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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-07-11 to 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Water samples and stock solution were collected from one test chamber of each treatment and control group three and four 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, 6, 13, 21, 28 and 33 (test termination) to determine concentrations of the test substance in the test chambers. Additional stock solution samples were collected three days prior to test initiation and on the day of test initiation to confirm the concentration of the stock solution. The stock solution and water samples (8.00 mL) were collected at mid-depth in the test chambers, placed in glass vials containing acetonitrile (2.00 mL), using glass volumetric pipettes, and processed immediately for analysis or stored refrigerated until analysis.
Vehicle:
no
Details on test solutions:
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.

Preparation of Test Concentrations
Test solutions were prepared daily during the test. At each preparation, a primary stock solution at concentrations of 5.0 mg/L was prepared. A calculated nominal amount of test substance (0.6500 g) was weighed into a weigh boat and was rinsed into a 175 L stainless steel drum using a pre-measured ~20 to 80 mL of HPLC-grade acetone. Once all test substance was transferred into the drum, acetone was allowed to evaporate for approximately 10 to 30 minutes. Then 130 L of UV sterilized well water was measured and added to the stainless steel drum using calibrated plastic buckets and the solutions were stirred overnight to mix using a top-down electric mixer. The stock solutions appeared clear and colorless at the beginning of the mixing period and clear and colorless with a slight oil slick at the water’s surface.

The 5.0 mg/L stock solution was delivered to each mixing chamber of each treatment concentration to prepare test solutions at concentrations of 0.063, 0.13, 0.25 and 0.50 mg/L using the FMI pump and at concentration of 1.0 mg/L using a peristaltic pump. All test solutions appeared clear and colorless after mixing with the dilution water. The volume of the 5.0 mg/L stock solution delivered to each mixing chamber to prepare each test concentration is as follows:

Target Test Concentration (mg/L) // Volume of Stock Solution (mL/min) // Volume of Dilution Water (mL/min)***
Negative Control (0.0) 0.0 200
0.063* 2.52 198
0.13* 5.20 195
0.25* 10.0 190
0.50* 20.0 180
1.0** 40.0 160
* An FMI pump was used to deliver the toxicant to the mixing chamber.
** A peristaltic pump was used to deliver the toxicant to each of the mixing chambers.
*** Rotameters were used to deliver the dilution water to the mixing chambers to mix with the stock solution to prepare the test solutions.


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. 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 neurula). Embryos collected for use in the test were from 16 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, Ogden, Utah. 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 0.02 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 0.22 g fish/L.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Test temperature:
23.62 - 24.90 °C
pH:
8.0 - 8.3
Dissolved oxygen:
>= 95% (7.8 mg/L)
Nominal and measured concentrations:
Nominal
Negative Control
0.063 mg/L
0.13 mg/L
0.25 mg/L
0.50 mg/L
1.0 mg/L

Mean Measured
0.033 mg/L
0.061 mg/L
0.11 mg/L
0.25 mg/L
0.49 mg/L
Details on test conditions:
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 liquid-in-glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a validated environmental monitoring system (PointView Central Monitoring System), which were 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 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 Thermo Orion Star A122 Portable Conductivity Meter.
Reference substance (positive control):
no
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: Wet weight, dry weight
Key result
Duration:
33 d
Dose descriptor:
EC10
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: Wet weight
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.49 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Hatching success, time to hatch, post-hatch and overall survival, length

Table 1 

Measured Concentrations of BADGEDA in Stock Solution Samples

 

Nominal

Concentration

(mg/L)

Sample

Number

(803A-105-)

Sampling

Time

(Day)

Measured

Concentration

(mg/L)1

Percent

of

Nominal1

5.0

S-1

-3

2.46

49.2

5.0

S-2

0

1.70

33.9

1  Results were generated using OpenLab CDS Rev.A.01.05. Manual calculations may differ slightly.

 

Table 2

 

Measured Concentrations of BADGEDA in Pretest Diluter Verification Samples

 

Nominal

Concentration

(mg/L)

Sample

Number

(803A-105-)

Sampling

Time

(Day)

Measured

Concentration

(mg/L)*

Percent

of

Nominal**

Negative Control

PT-1

-4

< LOQ

--

 

PT-7

-3

< LOQ

--

 

 

 

 

 

0.063

PT-2

-4

0.0292***

46.3

 

PT-8

-3

0.0276***

43.8

 

 

 

 

 

0.13

PT-3

-4

0.0586

45.1

 

PT-9

-3

0.0562

43.3

 

 

 

 

 

0.25

PT-4

-4

0.116

46.3

 

PT-10

-3

0.117

46.7

 

 

 

 

 

0.50

PT-5

-4

0.234

46.8

 

PT-11

-3

0.238

47.5

 

 

 

 

 

1.0

PT-6

-4

0.454

45.4

 

PT-12

-3

0.484

48.4

 

 

 

 

 

*   The limit of quantitation (LOQ) for this study was set at 0.0300 mg/L, defined as the lowest matrix fortification concentration in which a mean recovery of 70-110% was obtained.

