Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-27 to 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
DEVIATION TO STUDY PROTOCOL 1
Deviation: On August 29, 2019, the observations of the organisms in the 10 µg/L treatment concentration were inadvertently not recorded.
The deviation was discovered by the study director during the preparation of the report.
Reason: biologist oversight.
Impact: However, the observations of the treatment level were recorded on the day immediately prior to and after this day. Therefore, the omission of the data recorded on August 29, 2019 had no adverse impact on the study or the interpretation of the study results.

DEVIATION TO STUDY PROTOCOL 2
Deviation: The test solution samples of the negative and solvent control were not collected for analysis with the test solution samples.
The deviation was discovered by the auditor during the auditing of the draft report.
Reason: The samples collected from August 2, 2019 were originally intended to be used to confirm the test solutions concentrations; therefore, the negative and solvent control solutions were not collected for analysis. However, when the first trial of the study (524A-139) was terminated, the diluter was allowed to continue into the definitive test (524A-139A). Because no additional samples were collected for the pre-test diluter verification of the definitive test, the results from August 2, 2019 was used as a pre-test diluter verification samples for the definitive study.
Impact: The intent of the pre-test diluter verification samples was to verify the operation of the toxicant delivery system. Because no toxicant was delivered to the diluter system, the samples of the negative and solvent control samples did not confirm the diluter delivery of the toxicant. Therefore, the omission of the samples from the two controls during the pre-test had no adverse impact on the study or the interpretation of the study results.
Deviations:
yes
Remarks:
see "Version / remarks".
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
EC Number:
620-097-9
Cas Number:
54299-17-1
Molecular formula:
C32H22O4
IUPAC Name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water samples were collected from one test chamber of each treatment group six days prior to test initiation to confirm the operation of the diluter. Water samples were collected from alternating replicate test chambers of each treatment and control group on Days 0, 7, 14, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Additional samples were collected from one replicate of each treatment group on Day 6 to confirm the test concentrations when the interruption of the delivery of stock solution to all treatment concentrations was found at the first check of the diluter on Day 6 and from one replicate of one treatment group on Day 22 to confirm the measured concentration of the 0.64 µg/L treatment group following a delivery interruption on Day 21. All samples were collected at mid-depth in the test chambers, placed in glass vials and processed immediately for analysis or stored refrigerated until processed for analysis. Sample of each stock solution (~2 mL) was collected on Day -6 from the syringes, placed in glass vials and processed immediately for analysis to confirm the preparation of the stock solutions.

Test solutions

Vehicle:
yes
Details on test solutions:
Preparation of Test Concentrations
Stock solutions were prepared twelve times during the test. At each preparation, a primary stock solution was prepared in HPLC-grade DMF at a nominal concentration of 100 µg/mL. The primary stock solutions were sonicated for ~15 minutes 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 2.6, 6.4, 16 and 40 µg/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 ¿L/minute) where they were mixed with dilution water (at a target rate of 400 mL/minute) to achieve the desired test concentrations of 0.26, 0.64, 1.6, 4.0 and 10 µg/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 EKKE treatment groups was 0.1 mL/L.

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. 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.

Test organisms

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 on spawning substrates from cultures maintained by Eurofins-Easton, Easton, Maryland. 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 gastrula). Fathead minnow embryos used in the test were obtained on spawning substrates from cultures maintained by Eurofins-Easton, Easton, Maryland. 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 9 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 0.032 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 0.32 g fish/L.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d

Test conditions

Hardness:
140 - 152 mg/L as CaCO3
Test temperature:
23.73 - 25.34 °C
pH:
7.8 - 8.1
Dissolved oxygen:
7.5 - 8.2 mg/L
Nominal and measured concentrations:
Nominal Concentrations
Negative Control
Solvent Control
0.26 µg/L
0.64 µg/L
1.6 µg/L
4.0 µg/L
10 µg/L

Time-Weighted Mean Measured Concentrations
0.20 µg/L
0.50 µg/L
1.3 µg/L
3.5 µg/L
8.6 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 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 initial trial. Since the flow to the delivery system was maintained after the initial trial was terminated and the flow continued on until the initiation of the definitive test the syringe pumps were not re-calibrated but were verified at test termination. 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 21 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 15.5 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.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.5 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Compared to pooled control; post-hatch larval survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.2 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: Compared to negative control only; dry and wet weight
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
8.6 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: Compared to pooled control; total length
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
3.5 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: Compared to negative control only; hatching success
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
8.6 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
time to hatch
Remarks on result:
other: Compared to negative control only; mean time to hatch
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
8.6 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
larval development
Remarks on result:
other: Compared to solvent control only; larval survival, dry and wet weight, mean time to hatch, hatching success
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
8.6 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: Compared to solvent control only; hatching success
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.5 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Compared to negative control only; overall larval survival
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in the study were 0.26, 0.64, 1.6, 4.0 and 10 µg/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.

Samples of the stock solutions being delivered to the diluter system had measured concentrations that ranged from 97.1 to 104% of nominal concentrations (Table 1). The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 58.0 to 85.6% of nominal concentrations (Table 2). Samples of the test solutions collected during the test had measured concentrations that ranged from
Physical and Chemical Measurements of Water
Measurements of temperature, dissolved oxygen and pH in the test chambers are summarized in Table 4. 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 7.8 to 8.1 during the test. Weekly measurements of specific conductance, hardness and alkalinity in the negative control water and in the highest test concentration are summarized in Table 4. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by EKKE concentration. Light intensity at test initiation was 561 lux at the surface of the water of one representative test chamber.

