpm) ^14C-CDBC Equivalents Found^1, 2, 3 (µg/L)   Percent of Nominal Mean^14C-CDBC Equivalents Found (µg/L)/Standard Deviation/ %CV^1, 2, 3 Mean Percent of Nominal^1   1.0 9 0 25001 211 0.844 84.4 1.0 99.6     21 7 25001 262 1.05 105 SD = 0.209       31 14 25001 211 0.844 84.4 %CV = 21.0       46^4 21 25001 186 0.744 74.4         49^4 21 25001 177 0.708 70.8         51^4 22 25001 248 0.992 99.2           21 Average^4     0.815^5 81.5         60 28 25001 266 1.06 106         72^6 32 25001 325 1.30 130         BU-72^6 32 25001 247 0.988 98.8         76^6 32 25001 403 1.61 161         BU-76^6 32 25001 388 1.55 155           32 Average^6     1.36^5 136                           2.0 10 0 12500 225 1.80 90.0 1.6 81.7     22 7 12500 211 1.69 84.4 SD = 0.171       32 14 12500 211 1.69 84.4 %CV = 10.5       47 21 12500 221 1.77 88.4         61^4 28 12500 158 1.26 63.2         62^4 28 12500 163 1.30 65.2         63^4 29 12500 193 1.54 77.2           28 Average^4     1.37^5 68.5         73 32 12500 198 1.58 79.2         BU-73^7 32 12500 171 1.37 68.4           32 Average^7     1.48^5 74.0     ^1 Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly. ^2 ^14C-CDBC Equivalents = [(Total dpm found/Sample volume)/Specific activity] Sample volume = 0.0100 L ^3 The limit of quantification (LOQ) for^14C-CDBC in water is 50 dpm, based upon the LOQ of the liquid scintillation counter (LSC). ^4The original sample had lower than expected recoveries. An additional sample was collected on the same day, and confirmed the original results. Another additional sample was collected the next day. The average of the original sample, and the two additional samples, are reported here. ^5 Average used to calculate the mean^14C-CDBC equivalent concentration. ^6 Two additional samples (76 and BU-76) were collected on Day 32 due to the discrepancies in results between 72 and BU-72. The original sample and the back-up sample, as well as the two additional samples, are all averaged here for Day 32. ^7 Back-up samples were analyzed on Day 32. The average of the original sample and the back-up sample are reported here.   Measured Concentrations of^12C-CDBC in Test Solution Samples   Sample Number (524A-138-)   Sampling Interval (Day) CDBC Nominal Concentration  (µg/L) ^12C-CDBC Nominal Concentration (µg/L)^1 ^12C-CDBC Measured Concentration (µg/L)^1,2,3 ^12C-CDBC Percent of Nominal^3,4    11^5 0 0.13 0.030 < LOQ --   33 14 0.13 0.030 ND --   74 32 0.13 0.030 ND --                  12^5 0 2.0 1.9 0.873 45.9   34 14 2.0 1.9 1.02 53.6   75 32 2.0 1.9 0.531 27.9    ^1The stock solutions for the test system were prepared as a mixture of radiolabeled and non-labeled material. Nominal concentrations of CDBC in the samples were corrected for the ratio of^12C-CDBC in each test solution. The corrected nominal values were calculated as follows [Cold Material (mg)/(Hot Material (mg) + Cold Material (mg))] x CDBC Nominal Concentration. ^2Thelimit of quantification (LOQ) for^12C-CDBC in freshwater is 0.125 µg/L, based upon the lowest nominal matrix fortification concentration in which a mean recovery of 70-110% was obtained. The LOQ is higher than the lowest nominal^12C-CDBC sample concentration due to method limitations. ND is below the LOD (limit of detection) of 0.00229 µg/L. ^3Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly. ^4Calculated based on the nominal concentration of^12C-CDBC. ^5Sample was reanalyzed using a lower calibration curve range in effort to achieve better quantification, however, the calibration curve could not be lowered. The original result is presented here.   