**  Results were generated using OpenLab CDS Rev.A.01.05. Manual calculations may differ slightly.

***        Result is below LOQ but quantifiable.


Table 3

 

Measured Concentrations of BADGEDA in Test Solution Samples

 

 

Nominal Test

Concentration

(mg/L)

 

Sample

Number

(803A-105-)

 

Sampling

Time

(Day)

 

Measured

Concentration

(mg/L)*,**

 

Percent

of

Nominal**

Time-Weighted Mean ± SD

Measured

Concentration

(mg/L)***

Time-Weighted Mean

Measured

Percent of Nominal***

Negative

1

0

< LOQ

--

< LOQ

--

Control

7

6

< LOQ

--

 

 

 

13

13

< LOQ

--

 

 

 

19

21

< LOQ

--

 

 

 

25

28

< LOQ

--

 

 

 

31

33

< LOQ

--

 

 

 

 

 

 

 

 

 

0.063

2

0

0.0186****

29.6

0.033 ± 0.00208

53

 

8

6

0.0324

51.4

(%CV = 6.28)

 

 

14

13

0.0338

53.6

 

 

 

20

21

0.0363

57.6

 

 

 

26

28

0.0395

62.7

 

 

 

32

33

0.0256****

40.6

 

 

 

 

 

 

 

 

 

0.13

3

0

0.0383

29.4

0.061 ± 0.00379

47

 

9

6

0.0617

47.5

(%CV = 6.18)

 

 

15

13

0.0563

43.3

 

 

 

21

21

0.0621

47.8

 

 

 

27

28

0.0821

63.1

 

 

 

33

33

0.0508

39.1

 

 

 

 

 

 

 

 

 

0.25

4

0

0.0800

32.0

0.11 ± 0.0539

44

 

10

6

0.118

47.1

(%CV = 4.88)

 

 

16

13

0.0903

36.1

 

 

 

22

21

0.120

47.8

 

 

 

28

28

0.138

55.0

 

 

 

34

33

0.0930

37.2

 

 

 

 

 

 

 

 

 

0.50

5

0

0.176

35.2

0.25 ± 0.0131

50

 

11

6

0.250

50.0

(%CV = 5.21)

 

 

17

13

0.231

46.3

 

 

 

23

21

0.264

52.8

 

 

 

29

28

0.320

64.0

 

 

 

35

33

0.202

40.4

 

 

 

 

 

 

 

 

 

*   The limit of quantitation (LOQ) for this study was set at 0.0300 mg/L, defined as the lowest matrix fortification concentration in which a mean recovery of 70-110% was obtained.

**  Results were generated using OpenLab CDS Rev.A.01.05. Manual calculations may differ slightly.

***Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

****Result is below LOQ but quantifiable.


Table 3 (Continued)

 

Measured Concentrations of BADGEDA in Test Solution Samples

 

 

Nominal Test

Concentration

(mg/L)

 

Sample

Number

(803A-105-)

 

Sampling

Time

(Day)

 

Measured

Concentration

(mg/L)*,**

 

Percent

of

Nominal**

Time-Weighted Mean ± SD

Measured

Concentration

(mg/L)***

Time-Weighted Mean

Measured

Percent of Nominal***

1.0

6

0

0.308

30.8

0.49 ± 0.0263

49

 

12

6

0.510

51.0

(%CV = 5.31)

 

 

18

13

0.463

46.3

 

 

 

24

21

0.505

50.5

 

 

 

30

28

0.624

62.4

 

 

 

36

33

0.431

43.1

 

 

 

 

 

 

 

 

 

*   The limit of quantitation (LOQ) for this study was set at 0.0300 mg/L, defined as the lowest matrix fortification concentration in which a mean recovery of 70-110% was obtained.