Time to Hatch and Hatching Success
Mean time to hatch and hatching success of the fathead minnow embryos are summarized in Table 5. The LCx values for percent hatching success were reported in Table 6. Fathead minnow embryos in the control and treatment replicates began hatching on Day 2 of the test and the majority of fathead minnow embryos in the control and treatment replicates hatched on Day 5 of the test and all viable embryos hatched or died by Day 8 of the test. The mean time to hatch for each treatment replicate represents the mean time span, in days, between the introduction of fertilized embryos (day 0 of the test) and hatch within the replicate.
The mean time to hatch in the negative control and solvent control was 4.1 and 5.1 days, respectively. The mean time to hatch in the solvent control included or excluded the data from replicate D were the same, 5.1 days. Because there was a statistically significant difference found between the two controls, the data from the treatment groups were compared to the negative and solvent control separately. The mean time to hatch in the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 4.5, 4.1, 5.5, 5.7 and 5.5 days, respectively. There were no statistically significant difference in mean time to hatch in any of the EKKE treatment groups when compared to the negative control (Dunnett’s one-tailed test, p >0.05). Statistically significant differences were noted in mean time to hatch in the 0.20 and 0.50 µg/L treatment groups from the solvent control with and without the data from solvent control replicate D. However, the statistically significant differences noted in these treatment groups did not follow a dose response pattern. In addition, since the mean time to hatch in these treatment groups were comparable to that of the negative control the differences found from the solvent control could not be considered treatment related effect. Consequently, the NOEC for mean time to hatch was 8.6 µg/L and the LOEC was >8.6 µg/L, the highest concentration tested.

Majority of fathead minnow embryos in the control and treatment replicates hatched on Day 5 of the test, when the surviving larvae were released into their test chambers and the post-hatch period began. The remaining un-hatched embryos were kept in their incubation chambers until hatched or died by Day 8 of the test. The additional larvae that hatched were released into their respective growth chambers.
The percent hatching success in the negative control was 94%, while the percent hatching success in solvent control was 75% when the data from replicate D was included and was 87% when the data from replicate D was excluded. The data were analyzed with the data from replicate D of the solvent control included and excluded.

1. Hatching Success Include Data from Solvent Control Replicate D
Percent hatching success in the negative control and solvent control was 94 and 75%, respectively. Since a statistically significant difference was noted between the negative control and solvent control, the treatment data were compared to the negative control and solvent control separately. Hatching success in the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 94, 96, 91, 88 and 73%, respectively (Table 5). A statistically significant reduction in percent hatching success was noted in the 8.6 µg/L treatment group from the negative control (Fisher’s Exact test, p = 0.05). However, no statistically significant difference was noted in any treatment groups from the solvent control (Fisher’s Exact test, p > 0.05). Since the percent hatching success in the 8.6 µg/L treatment group met the control validity criteria of >70%, the statistically significant reduction in this treatment group from the negative control was not considered to be biologically meaningful. Consequently, the NOEC for hatching success was 8.6 µg/L and the LOEC was >8.6 µg/L.

2. Hatching Success Exclude Data from Solvent Control Replicate D
Percent hatching success in the negative control and solvent control was 94 and 87%, respectively. Since no statistically significant difference was noted between the negative control and solvent control, the control data were pooled for comparison with the treatment data. Hatching success in the pooled control and the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 91, 94, 96, 91, 88 and 73%, respectively (Table 5). A statistically significant reduction in percent hatching success was noted in the 8.6 µg/L treatment group from the pooled control (Fisher’s Exact test, p = 0.05). Since the percent hatching success in the 8.6 µg/L treatment group met the control validity criteria of >70%, the statistically significant reduction in this treatment group from the pooled control was not considered to be biologically meaningful. Consequently, the NOEC for hatching success was 8.6 µg/L and the LOEC was >8.6 µg/L.

Larval Survival and Clinical Observations
Survival of the fathead minnow larvae through Day 28 post-hatch and overall survival are summarized in Table 5. The LCx values for post hatch and overall survival were reported in Table 6.

1. Larval Survival Include Data from Solvent Control Replicate D
The post-hatch survival in the negative control and solvent control was 81 and 67%, respectively. Because no statistically significant difference was found between the two controls, the control data were pooled for comparison with the treatment groups. The post-hatch survival in the pooled control and the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 75, 88, 87, 62, 40 and 67%, respectively (Table 5). Fisher’s Exact test indicated statistically significant decreases in post-hatch survival in the 1.3 and 3.5 µg/L treatment groups in comparison to the pooled controls (p = 0.05). However, the percent post-hatch survival in the 8.6 µg/L treatment group did not meet the control validity criteria and was considered to be treatment related. Consequently, the NOEC for post-hatch survival was 0.50 µg/L and the LOEC was 1.3 µg/L.

Overall survival in the negative control and solvent control was 76 and 50%, respectively. Statistically significant difference was noted between the two controls; therefore, the data from the treatment groups were compared to the negative control and solvent control separately. The overall survival in the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 83, 84, 56, 35 and 49%, respectively (Table 5). Fisher’s Exact test indicated statistically significant reductions (p = 0.05) in overall survival in the 1.3, 3.5 and 8.6 µg/L treatment groups from the negative control and in the 3.5 µg/L treatment group from the solvent control. Conservatively, the NOEC for overall survival was 0.50 µg/L and the LOEC was 1.3 µg/L.

2. Larval Survival Exclude Data from Solvent Control Replicate D
The post-hatch survival in the negative control and solvent control was 81 and 71%, respectively. Because no statistically significant difference was found between the two controls, the control data were pooled for comparison with the treatment groups. The post-hatch survival in the pooled control and the 0.20, 0.50, 1.3 and 3.5 µg/L treatment groups was 77, 88, 87, 62, 40 and 67%, respectively (Table 5). Fisher’s Exact test indicated statistically significant decreases in post-hatch survival in the 1.3 and 3.5 µg/L treatment groups in comparison to the pooled controls (p = 0.05). Consequently, the NOEC for post-hatch survival was 0.50 µg/L and the LOEC was 1.3 µg/L.

Overall survival in the negative control and solvent control was 76 and 62%, respectively. Since no statistically significant difference was noted between the two controls; the data from the controls were pooled for comparison to the treatment data. The overall survival in the pooled control and the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups was 70, 83, 84, 56, 35 and 49%, respectively (Table 5). Fisher’s Exact test indicated statistically significant reductions (p = 0.05) in overall survival in the 1.3, 3.5 and 8.6 µg/L treatment groups from the pooled control. Consequently, the NOEC for overall survival was 0.50 µg/L and the LOEC was 1.3 µg/L.