Means and Ranges of Water Quality Measurements Taken During the 32-Day Exposure to CDBC   Mean Measured Concentration (µg/L) Mean ± SD and Range of Measured Parameters Temperature^1 (°C) DO^2 (mg/L)   pH Hardness^3 (mg/L as CaCO3) Alkalinity^3 (mg/L as CaCO3) Conductivity^3 (mS/cm)               Negative Control 25.0 ± 0.17 7.9 ± 0.29 8.0 ± 0.06 140 ± 4 174 ± 5 326 ± 10   (24.7 – 25.2) (7.5 – 8.2) (7.9 – 8.1) (132 – 144) (166 – 180) (316 – 340)               Solvent Control 24.8 ± 0.20 8.0 ± 0.27 8.1 ± 0.05 -- -- --   (24.4 – 25.1) (7.6 – 8.2) (8.0 – 8.1) -- -- --               0.14 24.8 ± 0.19 7.9 ± 0.25 8.1 ± 0.05 -- -- --   (24.5 – 25.1) (7.7 – 8.2) (8.0 – 8.1) -- -- --               0.23 24.7 ± 0.23 7.9 ± 0.23 8.1 ± 0.05 -- -- --   (24.2 – 24.9) (7.6 – 8.2) (8.0 – 8.1) -- -- --               0.44 24.8 ± 0.18 7.9 ± 0.29 8.1 ± 0.05 -- -- --   (24.5 – 25.0) (7.5 – 8.2) (8.0 – 8.1) -- -- --               1.0 24.9 ± 0.15 7.9 ± 0.33 8.1 ± 0.08 -- -- --   (24.7 – 25.1) (7.5 – 8.2) (8.0 – 8.2) -- -- --               1.6 24.8 ± 0.17 7.9 ± 0.30 8.1 ± 0.04 141 ± 3 175 ± 4 329 ± 10   (24.6 – 25.0) (7.5 – 8.2) (8.0 – 8.1) (136 – 144) (168 – 180) (315 – 338)               ^1Temperature measured continuously during the test ranged from 24.59 to 25.20°C, measured to the nearest 0.01°C. ^2A dissolved oxygen concentration of 4.9 mg/L represents 60% of air saturation at 25ºC in freshwater. ^3-- = no measurements scheduled. Summary of Time to Hatch, Hatching Success, Post-Hatch Larval Survival and Overall Survival of Fathead Minnows Exposed toCDBC   Mean Measured Concentration  (µg/L)   Number Exposed Total Number Hatched Hatching Success (%)^1 Mean Time to Hatch ± Std. Dev. (Days) Number Surviving to Termination Post-Hatch Survival (%)^1 Overall Survival (%)^1 Negative Control 80 78 97.5 3.96 ± 0.125 72 92.4 90.0 Solvent Control 80 77 96.3 4.07 ± 0.132 75 97.4 93.8 Pooled Control 160 155 96.9 4.01 ± 0.131 147 94.9 91.9 0.14 80 79 98.8 4.01 ± 0.025 73 92.3 91.3 0.23 80 75 93.8 4.01 ± 0.064 74 98.7 92.5 0.44 80 77 96.3 4.08 ± 0.029 75 97.4 93.8 1.0 80 77 96.3 4.07 ± 0.078 73 94.4 91.3 1.6 80 77 96.3  4.16 ± 0.125 * 69 89.6 86.3 LC10(µg/L): -- > 1.6^2 -- -- > 1.6^2 > 1.6^2 LC20(µg/L): -- > 1.6^2 -- -- > 1.6^2 > 1.6^2 ^1       There was not a statistically significant difference in hatching success, post-hatch survival or overall survival in any treatment group when compared to the pooled control (Jonckheere-Terpstra step-down trend test,p> 0.05). Both negative and solvent control groups met the validity criteria of >70% hatching success and =75% post-hatch larval survival. ^2       Empirically estimated to be greater than the highest test concentration, since there was less than a 10% decrease in any treatment group in comparison to the pooled control. *       According to Dunnett’s test, statistically significant increase evident in the treatment group data in comparison to the pooled control group (p= 0.05). However, this is not considered to be biologically meaningful since the increase was very slight (3.7%) in comparison to the pooled control.     Summary of Larval Growth of Fathead Minnows Exposed toCDBC   Mean Measured Concentration (µg/L) Mean Total Length ± Std. Dev. (mm) Mean Wet Weight ± Std. Dev. (mg)^1 Mean Dry Weight ± Std. Dev. (mg)^2 Negative Control 22.6 ± 0.19 85.8 ± 2.28 17.6 ± 0.56 Solvent Control 22.4 ± 0.16 79.9 ± 1.28 16.5 ± 0.25 Pooled Control 22.5 ± 0.20 --^3 --^3 0.14   22.1 ± 0.23 * 79.0 ± 3.69 16.0 ± 0.49 0.23   21.8 ± 0.24 * 77.2 ± 2.20 16.2 ± 0.64 0.44   22.0 ± 0.12 * 77.1 ± 2.36 15.8 ± 0.66 1.0   21.8 ± 0.29 *  72.2 ± 0.71^¦  15.0 ± 0.60^¦ 1.6 22.3 ± 0.24 79.9 ± 2.12 16.1 ± 0.39 IC10(µg/L): > 1.6^4 > 1.6^4 > 1.6^4 IC20(µg/L): > 1.6^4 > 1.6^4 > 1.6^4 ^1       The data failed the assumption of homogeneity of variance (normality (p= 0.01). Log transformation corrected the condition for the comparison with the solvent control. ^2       The data failed the assumption of normality (p= 0.01). Log transformation did corrected the condition for the comparison with the solvent control. ^3       The negative control and solvent control data for dry weight were significantly