**  Results were generated using OpenLab CDS Rev.A.01.05. Manual calculations may differ slightly.

***Results were generated using Excel 2010 in full precision mode. Manual calculations may differ slightly.

 

Table 4

 

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

 

Time-Weighted

Mean Measured

Concentration

(mg/L)

Mean ± SD and Range of Measured Parameters

Temperature1

(°C)

DO2

(mg/L)

 

pH

Hardness3

(mg/L as CaCO3)

Alkalinity3

(mg/L as CaCO3)

Conductivity3

(mS/cm)

 

 

 

 

 

 

 

Negative Control

24.9 ± 0.11

8.1 ± 0.13

8.1 ± 0.12

139 ± 14

179 ± 1

329 ± 19

 

(24.7 – 25.2)

(7.9 – 8.2)

(8.0 – 8.3)

(112 – 148)

(178 – 180)

(307 – 354)

 

 

 

 

 

 

 

0.033

25.2 ± 0.15

8.2 ± 0.08

8.0 ± 0.08

--

--

--

 

(25.0 – 25.6)

(8.0 – 8.2)

(8.0 – 8.2)

--

--

--

 

 

 

 

 

 

 

0.061

24.7 ± 0.10

8.1 ± 0.13

8.1 ± 0.08

--

--

--

 

(24.5 – 24.8)

(7.9 – 8.2)

(8.0 – 8.2)

--

--

--

 

 

 

 

 

 

 

0.11

25.1 ± 0.11

8.1 ± 0.16

8.1 ± 0.06

--

--

--

 

(24.9 – 25.3)

(7.8 – 8.2)

(8.0 – 8.2)

--

--

--

 

 

 

 

 

 

 

0.25

24.8 ± 0.14

8.1 ± 0.13

8.1 ± 0.04

--

--

--

 

(24.6 – 25.1)

(7.9 – 8.2)

(8.1 – 8.2)

--

--

--

 

 

 

 

 

 

 

0.49

25.2 ± 0.07

8.1 ± 0.13

8.1 ± 0.04

142 ± 9

179 ± 3

331 ± 21

 

(25.1 – 25.3)

(7.9 – 8.2)

(8.1 – 8.2)

(124 – 148)

(176 – 184)

(304 – 358)

 

 

 

 

 

 

 

1 Temperature measured continuously during the test ranged from approximately 23.62 to 24.90°C.

2 A dissolved oxygen concentration of 4.9 mg/L represents 60% saturation at 25ºC in freshwater.

3 -- = no measurements scheduled.


Table 5

 

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

 

Time-Weighted

Mean Measured

Concentration

(mg/L)

 

 

Number

Exposed

 

Total

Number

Hatched

 

Hatching

Success

(%)1

Mean ± SD.

Time to

Hatch

(day)1

 

Number

Surviving to

Termination

 

Post-Hatch

Survival

(%)1

 

Overall

Survival

(%)1

 

Mean ± SD.

Total Length

(mm)

 

Mean ± SD.

Wet Weight

(mg)

 

Mean ± SD.

Dry Weight

(mg)

Negative Control

80

79

99

4.2 ± 0.27

75

95

94

22.1 ± 0.25

77.2 ± 2.4

15.8 ± 0.40

0.033

80

78

98

4.0 ± 0.030

74

95

93

21.8 ± 0.25

74.0 ± 1.9

15.1 ± 0.72

0.061

80

77

96

4.2 ± 0.12

70

91

88

21.7 ± 0.54

71.4 ± 2.5¿

14.4 ± 0.40¿

0.11

80

79

99

4.1 ± 0.12

74

94

93

21.9 ± 0.10

74.8 ± 2.8

15.2 ± 0.30

0.25

80

78

98

4.2 ± 0.23

70

90

88

21.6 ± 0.27

71.7 ± 3.7¿

14.6 ± 0.71¿

0.49

80

77

96

4.1 ± 0.18

74

96

93

21.6 ± 0.099§

68.0 ± 0.88¿

14.2 ± 0.20¿

1 There were no statistically significant differences (p>0.05) in mean time to hatch (Dunnett’s one-tailed test, p>0.05), in percent hatching success, percent post-hatch survival or percent overall survival (Fisher’s Exact test, p>0.05) from the negative control..

*  Indicates statistically significant differences in mean dry weight (Dunnett’s one-tailed test, p=0.05) in the 0.49 mg/L treatment group from the negative control.

§   Indicates statistically significant differences in mean total length (Dunnett’s one-tailed test, p=0.05) in the 0.49 mg/L treatment group from the negative control. However, the mean total length of the 0.49 mg/L treatment group did not follow a dose-response pattern and therefore was not considered to be biologically meaningful.

¿ Indicates statistically significant differences in mean wet weight and dry weight from the negative control (Dunnett’s one-tailed test, p=0.05). However, the mean wet weight and dry weight in the 0.061 and 0.25 mg/L treatment groups did not follow a dose response pattern, the statistically significant reductions noted in these levels were not considered to be biologically meaningful.