The fish in the control group and in the 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups were observed during the test for sub-lethal effects during the 28-day post hatch period. There were some observations of organisms that appeared smaller in comparison to the fish in the control replicates, weak, curled, crooked spine, discolored (pale), loss of equilibrium, lying on the bottom of the test chamber, swimming erratically, noted hemorrhaging on top of the head, noted with air bubble in abdomen (causing fish to rise to the surface), noted with inflamed opercula and / or noted with a protrusion on the left side of abdomen. However, these observations were infrequent and were comparable in the control.

Growth
Growth measurements at the end of the 28-day post-hatch period are summarized in Table 5. The ICx values for total length, wet and dry weight were reported in Table 6.

1. Growth Include Data from Solvent Control Replicate D
Because no statistically significant difference was found between the mean total length of the negative control and solvent control, the control data were pooled for comparison with the treatment data. No statistically significant difference in mean total length in any of the treatment groups when compared to the pooled control (Dunnett’s one-tailed test, p > 0.05).

Since significant differences were found between the negative control and solvent control mean wet and dry weight, the treatment data were compared to the negative control and solvent control data separately. No statistically significant reductions were found in any treatment group wet or dry weight data when compared to the solvent control (Dunnett’s one-tailed test, p > 0.05). Dunnett’s one-tailed test indicated statistically significant reductions (p = 0.05) in mean wet weight in the 0.50 and 8.6 µg/L treatment groups and in mean dry weight in the 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups from the negative control. Since the mean wet weight in the 0.50 µg/L treatment group did not follow a dose response pattern, the significant reduction noted in this treatment group was not considered to be biologically meaningful.

2. Growth Exclude Data from Solvent Control Replicate D
The control total length data were pooled for comparison with the treatment data because no statistically significant difference was found between the mean total length of the negative control and solvent control. No statistically significant difference in mean total length in any of the treatment groups when compared to the pooled control (Dunnett’s one-tailed test, p > 0.05).

Because statistically significant differences were found between the negative control and solvent control mean wet and dry weight, the treatment data were compared to the negative control and solvent control data separately. There were no statistically significant reductions noted in any treatment groups wet or dry weight data when compared to the solvent control (Dunnett’s one-tailed test, p > 0.05). Dunnett’s one tailed test indicated statistically significant reductions (p = 0.05) in mean wet weight in the 0.50 and 8.6 µg/L treatment groups from the negative control. Since the mean wet weight in the 0.50 µg/L treatment group did not follow a dose response pattern, the significant reduction noted was not considered to be biologically meaningful. The mean dry weight in the 0.50, 1.3, 3.5 and 8.6 µg/L treatment groups were statistically significant differences from the negative control (Dunnett’s one-tailed test, p = 0.05). Consequently, the NOEC and LOEC based on growth (mean total length, wet and dry weight) was 0.20 and 0.50 µg/L, respectively.

Percent Inhibition
The percent inhibitions for all endpoints were calculated with the data from solvent control replicate D included and excluded and the results are summarized in Table 7.

Any other information on results incl. tables

Table 1

 

Measured Concentrations of EKKE in Stock Solution Samples

 

Nominal Test

Concentration

(µg/mL)

Sample

Number

(524A-139-)*

Sampling

Time

(Day)

Measured

Concentration

(µg/mL)**

Percent

of

Nominal*,**

2.6

S1

-6

2.56

98.6

6.4

S2

-6

6.21

97.1

16

S3

-6

16.4

102

40

S4

-6

41.6

104

100

S5

-6

99.3

99.3

*  Sample numbers are reported as listed in the raw data but are associated with study number 524A-139A.

**  Results were generated using Analyst version 1.6.3. Manual calculations may differ slightly.

Table 2

 

Measured Concentrations of EKKE in Pre-Test Diluter Verification Samples

 

 

Nominal Test

Concentration

(µg/L)

 

Sample

Number

(524A-139-)*

 

Sampling

Time

(Day)

 

Measured

Concentration

(µg/L)**,***

 

Percent

of

Nominal***

 

0.26

8

-6

0.189

72.8

 

 

 

 

 

 

 

0.64

9

-6

0.371

58.0

 

 

 

 

 

 

 

1.6

10

-6

1.33

83.3

 

 

 

 

 

 

 

4.0

11

-6

3.05

76.4

 

 

 

 

 

 

 

10

12

-6

8.56

85.6

 

* Sample numbers are reported as listed in the raw data but are associated with study number 524A-139A.

**The method limit of quantitation (LOQ) for these analyses was set at 0.170 µg/L, based upon the lowest analyte concentration in a fortified sample that obtained a mean recovery of 70-110%.

***Results were generated using Analyst version 1.6.2. Manual calculations may differ slightly.

Table 3

 

Measured Concentrations of EKKE in Test Solution Samples

 

 

 

Nominal Test

Concentration

(µg/L)

 

 

Sample

Number

(524A-139A-)

 

 

Sampling

Time

(Day)

 

 

Measured

Concentration

(µg/L)*,**

 

 

Percent

of

Nominal**

Time-weighted

Mean ± SD

Measured

Concentration

(µg/L)***

Time-weighted

Mean

Measured

Percent of

Nominal***

Negative Control

1

0

< LOQ

--

--

--

(0.0)

13

7

< LOQ

--

20

14

< LOQ

--

27

21

< LOQ

--

35

28

< LOQ

--

42

33

< LOQ

--

 

 

 

 

 

 

 

Solvent Control

2

0

< LOQ

--

--

--

(0.0)

14

7

< LOQ

--

21

14

< LOQ

--

28

21

< LOQ

--

36

28

< LOQ

--

43

33

< LOQ

--

 

 

 

 

 

 

 

0.26

3

0

0.190

73.2

0.20 ± 0.0196

78.4

8****

6

< LOQ*****

--

(%CV = 9.62)

 

15

7

0.190

73.1

 

 

22

14

0.223

85.8

29

21

0.258

99.4

37

28

0.180

69.3

44

33

0.260

99.8

 

 

 

 

 

 

 

0.64

4

0

0.585

91.4

0.50 ± 0.0380

78.4

 

9****

6

0.239

37.4

(%CV = 7.58)

 

 

16

7

0.566

88.4

 

 

 

23

14

0.679

106

 

 

 

30

21

0.313******

48.9

 

 

 

30-RE

21

0.325******

50.8

 

 

 

34

22

0.418

65.4

 

 

 

38

28

0.494

77.1

 

 

 

45

33

0.594

92.8

 

 

*   The method limit of quantitation (LOQ) for these analyses was set at 0.170 µg/L, based upon the lowest analyte concentration in a fortified sample that obtained a mean recovery of 70-110%.