different (p= 0.05). Therefore, the treatment data for dry weight were compared to the solvent control. ^4       Empirically estimated to be greater than the highest test concentration, since there was less than a 10% decrease in any treatment group in comparison to the pooled control. *       According to Dunnett’s test, statistically significant decrease evident in the treatment group data in comparison to the pooled control group (p= 0.05). However, the statistically significant decrease is not considered to be treatment-related since the decrease was not dose-responsive. ^¦       According to Dunnett’s test, statistically significant decrease evident in the treatment group data in comparison to the solvent control group (p= 0.05). However, the statistically significant decrease is not considered to be treatment-related since the decrease was not dose-responsive. Percent Inhibition as Compared to the Control Groups   Mean Measured Concentration (µg/L) Percent Inhibition (%) Compared to the Pooled or Solvent Control Groups^1, 2 Time to Hatch Hatching Success Post-Hatch Larval Survival Overall Survival Mean Total Length Mean Wet Weight Mean Dry Weight PC PC PC PC PC SC SC 0.14 0.0 -1.9 2.7 0.7 1.8 1.2 3.0 0.23 0.1 3.3 -4.0 -0.7 3.1 3.4 1.6 0.44 -1.6 0.7 -2.6 -2.0 2.3 3.5 4.0 1.0 -1.3 0.7 0.5 0.7 3.0 9.6 8.6 1.6 -3.7 0.7 5.6 6.1 1.1 0.0 2.4 ^1      SC = solvent control group; PC = pooled control group. ^2      Stimulation or a greater response in the test substance treatment than the controls is reported as a negative percent inhibition. Quality Control Samples of^12C-CDBC inFreshwater     Sample Number (524A-138-)   Sampling Time (Days)     Sample Type Concentration of ^12C-CDBC     Percent Recovery^2 Fortified (µg/L) Measured^1, 2 (µg/L) MAB-1 0 Matrix Blank 0.0 < LOQ -- MAB-2 14 Matrix Blank 0.0 < LOQ -- MAB-3 32 Matrix Blank 0.0 < LOQ --             MAS-1 0 Matrix Fortification 0.125 0.143 114 MAS-2 0 Matrix Fortification 2.00 1.63 81.4             MAS-3 14 Matrix Fortification 0.125 0.138 110 MAS-4 14 Matrix Fortification 2.00 1.93 96.7             MAS-5 32 Matrix Fortification 0.125 0.100^4 80.0                   Mean^3= Standard Deviation^3= CV^3= 96.4 15.7 16.3% ^1Thelimit of quantification (LOQ) for^12C-CDBC in freshwater is 0.125 µg/L, based upon the lowest nominal matrix fortification concentration in which a mean recovery of 70-110% was obtained. ^2 Results were generated using Analyst Version 1.6.3. Manual calculations may differ slightly. ^3Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly. ^4Extrapolated value due to exclusion of the low level standard but quantifiable based upon the range of the calibration curve. The associated high level matrix fortification for the interval is not reported due to preparation error. Hatching Success of Fathead Minnow Embryos Exposed toCDBC   Mean Measured Concentration (µg/L)     Rep Number of Embryos Exposed Cumulative No. of Dead Embryos Cumulative Number Hatched by Day of Test Replicate % Hatching Success Treatment % Hatching Success Day 1 Day 2 Day 3 Day 4^1 Day 5   Negative Control A 20 0 0 0 4 20 20   100 97.5   B 20 2 0 0 1 17 18   90     C 20 0 0 0 0 18 20   100     D 20 0 0 0 1 20 20   100                           Solvent Control A 20 1 0 0 0 14 19   95 96.3   B 20 2 0 0 0 18 18   90     C 20 0 0 0 0 20 20   100     D 20 0 0 0 0 20 20   100                           0.14 A 20 0 0 0 0 20 20   100 98.8   B 20 1 0 0 0 19 19   95     C 20 0 0 0 1 18 20   100     D 20 0 0 0 0 20 20   100                           0.23 A 20 1 0 0 0 19 19   95 93.8   B 20 3 0 0 0 17 17   85     C 20 0 0 0 1 17 20   100     D 20 1 0 0 1 19 19   95   ^1Day 4 of test = Day 0 post-hatch. Any embryos remaining after Day 4 of the test were kept in the egg cup until death occurred, or until hatching, at which time the newly hatched larva was added to the larval growth chamber.