** Results were generated using Analyst version 1.6.2. Manual calculations may differ slightly.

***Results were generated using Excel 2010. Manual calculations may differ slightly.

****         Samples collected due to toxicant delivery interruption.

*****        Half of the LOQ (0.0850 µg/L) was used to calculate the time-weighted mean measured concentration for sample 524A-139A-8.

******       Sample 524A-139A-30 was reanalyzed on Day 22 (as sample 524A-139A-30-RE) and the result of the reanalysis confirmed the original. The time-weighted mean measured concentration for the 0.64 test concentration was calculated using the mean result of the original and reanalysis.


Table 3 (Continued)

 

Measured Concentration of EKKE in Test Solution Samples

 

 

 

Nominal Test

Concentration

(µg/L)

 

 

Sample

Number

(524A-139A-)

 

 

Sampling

Time

(Day)

 

 

Measured

Concentration

(µg/L)*,**

 

 

Percent

of

Nominal**

Time-weighted

Mean ± SD

Measured

Concentration

(µg/L)***

Time-weighted

Mean

Measured

Percent of

Nominal***

1.6

5

0

0.944

59.0

1.3 ± 0.154

81.2

 

10****

6

0.470

29.4

(%CV = 11.8)

 

 

17

7

1.24

77.4

 

 

 

24

14

1.63

102

 

 

 

31

21

1.37

85.5

 

 

 

39

28

1.47

91.7

 

 

 

46

33

1.44

90.1

 

 

 

 

 

 

 

 

 

4.0

6

0

3.45

86.2

3.5 ± 0.505

88.2

 

11****

6

< LOQ*****

--

(%CV = 14.3)

 

 

18

7

3.53

88.2

 

 

 

25

14

4.51

113

 

 

 

32

21

3.97

99.4

 

 

 

40

28

3.58

89.4

 

 

 

47

33

4.35

109

 

 

 

 

 

 

 

 

 

10

7

0

9.65

96.5

8.6 ± 0.735

85.8

12****

6

2.81

28.1

(%CV = 8.6)

 

19

7

8.87

88.7

 

 

26

14

10.3

103

33

21

8.02

80.2

 

41

28

9.66

96.6

 

 

48

33

9.10

91.0

*   The method limit of quantitation (LOQ) for these analyses was set at 0.170 µg/L, based upon the lowest analyte concentration in a fortified sample that obtained a mean recovery of 70-110%.

** Results were generated using Analyst version 1.6.2. Manual calculations may differ slightly.

***Results were generated using Excel 2010. Manual calculations may differ slightly.

****         Samples collected due to toxicant delivery interruption.

*****        Half of the LOQ (0.0850 µg/L) was used to calculate the time-weighted mean measured concentration for sample 524A-139A-11.

Table 4

 

Means and Ranges of Water Quality Measurements Taken During the 33-Day Exposure to EKKE

 

Mean Measured

Concentration

(µg/L)

Mean ± SD and Range of Measured Parameters

Temperature*

(°C)

DO**

(mg/L)

 

pH***

Hardness****

(mg/L as CaCO3)

Alkalinity****

(mg/L as CaCO3)

Conductivity****

(mS/cm)

 

 

 

 

 

 

 

Negative Control

24.8 ± 0.28

8.1 ± 0.12

8.0 ± 0.10

147 ± 3

179 ± 1

325 ± 10

 

(24.3 – 25.2)

(7.9 – 8.2)

(7.8 – 8.1)

(144 – 152)

(178 – 180)

(313 – 337)

 

 

 

 

 

 

 

Solvent Control

24.5 ± 0.27

7.9 ± 0.21

8.0 ± 0.08

--

--

--

 

(24.0 – 24.8)

(7.6 – 8.2)

(7.8 – 8.0)

--

--

--

 

 

 

 

 

 

 

0.20

24.6 ± 0.27

7.9 ± 0.22

8.0 ± 0.08

--

--

--

 

(24.2 – 25.0)

(7.6 – 8.2)

(7.9 – 8.1)

--

--

--

 

 

 

 

 

 

 

0.50

24.4 ± 0.26

7.9 ± 0.21

8.0 ± 0.06

--

--

--

 

(23.9 – 24.7)

(7.7 – 8.2)

(7.9 – 8.1)

--

--

--

 

 

 

 

 

 

 

1.3

24.4 ± 0.25

7.9 ± 0.19

8.0 ± 0.04

--

--

--

 

(24.0 – 24.8)

(7.7 – 8.2)

(7.9 – 8.0)

--

--

--

 

 

 

 

 

 

 

3.5

24.6 ± 0.20

7.9 ± 0.26

8.0 ± 0.08

--

--

--

 

(24.3 – 24.9)

(7.5 – 8.2)

(7.9 – 8.1)

--

--

--

 

 

 

 

 

 

 

8.6

24.5 ± 0.21

7.9 ± 0.15

8.0 ± 0.04

146 ± 3

179 ± 2

326 ± 12

 

(24.2 – 24.8)

(7.7 – 8.1)

(7.9 – 8.0)

(140 – 148)

(174 – 180)

(314 – 346)

 

 

 

 

 

 

 

*Temperature measured continuously during the test ranged from 23.73 to 25.34°C. Temperature measured when 100% mortality occurred in the replicate was within the 25 ± 1ºC.

**A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation at 25ºC in freshwater. A dissolved oxygen when 100% mortality occurred in the replicate was 8.2 mg/L.

***                                                                                           pH measured when 100% mortality occurred in the replicate was 7.9.

****                                                                                                                                                                                     -- = no measurements scheduled.

Table 5

 

Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed toEKKE

 

Results Include Data from Solvent Control Replicate D

 

Time-Weighted

Mean Measured

Concentration

(µg/L)

 

Hatching

Success

(%)*

Mean ± SD.

Time to

Hatch

(day)*

 

Post-Hatch

Survival

(%)

 

Overall

Survival

(%)

Growth at Day 28 Post-Hatch

Mean ± SD.

Total Length

(mm)*

Mean ± SD.

Wet Weight

(mg)***

Mean ± SD.

Dry Weight

(mg)***

Negative Control

94

4.1 ± 0.22

81

76

24.0 ± 0.32

113 ± 0.82

23.3 ±0.62

Solvent Control

75

5.1 ± 0.17

67

50

22.9 ± 0.74

96.8 ± 9.4

18.3 ± 3.0

Pooled Control

--**

--**

75

--**

23.5 ± 0.76

--**

--**

0.20

94

4.5 ± 0.26ß

88

83

24.1 ± 0.094

116 ± 8.7

22.7 ± 2.2

0.50

96

4.1 ± 0.24ß

87

84

23.4 ± 0.54

101 ± 6.8¿

19.9 ± 1.4¿

1.3

91

5.5 ± 0.16

62

56¿

23.5 ± 0.12

102 ± 3.2

19.9 ± 0.95¿

3.5

88

5.7 ± 0.33

40

35¿,±

22.8 ± 0.65

103 ± 6.6

19.6 ± 1.4¿

8.6

73§

5.5 ± 0.23

67

49¿

22.9 ± 0.91

96.7 ± 11¿

18.4 ± 2.8¿

* There were no statistically significant differences (p>0.05) in mean length in any treatment groups from the pooled control (Dunnett’s one-tailed test), in mean time to hatch from the negative control (Dunnett’s one-tailed test, p > 0.05) or in mean percent hatching success from the solvent control (Fisher’s Exact test, p > 0.05).

**                                                   Due tostatistically significant differences found between the negative control and solvent control, mean time to hatch, percent overall survival, wet and dry weight data, the treatment data were compared to the negative control and solvent control separately.

***                                                                             No statistically significant reduction in mean wet and dry weight in any of the treatment groups in comparison to the solvent control (Dunnett’s one-tailed test, p>0.05).

§   Indicates statistically significant difference in percent hatching success in the 8.6 µg/L treatment group from the negative control (Fisher’s Exact test, p=0.05). However, the percent hatching success in this treatment group met the control validity criteria of >70% and was similar to that of the solvent control; therefore, it was not considered to be biologically meaningful.

ß   Indicated statistically significant difference in mean time to hatch from the solvent control (Dunnett’s one-tailed test, p=0.05). However, the differences were approximately 14.4 to 24 hours and were not considered to be biologically significance as these time were comparable to the negative control.

   Indicates statistically significant reductions in mean percent post-hatch survival from the pooled control (Fisher’s Exact test, p=0.05).

±   Indicates statistically significant difference in percent overall survival from the solvent control (Fishers Exact test, p=0.05).

¿ Indicates statistically significant differences (p=0.05) in percent overall survival and mean wet weight, dry weight from the negative control (Fisher’s Exact test for percent overall survival and Dunnett’s one-tailed test for wet and dry weight).


Table 5 (Continued)

 

Summary of Hatching Success, Larval Survival and Growth of Fathead Minnows Exposed toEKKE

 

Results Exclude Data from Solvent Control Replicate D

 

Time-Weighted

Mean Measured

Concentration

(µg/L)

 

Hatching

Success

(%)

Mean

Time to

Hatch ± SD.

(day)*

 

Post-Hatch

Survival

(%)

 

Overall

Survival

(%)

Growth at Day 28 Post-Hatch

Mean ± SD.

Total Length

(mm)*

Mean ± SD.

Wet Weight

(mg)***

Mean ± SD.

Dry Weight

(mg)***

Negative Control

94

4.1 ± 0.22

81

76

24.0 ± 0.32

113 ± 0.82

23.3 ±0.62

Solvent Control4

87****

5.1 ± 0.20

71

62

23.2 ± 0.49

101 ± 4.5

19.8 ± 0.41

Pooled Control

91

--**

77

70

23.7 ± 0.53

--**

--**

0.20

94

4.5 ± 0.26ß

88

83

24.1 ± 0.094

116 ± 8.7

22.7 ± 2.2

0.50

96

4.1 ± 0.24ß

87

84

23.4 ± 0.54

101 ± 6.8¿

19.9 ± 1.4¿

1.3

91

5.5 ± 0.16

62

56§

23.5 ± 0.12

102 ± 3.2

19.9 ± 0.95¿

3.5

88

5.7 ± 0.33

40

35§

22.8 ± 0.65

103 ± 6.6

19.6 ± 1.4¿

8.6

73§

5.5 ± 0.23

67

49§

22.9 ± 0.91

96.7 ± 11¿

18.4 ± 2.8¿

* There were no statistically significant differences (p>0.05) in mean total length in any treatment groups from the pooled control (Dunnett’s one-tailed test) or in mean time to hatch when compared to the negative control (Dunnett’s one-tailed test, p > 0.05).

**                                                   Due tostatistically significant differences found between the negative control and solvent control, mean time to hatch, percent overall survival, wet and dry weight data, the treatment data were compared to the negative control and solvent control separately.

***                                                                             No statistically significant reduction in mean wet and dry weight in any of the treatment groups in comparison to the solvent control (Dunnett’s one-tailed test, p>0.05).

****                                               Because the percent mortality of embryos and larvae of the solvent control replicate D were unusually high, data from solvent control replicate D were excluded from data analyses of all endpoints.

ß   Indicated statistically significant difference in mean time to hatch from the solvent control (Dunnett’s one-tailed test, p=0.05). However, the differences were approximately 14.4 to 24 hours and were not considered to be biologically significance.

§   Indicates statistically significant differences in percent hatching success from the pooled control (Fisher’s Exact test, p=0.05).

   Indicates statistically significant reductions in mean percent post-hatch survival from the pooled control (Fisher’s Exact test, p=0.05). However, the mean percent post-hatch survival did not follow a dose response pattern and therefore, were not considered to be biologically meaningful.

¿ Indicates statistically significant differences (p=0.05) in mean wet weight or mean dry weight from the negative control (Dunnett’s one-tailed test for wet and dry weight).