Hatching Success of Fathead Minnow Embryos Exposed to CDBC

 

Mean Measured Concentration (µg/L)	Rep	Number of Embryos Exposed	Cumulative No. of Dead Embryos	Cumulative Number Hatched by Day of Test						Replicate % Hatching Success	Treatment % Hatching Success
				Day 1	Day 2	Day 3	Day 4^1	Day 5
0.44	A	20	1	0	0	0	17	19		95	96.3
	B	20	0	0	0	0	19	20		100
	C	20	0	0	0	0	18	20		100
	D	20	2	0	0	0	17	18		90
1.0	A	20	0	0	0	0	17	20		100	96.3
	B	20	0	0	0	0	20	20		100
	C	20	3	0	0	0	15	17		85
	D	20	0	0	0	0	20	20		100
1.6	A	20	0	0	0	0	14	20		100	96.3
	B	20	0	0	0	1	16	20		100
	C	20	3	0	0	0	17	17		85
	D	20	0	0	0	0	16	20		100
^1Day 4 of test = Day 0 post-hatch. Any embryos remaining after Day 4 of the test were kept in the egg cup until death occurred, or until hatching, at which time the newly hatched larva was added to the larval growth chamber.

Survival to Day 28 Post-Hatch of Fathead Minnow Larvae Exposed toCDBC

 

Nominal Concentration (µg/L)	Rep.	Number of Larvae Hatched	Live Counts by Day Post-Hatch^1									Replicate Percent Post-Hatch Survival^2	Treatment Percent Post-Hatch Survival^2	Replicate Percent Overall Survival^2	Treatment Percent Overall Survival^2
			Day 0	Day 3	Day 7	Day 10	Day 14	Day 17	Day 21	Day 24	Day 28
Negative Control	A	20	20	20	19	18	18	18	18	18	18	90.0	92.3	90.0	90.0
	B	18	17^3	18	17	17	17	17	17	17	17	94.4		85.0
	C	20	18^3	20	19	19	19	19	19	19	19	95.0		95.0
	D	20	20	18	18	18	18	18	18	18	18	90.0		90.0
Solvent Control	A	19	14^3	18	18	18	18	18	18	18	18	94.7	97.4	90.0	93.8
	B	18	18	18	18	18	18	18	18	18	18	100		90.0
	C	20	20	20	20	20	20	20	20	20	20	100		100
	D	20	20	20	20	20	20	20	19	19	19	95.0		95.0
0.14	A	20	20	18	18	18	18	18	18	18	18	90.0	92.4	90.0	91.3
	B	19	19	19	18	16	16	16	16	16	16	84.2		80.0
	C	20	18^3	20	20	20	20	20	20	20	20	100		100
	D	20	20	20	19	19	19	19	19	19	19	95.0		95.0
0.23	A	19	19	19	18	18	18	18	18	18	18	94.7	98.7	90.0	92.5
	B	17	17	17	17	17	17	17	17	17	17	100		85.0
	C	20	17^3	20	20	20	20	20	20	20	20	100		100
	D	19	19	19	19	19	19	19	19	19	19	100		95.0

 

Survival to Day 28 Post-Hatch of Fathead Minnow Larvae Exposed toCDBC

 

Nominal Concentration (µg/L)	Rep.	Number of Larvae Hatched	Live Counts by Day Post-Hatch^1									Replicate Percent Post-Hatch Survival^2	Treatment Percent Post-Hatch Survival^2	Replicate Percent Overall Survival^2	Treatment Percent Overall Survival^2
			Day 0	Day 3	Day 7	Day 10	Day 14	Day 17	Day 21	Day 24	Day 28
0.44	A	19	17^3	19	19	19	19	19	19	18	18	94.7	97.4	90.0	93.8
	B	20	19^3	20	20	20	20	20	20	20	20	100		100
	C	20	18^3	19	19	19	19	19	19	19	19	95.0		95.0
	D	18	17^3	18	18	18	18	18	18	18	18	100		90.0
1.0	A	20	17^3	20	20	20	20	20	20	20	20	100	94.8	100	91.3
	B	20	20	20	20	20	20	20	20	20	20	100		100
	C	17	15^3	14	14	14	14	14	14	14	14	82.4		70.0
	D	20	20	19	19	19	19	19	19	19	19	95.0		95.0
1.6	A	20	14^3	20	19	19	19	19	19	19	19	95.0	89.6	95.0	86.3
	B	20	16^3	18	17	17	17	17	17	17	17	85.0		85.0
	C	17	17	16	16	16	16	16	15	15	15	88.2		75.0
	D	20	16^3	18	18	18	18	18	18	18	18	90.0		90.0
^1 Day 0 post-hatch = Day 4 of test. Live counts made on Days 0 through 28 post-hatch were actual counts made at transfer to clean test chambers and at test termination. ^2 Replicate and treatment group means were calculated using Excel in full-precision. Manual calculations may differ slightly. ^3 Additional larvae hatched on Day 1 post-hatch and were released into the larval growth chamber.

Summary of Clinical Observations of Fathead Minnow Larvae Exposed toCDBCDuring the 28-Day Post-Hatch Period

 

Mean Measured Concentration (µg/L)	Rep.	Clinical Observations by Day Post-Hatch^1
		Day 0	Day 3	Day 7	Day 10
Negative Control	A	AN	20 AN	1 S,MC,R,D; 18 AN	1 M; 18 AN
	B	AN	18 AN	17 AN	17 AN
	C	AN	20 AN	1 M; 19 AN	19 AN
	D	AN	1 X; 1 M; 18 AN	18 AN	18 AN
Solvent Control	A	AN	1 M; 18 AN	18 AN	18 AN
	B	AN	18 AN	18 AN	18 AN
	C	AN	20 AN	1 S,W; 19 AN	1 S,W; 19 AN
	D	AN	20 AN	1 S; 19 AN	1 S; 19 AN
0.14	A	1 CU,W; rest AN	1 X; 1 M; 18 AN	18 AN	18 AN
	B	1 W; rest AN	19 AN	1 X; 1 S,W; 17 AN	2 M; 16 AN
	C	AN	20 AN	20 AN	20 AN
	D	1 CU,R; rest AN	1 CU,W; 19 AN	19 AN	19 AN
0.23	A	AN	19 AN	1 M; 18 AN	18 AN
	B	AN	17 AN	17 AN	17 AN
	C	AN	20 AN	20 AN	20 AN
	D	AN	19 AN	19 AN	19 AN

Summary of Clinical Observations of Fathead Minnow Larvae Exposed to CDBC During the 28-Day Post-Hatch Period

 

Mean Measured Concentration (µg/L)	Rep.	Clinical Observations by Day Post-Hatch^1
		Day 0	Day 3	Day 7	Day 10
0.44	A	AN	19 AN	19 AN	19 AN
	B	AN	20 AN	20 AN	20 AN
	C	AN	1 M; 19 AN	19 AN	19 AN
	D	AN	18 AN	1 S; 17 AN	1 S; 17 AN
1.0	A	AN	20 AN	20 AN	20 AN
	B	AN	20 AN	20 AN	20 AN
	C	AN	3 M; 14 AN	14 AN	14 AN
	D	AN	1 M; 19 AN	19 AN	19 AN
1.6	A	AN	20 AN	1 M; 19 AN	19 AN
	B	1 W; rest AN	2 M; 18 AN	1 M; 17 AN	17 AN
	C	1 CU,R; rest AN	1 M; 16 AN	16 AN	16 AN
	D	AN	1 M; 18 AN	18 AN	18 AN