Table 6

 

10% and 20% Effective Concentrations and 95% Confidence Intervals

 

Results Include Data from Solvent Control Replicate D

 

 

Parameter

LC/IC10

(95% Confidence Interval)

(µg/L)

LC/IC20

(95% Confidence Interval)

(µg/L)

 

NOEC

(µg/L)

 

LOEC

(µg/L)

Compared to pooled control

 

 

 

 

Post-hatch larval survival

1.1***

4.***

0.50

1.3

Total length

>8.6***

>8.6***

8.6

>8.6

Compared to negative control*

 

 

 

 

Hatching success

4.2 (1.6 - >8.6)

<0.20**, ***

3.5

8.6

Mean time to hatch

NA

NA

8.6

>8.6

Overall larval survival

<0.20***

0.51***

0.50

1.3

Wet weight

2.0***

<0.20**, ***

0.20

0.50

Dry weight

0.42***

5.6 (0.90 - >8.6)

0.20

0.50

Compared to solvent control*

 

 

 

 

Hatching success

6.5 (2.0 – >8.6)

>8.6***

8.6

>8.6

Mean time to hatch

NA

NA

8.6

>8.6

Overall larval survival

2.3***

2.4***

8.6

>8.6

Wet weight

>8.6***

>8.6***

8.6

>8.6

Dry weight

>8.6***

>8.6***

8.6

>8.6

*       Due to a statistically significant difference between the negative control and solvent control percent overall survival, mean wet and dry weight, the percent inhibition is calculated as compared to the negative control and solvent control separately.

**  The calculated LCx values were not realistic and were not reported.

***   The 95% confidence intervals were overly wide and were not reported.

      NA = not applicable; LC/ICx not required.

Table 6 (Continued)

 

10% and 20% Effective Concentrations and 95% Confidence Intervals

 

Results Exclude Data from Solvent Control Replicate D

 

 

 

Parameter

LC/IC10

(95% Confidence Interval)

(µg/L)

LC/IC20

(95% Confidence Interval)

(µg/L)

 

NOEC

(µg/L)

 

LOEC

(µg/L)

Compared to pooled control

 

 

 

 

Hatching success

4.8 (2.1 - >8.6)

8.0 (5.3 - >8.6)

3.5

8.6

Post-hatch larval survival

0.63***

2.8***

0.50

1.3

Overall larval survival**

<0.20***

>8.6***

0.50

1.3

Total length

>8.6***

>8.6***

8.6

>8.6

Compared to negative control*

 

 

 

 

Mean time to hatch

NA

NA

8.6

>8.6

Wet weight

2.0***

<0.20**, ***

0.20

0.50

Dry weight

0.42***

5.6 (0.90 - >8.6)

0.20

0.50

Compared to solvent control*

 

 

 

 

Mean time to hatch

NA

NA

8.6

>8.6

Wet weight

>8.6***

>8.6***

8.6

>8.6

Dry weight

6.8 (2.0 - >8.6)

>8.6***

8.6

>8.6

* Due to statistically significant differences in wet and dry weight between the negative control and solvent control, the treatment data were compared to the negative and solvent control separately.

**The calculated LCx values did not meet the criteria and were not realistic. Therefore, the values were not reported.

***                 The 95% confidence intervals were overly wide and were not reported

   NA = not applicable; LC/ICx not required.

Table 7

 

Percent Inhibition from the Negative Control, Solvent Control or Pooled Control

 

Results Include Data from Solvent Control Replicate D

 

 

Time-Weighted

Mean Measured

Concentration

(µg/L) 

 

 

Percent

Hatching

Success

%

Inhibition

From

Negative

Control

%

Inhibition

From

Solvent

Control

 

Mean

Time

to Hatch

(Day)

%

Inhibition

From

Negative

Control

%

Inhibition

From

Solvent

Control

 

Percent

Post-

Hatch

Survival

%

Inhibition

From

Pooled

Control

 

 

Percent

Overall

Survival*

%

Inhibition

From

Negative

Control

%

Inhibition

From

Solvent

Control

Negative Control

94

--

--

4.1

--

--

81

--

76

--

--

Solvent Control

75

--

--

5.1

--

--

67

--

50

--

--

Pooled Control

--*

--

--

--*

--

--

75

--

--*

--

--

0.20

94

0.0

-25

4.5

-9.8

12

88

-17

83

-9.2

-66

0.50

96

-2.1

-28

4.1

0.0

20

87

-16

84

-11

-68

1.3

91

3.2

-21

5.5

-34

-7.8

62

17

56

26

-12

3.5

88

6.4

-17

5.7

-39

-12

40

47

35

54

30

8.6

73

22

2.7

5.5

-34

-7.8

67

11

49

36

2.0

*     Due to a statistically significant difference between the negative control and solvent control percent overall survival, the percent inhibition is calculated as compared to the negative control and solvent control separately.

Note: Stimulate or a greater response in the test substance treatment than the control is reported as negative % inhibition.

 

 

Time-Weighted

Mean Measured Concentration

(µg/L) 

 

Mean Total

Length

(mm)

 

% Inhibition

From Pooled

Control

 

Mean Wet

Weight

(mg)*

 

% Inhibition

From Negative

Control

 

% Inhibition

From Solvent

Control

 

Mean Dry

Weight

(mg)*

 

% Inhibition

From Negative

Control

 

% Inhibition

From Solvent

Control

Negative Control

24.0

--

116

--

--

23.3

--

--

Solvent Control

22.9

--

96.8

--

--

18.3

--

--

Pooled Control

23.5

--

--*

--

--

--*

--

--

0.20

24.1

-2.6

116

0.0

-20

22.7

2.6

-

0.50

23.4

0.4

101

13

-4.3

19.9

15

-8.7

1.3

23.5

0.0

102

12

-5.4

19.9

15

-8.7

3.5

22.8

3.0

103

11

-6.4

19.6

16

-7.1

8.6

22.9

2.6

96.7

17

0.10

18.4

21

-0.5

*     Due to a statistically significant difference between the negative control and solvent control mean wet and dry weight, the percent inhibition is calculated as compared to the negative control and solvent control separately.

Note: Stimulate or a greater response in the test substance treatment than the control is reported as negative % inhibition.