Summary of Clinical Observations of Fathead Minnow Larvae Exposed to CDBC During the 28-Day Post-Hatch Period

 

Mean Measured Concentration (µg/L)	Rep.	Clinical Observations by Day Post-Hatch^1
		Day 14	Day 17	Day 21	Day 24	Day 28
Negative Control	A	18 AN	18 AN	18 AN	18 AN	18 AN
	B	17 AN	17 AN	17 AN	17 AN	17 AN
	C	19 AN	19 AN	19 AN	19 AN	19 AN
	D	18 AN	18 AN	18 AN	18 AN	18 AN
Solvent Control	A	18 AN	18 AN	18 AN	18 AN	18 AN
	B	18 AN	18 AN	18 AN	18 AN	18 AN
	C	1 S; 19 AN	1 S,P; 19 AN	1 S,P; 19 AN	1 SA; 19 AN	1 S,MC,SA; 19 AN
	D	1 S; 19 AN	1 S,W; 19 AN	1 M; 19 AN	19 AN	19 AN
0.14	A	18 AN	1 S; 17 AN	1 S; 17 AN	1 S; 17 AN	1 S; 17 AN
	B	16 AN	2 S; 14 AN	1 S; 15 AN	16 AN	2 S; 14 AN
	C	20 AN	1 MC; 19 AN	1 MC; 19 AN	20 AN	1 S; 19 AN
	D	19 AN	19 AN	19 AN	19 AN	19 AN
0.23	A	18 AN	18 AN	18 AN	18 AN	18 AN
	B	17 AN	17 AN	17 AN	17 AN	17 AN
	C	20 AN	20 AN	20 AN	20 AN	20 AN
	D	19 AN	19 AN	19 AN	19 AN	19 AN

Summary of Clinical Observations of Fathead Minnow Larvae Exposed to CDBC During the 28-Day Post-Hatch Period

 

Mean Measured Concentration (µg/L)	Rep.	Clinical Observations by Day Post-Hatch^1
		Day 14	Day 17	Day 21	Day 24	Day 28
0.44	A	19 AN	19 AN	19 AN	18 AN	18 AN
	B	20 AN	20 AN	20 AN	20 AN	20 AN
	C	19 AN	19 AN	19 AN	19 AN	19 AN
	D	1 S; 17 AN	1 S; 17 AN	1 S; 17 AN	1 S; 17 AN	1 S; 17 AN
1.0	A	20 AN	20 AN	20 AN	20 AN	20 AN
	B	20 AN	20 AN	20 AN	20 AN	20 AN
	C	14 AN	14 AN	14 AN	14 AN	14 AN
	D	19 AN	19 AN	19 AN	19 AN	19 AN
1.6	A	19 AN	19 AN	19 AN	19 AN	19 AN
	B	17 AN	17 AN	17 AN	17 AN	17 AN
	C	16 AN	16 AN	1 X; 15 AN	15 AN	15 AN
	D	18 AN	18 AN	18 AN	18 AN	18 AN
^1 Day 0 post-hatch = Day 4 of test. Observations made on Days 3 through 28 post-hatch were made at transfer to clean test chambers and at test termination. Observations: AN = appear normal; S = small; X = dead; P = protrusion on left side of abdomen; W = weak; M = missing and assumed dead; SA = swollen abdomen; MC= crooked spine; CU = curled; R = lying on bottom with little motion other than gill or appendage movement; D = discoloration (pale).

Validity criteria fulfilled:

yes

Conclusions:

CONCLUSIONS
Fathead minnows (Pimephales promelas) were exposed to CDBC at mean measured concentrations of 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period). The highest concentration selected for the test was based on preliminary solubility work conducted in dilution water. There were no biologically meaningful, statistically significant treatment-related effects on time to hatch, hatching success, post-hatch larval survival, overall survival or growth at concentrations = 1.6 µg/L. Consequently, the NOEC and LOEC were 1.6 µg/L and > 1.6 µg/L, respectively.

Lethal concentrations (LC10 and LC20) for the hatching success, post-hatch larval survival and overall survival endpoints were empirically estimated to be greater than the highest test concentrations, since there was less than a 10% reduction in any treatment group when compared to the pooled control group. Inhibition concentrations (IC10 and IC20) for the total length, wet weight and dry weight endpoints could not be estimated, since there was a less than a 10% inhibition between the treatment group means and the pooled or solvent control.

In conclusion, no effect was measured up to the limit of solubility of the test item.

Executive summary:

The objective of this study, conducted according to the OECD guideline 210, was to determine the effects of copper bis(dibutyldithiocarbamate) (CDBC) on the time to hatch, hatching success, survival, and growth of fathead minnows,Pimephales promelas, during early life-stage development.

Fathead minnow embryos were exposed to a geometric series of five test concentrations, a negative (dilution water) control and a solvent control (0.1 mL/L HPLC-grade methanol) under flow-through conditions. The exposure period included a 4-day embryo hatching period, and a 28-day post-hatch juvenile growth period. Nominal test concentrations were 0.13, 0.25, 0.50, 1.0 and 2.0 µg/L. The concentrations were selected in consultation with the Sponsor and were based on the solubility of the test substance in water. The highest concentration selected, 2 µg/L, was selected based on preliminary solubility work conducted in dilution water. 

There were no biologically meaningful, statistically significant treatment-related effects on time to hatch, hatching success, post-hatch larval survival, overall survival or growth at concentrations = 1.6 µg/L. Consequently, the NOEC and LOEC were 1.6 µg/L and > 1.6 µg/L, respectively.

Lethal concentrations (LC10 and LC20) for the hatching success, post-hatch larval survival and overall survival endpoints were empirically estimated to be greater than the highest test concentrations, since there was less than a 10% reduction in any treatment group when compared to the pooled control group.  Inhibition concentrations (IC10 and IC20) for the total length, wet weight and dry weight endpoints could not be estimated, since there was a less than a 10% inhibition between the treatment group means and the pooled or solvent control.

 

 

LENGTH OF EXPOSURE:     32 Days (4-Day Hatch and 28-Day Post-Hatch)

 

TEST ORGANISMS:                          Fathead Minnow (Pimephales promelas)

  

AGE OF TEST ORGANISMS:            Embryos (<24 hours old)

 

TEST CONCENTRATIONS:                          Nominal                  Mean Measured

                                                               Negative Control                   <LOQ

                                                                Solvent Control                    <LOQ

                                                                    0.13 µg/L                      0.14 µg/L

                                                                    0.25 µg/L                      0.23 µg/L

                                                                    0.50 µg/L                      0.44 µg/L

                                                                     1.0 µg/L                        1.0 µg/L

                                                                     2.0 µg/L                        1.6 µg/L

 

 

RESULTS:      Based on mean measured test concentrations

Endpoint	LC10/IC10 (µg/L)	LC20/IC20 (µg/L)	NOEC (µg/L)	LOEC (µg/L)
Time to Hatch	--	--	1.6 µg/L	> 1.6 µg/L
Hatching Success	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
Post-Hatch Larval Survival	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
Overall Survival	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
Total Length	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
Wet Weight	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
Dry Weight	> 1.6 µg/L^1	> 1.6 µg/L^1	1.6 µg/L	> 1.6 µg/L
No-Observed-Effect Concentration:			1.6 µg/L
Lowest-Observed-Effect-Concentration:			> 1.6 µg/L
^1       Empirically estimated to be greater than the highest test concentration, since there was less than a 10% decrease in any treatment group in comparison to the pooled or solvent control.

 

Description of key information

Fathead minnows (Pimephales promelas) were exposed to CDBC at mean measured concentrations of 0.14, 0.23, 0.44, 1.0 and 1.6 µg/L under flow-through conditions for 32 days (a 4-day hatching period plus a 28-day post-hatch growth period).  The highest concentration selected for the test was based on preliminary solubility work conducted in dilution water.  There were no biologically meaningful, statistically significant treatment-related effects on time to hatch, hatching success, post-hatch larval survival, overall survival or growth at concentrations = 1.6 µg/L.  Consequently, the NOEC and LOEC were 1.6 µg/L and > 1.6 µg/L, respectively.

In conclusion, no effect was measured up to the limit of solubility of the test item.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

1.6 µg/L