Table 7 (Continued)

 

Percent Inhibition from the Negative Control, Solvent Control or Pooled Control

 

Results Exclude Data from Solvent Control Replicate D

 

Time-Weighted

Mean Measured

Concentration

(µg/L) 

 

Percent

Hatching

Success

 

% Inhibition

From Pooled

Control

 

Mean Time

to Hatch

(Day)

%

Inhibition

From Negative

Control

%

Inhibition

From Solvent

Control

 

Percent

Post-Hatch

Survival

%

Inhibition

From Pooled

Control

 

Percent

Overall

Survival*

 

% Inhibition

From Pooled

Control

Negative Control

94

--

4.1

--

--

81

--

76

--

Solvent Control

87

--

5.1

--

--

71

--

62

--

Pooled Control

91

--

--*

--

--

77

--

70

--

0.20

94

-3.3

4.5

-9.8

12

88

-14

83

-19

0.50

96

-5.5

4.1

0.0

20

87

-13

84

-20

1.3

91

0.0

5.5

-34

-7.8

62

19

56

20

3.5

88

3.3

5.7

-39

-12

40

48

35

50

8.6

73*

19.8

5.5

-34

-7.8

67

13

49

30

*  Due to a statistically significant difference between the negative control and solvent control mean percent inhibition values were derived as compared to the negative control and solvent control separately.

Note: Stimulate or a greater response in the test substance treatment than the control is reported as negative % inhibition.

 

 

Time-Weighted

Mean Measured Concentration

(µg/L) 

 

Mean Total

Length

(mm)

 

% Inhibition

From Pooled

Control

 

Mean Wet

Weight

(mg)*

 

% Inhibition

From Negative

Control

 

% Inhibition

From Solvent

Control

 

Mean Dry

Weight

(mg)*

 

% Inhibition

From Negative

Control

 

% Inhibition

From Solvent

Control

Negative Control

24.0

--

113

--

--

23.3

--

--

Solvent Control

23.2

--

101

--

--

19.8

--

--

Pooled Control

23.7

--

--*

--

--

--*

--

--

0.20

24.1

-1.7

116

-2.7

-15

22.7

2.6

-15

0.50

23.4

1.3

101

11

0.0

19.9

15

-0.51

1.3

23.5

0.84

102

9.7

-1.0

19.9

15

-0.51

3.5

22.8

3.8

103

8.8

-2.0

19.6

16

1.0

8.6

22.9

3.4

96.7

14

4.3

18.4

21

7.1

*  Due to a statistically significant difference between the negative control and solvent control mean percent inhibition values were derived as compared to the negative control and solvent control separately.

Note: Stimulate or a greater response in the test substance treatment than the control is reported as negative % inhibition.

 

Temperature of Water in the Test Chambers During Exposure to EKKE

 

Time-Weighted

Mean Measured

Concentration

(µg/L)

 

 

 

Replicate

Temperature (°C) by Test Day*

0

7

14

21

28

33

 

 

 

 

 

 

 

 

Negative Control

A

24.6

24.4

25.0

25.0

25.2

25.0

 

B

24.5

24.3

24.9

24.9

25.1

24.9

 

C

24.4

24.3

24.8

24.8

25.0

24.9

 

D

24.4

24.3

24.9

24.8

25.0

24.9

 

 

 

 

 

 

 

 

Solvent Control

A

24.2

24.2

24.8

24.5

24.8

24.7

 

B

24.1

24.1

24.6

24.6

24.7

24.6

 

C

24.1

24.1

24.6

24.6

24.7

24.6

 

D

24.1

24.0

24.6

24.6

24.7

24.7

 

 

 

 

 

 

 

 

0.20

A

24.4

24.2

24.9

24.6

25.0

24.9

 

B

24.4

24.2

24.7

24.7

24.9

24.8

 

C

24.3

24.2

24.7

24.7

24.9

24.7

 

D

24.3

24.2

24.8

24.7

24.9

24.8

 

 

 

 

 

 

 

 

0.50

A

24.2

24.1

24.6

24.5

24.7

24.7

 

B

24.1

24.0

24.4

24.5

24.6

24.5

 

C

24.0

23.9

24.5

24.5

24.5

24.5

 

D

24.0

24.0

24.7

24.5

24.6

24.5

 

 

 

 

 

 

 

 

1.3

A

24.2

24.1

24.8

24.5

24.7

24.6

 

B

24.1

24.0

24.6

24.4

24.6

24.5

 

C

24.1

24.1

24.6

24.4

24.6

24.6

 

D

24.1

24.1

24.7

24.4

24.6

24.7

 

 

 

 

 

 

 

 

3.5

A

24.5

24.4

24.9

24.7

24.8

24.7

 

B

24.4

24.4

24.7

24.7

24.7

24.8

 

C

24.4

24.3

24.8

24.7

24.7

24.8

 

D

24.4

24.3

24.9

--

--

--

 

 

 

 

 

 

 

 

8.6

A

24.3

24.2

24.8

24.6

24.6

24.6

 

B

24.3

24.2

24.7

24.5

24.6

24.6

 

C

24.2

24.2

24.7

24.6

24.7

24.6

 

D

24.2

24.2

24.8

24.5

24.6

24.7

 

 

 

 

 

 

 

 

*  Temperature measured continuously during the test ranged from 23.73 to 25.34°C. Temperature measured when 100% mortality occurred in the replicate was within the 25 ± 1ºC.

    -- = No data due to 100% mortality.

 

Dissolved Oxygen Content of Water in the Test ChambersDuring Exposure to EKKE

 

Time-Weighted

Mean Measured

Concentration

(µg/L)

Dissolved Oxygen (mg/L) by Test Day*

0

7

14

21

28

33

Negative Control

8.0

7.9

8.0

8.2

8.1

8.2

Solvent Control

7.9

7.6

7.8

8.2

8.1

7.9

0.20

8.2

7.6

7.8

8.1

7.8

7.8

0.50

8.2

7.7

7.9

8.0

7.7

7.7

1.3

8.2

7.8

7.7

8.1

7.8

7.9

3.5

8.2

7.5

7.8

8.1**

7.9

8.1

8.6

8.0

7.7

8.0

8.1

7.8

7.9

Replicate

A

B

C

D

A

B

* A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation in well water at a temperature of 25°C.

**Due to 100% mortality in replicate D, measurement was made in replicate A. A dissolved oxygen when 100% mortality occurred in the replicate was 8.2 mg/L.

pH of Water in the Test Chambers During Exposure to EKKE

 

Time-Weighted

Mean Measured

Concentration

(µg/L)

pH by Test Day*

0

7

14

21

28

33

Negative Control

8.0

7.8

8.0

7.9

7.9

8.1

Solvent Control

8.0

7.8

8.0

7.9

8.0

8.0

0.20

8.0

7.9

8.0

8.1

8.0

8.1

0.50

8.0

7.9

8.0

8.0

8.0

8.1

1.3

8.0

7.9

8.0

8.0

8.0

8.0

3.5

8.0

7.9

8.0

7.9**

8.0

8.1

8.6

8.0

7.9

8.0

8.0

8.0

8.0

Replicate

A

B

C

D

A

B

*   pH measurement when 100% mortality occurred in the replicate was 7.9.

**    Measurement was made in replicate A due to 100% mortality in replicate D.


Specific Conductance, Hardness and Alkalinity in the Negative Control

and Highest Concentration Treatment GroupDuring Exposure to EKKE

 

 

Negative Control

 

 

Parameter

Test Day

0

7

14

21

28

33

 

 

 

 

 

 

 

Specific Conductance

323

331

313

337

315

331

  (mS/cm)

 

 

 

 

 

 

 

 

 

 

 

 

 

Hardness

148

148

144

152

144

148

  (mg/L as CaCO3)

 

 

 

 

 

 

 

 

 

 

 

 

 

Alkalinity

178

180

180

180

178

180

  (mg/L as CaCO3)

 

 

 

 

 

 

 

 

 

 

 

 

 

Replicate

A

B

C

D

A

B

 

 

 

The Highest Treatment Group

 

 

Parameter

Test Day

0

7

14

21

28

33

 

 

 

 

 

 

 

Specific Conductance

325

333

314

346

322

318

  (mS/cm)

 

 

 

 

 

 

 

 

 

 

 

 

 

Hardness

148

148

148

148

144

140

  (mg/L as CaCO3)

 

 

 

 

 

 

 

 

 

 

 

 

 

Alkalinity

180

180

178

180

180

174

  (mg/L as CaCO3)

 

 

 

 

 

 

 

 

 

 

 

 

 

Time-Weighted

Mean Measured Test Concentration (µg/L)

8.6

8.6

8.6

8.6

8.6

8.6

Replicate

A

B

C

D

A

B


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Conditions for the Validity of the Test
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 = 91% (7.5 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 23.73 to 25.34°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 time-weighted mean measured value. The coefficients of variation for all treatment groups ranged from 7.58 to 14.3% 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 control group was 94%. At test termination, post-hatch larval survival in the negative control was 81%.
Percent hatching success in the solvent control was lower than typically seen in historical control data for the solvent control (97.9% with the range of 88 to 100%) due to an unusually low survival of the embryos in replicate D (40%). When the data of this replicate was included in the calculation of the percent hatching success, the percent hatching success of the solvent control was 75%, but was 87% when the data from this replicate was excluded. Percent post-hatch survival of the solvent control was 67% when the data from replicate D was included and was 71% when the data from this replicate was excluded. Although the percent survival of the larvae in the solvent control did not meet the control criteria, the percent post-hatch survival of the negative control met with criteria and the study results were conservatively based on both the negative control and solvent control.

CONCLUSIONS
Fathead minnows (Pimephales promelas) were exposed to EKKE at mean measured concentrations of 0.20, 0.50, 1.3, 3.5 and 8.6 µg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28 day post-hatch growth period). Because the hatching of embryos in the solvent control replicate D was unexpectedly low (40%) the results of the study were analyzed including and excluding the data from this replicate.

1. Results Include Data from Solvent Control Replicate D
There was no biologically meaningful treatment related decrease in hatching success at concentrations =8.6 µg/L in comparison to the negative control or solvent control. There were significant treatment related effect noted in post-hatch survival and overall survival at concentrations =1.3 µg/L in comparison to the negative and solvent control. Significant reductions in mean wet and dry weight were observed at concentration =0.50 µg/L when compared to the negative control. Due to the poor performance of the solvent control, there were no treatment related effects noted in wet and dry weight when compared to the solvent control.

2. Results Exclude Data from Solvent Control Replicate D
There were no biologically meaningful treatment-related effects on hatching success at concentrations =8.6 µg/L treatment group from the pooled control and no biologically significant treatment-related effects on mean time to hatch from the negative control or solvent control at concentration =8.6 µg/L. There were no biologically significant decreases in post-hatch survival noted at concentration =0.50 µg/L in comparison to the pooled controls and in overall survival at concentration =0.50 µg/L from the pooled control. There were no significant reductions in mean total length in any treatment group when compared to the pooled control. There were significant reductions noted in wet and dry weight data noted at concentrations =8.6 µg/L when compared to the solvent control, and at concentrations =0.20 µg/L when compared to the negative control. Due to the poor performance of the solvent control, there were no biologically meaningful effects noted in any endpoints when compared to the solvent control.

Based on the results with the data from replicate D included and excluded, growth (measured as dry weight) was the most sensitive biological endpoint measured in comparison to the negative control. The NOEC, based on growth, was 0.20 µg/L and the LOEC was 0.50 µg/L.

Executive summary:

This study was designed to determine the effects of the test item on the development of the early-life stages of the fathead minnow Pimephales promelas in a 33 days test according to the OECD 210 Guideline.

 

The total test period was 33 days. A continuous-flow diluter system was used to provide the test substance at five concentrations, a negative control (dilution water) and a solvent control (100 µL/L DMF).

The nominal concentrations were 0.0 (negative and solvent controls), 0.26, 0.64, 1.6, 4.0 and 10 µg/L. The exposure concentration was based on the time-weighted average of measured concentrations.

 

Based on the results with the data from replicate D included and excluded, growth (measured as dry weight) was the most sensitive biological endpoint measured in comparison to the negative control. The NOEC, based on growth, was 0.20 µg/L and the LOEC was 0.50 µg/L.