Table 6

 

Percent Inhibition of Each Endpoint from the Negative Control

 

Time-Weighted

Mean Measured

Concentration

(mg/L) 

 

Percent

Hatching

Success

 

% Inhibition

From

Control

 

Mean Time

to Hatch

(Day)

 

% Inhibition

From

Control

 

Percent

Post-Hatch

Survival

 

% Inhibition

From

Control

 

Percent

Overall

Survival

 

% Inhibition

From

Control

Negative Control

99

--

4.2

--

95

--

94

--

0.033

98

1.0

4.0

4.8

95

0.0

93

1.1

0.061

96

3.0

4.2

0.0

91

4.2

88

6.4

0.11

99

0.0

4.1

2.4

94

1.1

93

1.1

0.25

98

1.0

4.2

0.0

90

5.3

88

6.4

0.49

96

3.0

4.1

2.4

96

-1.1

93

1.1

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

 

 

 

Time-Weighted

Mean Measured Concentration

(mg/L) 

 

Mean Total

Length

(mm)

 

% Inhibition

From

Control

 

Mean Wet

Weight

(mg)

 

% Inhibition

From

Control

 

Mean Dry

Weight

(mg)

 

% Inhibition

From

Control

Negative Control

22.1

--

77.2

--

15.8

--

0.033

21.8

1.4

74.0

4.1

15.1

4.4

0.061

21.7

1.8

71.4

7.5

14.4

8.9

0.11

21.9

0.9

74.8

3.1

15.2

3.8

0.25

21.6

2.3

71.7

7.1

14.6

7.6

0.49

21.6

2.3

68.0

12

14.2

10

 

Table 7

 

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

 

 

Parameter

EC/IC10

(95% Confidence Interval)

(mg/L)

EC/IC20

(95% Confidence Interval)

(mg/L)

 

NOEC

(mg/L)

 

LOEC

(mg/L)

Hatching success

>0.49

>0.49

0.49

>0.49

Mean time to hatch

>0.49

>0.49

0.49

>0.49

Post-hatch larval survival

>0.49

>0.49

0.49

>0.49

Overall larval survival

>0.49

>0.49

0.49

>0.49

Total length

>0.49

>0.49

0.49

>0.49

Wet weight

0.43 (0.14 - >0.49)

>0.49

0.25

0.49

Dry weight

>0.49

>0.49

0.25

0.49

When less than 10% reduction was noted, the EC/IC10and EC/IC20values were empirically estimated to be greater than the highest concentration tested.

Appendix 4

 

Dilution Water Chemistry Parameters Measured During the

Approximate 4-Week Period Immediately Preceding the Test

 

Parameter

Mean

Range

 

 

 

Specific Conductance

327

301 - 357

      (µS/cm)

(N = 4)

 

 

 

 

Hardness

141

140 - 144

      (mg/L as CaCO3)

(N = 4)

 

 

 

 

Alkalinity

176

172 - 178

      (mg/L as CaCO3)

(N = 4)

 

 

 

 

pH

7.9

 7.8 – 8.0

 

(N = 4)

 

 

 

 

Total Organic Carbon

< 1

--1

      (mg C/L)

(N = 1)1

 

 

 

 

1 TOC is measured monthly.

Measurement of Test Concentrations

Nominal concentrations selected for use in the study were 0.063, 0.13, 0.25, 0.50 and 1.0 mg/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.

 

The measured concentrations of the stock solutions collected on Day -3 and Day 0 were 49.2 and 33.9% of nominal stock concentration (Table 1) and confirm the measured concentrations of the test solutions. The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 43.3 to 48.4% of nominal concentrations (Table 2). Samples of the test solutions collected during the test had measured concentrations that ranged from 29.4 to 64.0% of nominal concentrations (Table 3). Time-weighted mean measured concentrations of test solution samples collected on Days 0, 6, 13, 21, 28 and 33 of the test were calculated for each treatment group, the time-weighted mean measured test concentrations were0.033, 0.061, 0.11, 0.25 and 0.49mg/L, which represented 53, 47, 44, 50 and 49% of nominal concentrations, respectively. The results of the study were based on the time-weighted mean measured concentrations.

Physical and Chemical Measurements of Water

Measurements of temperature, dissolved oxygen and pH in the test chambers are summarized in Table 4, and individual measurements are presented in Appendices 7, 8 and 9, respectively. Water temperatures were within the 25 ± 1°C range established for the test. Dissolved oxygen concentrations remained = 95% of saturation (7.8 mg/L). Measurements of pH ranged from 8.0 to 8.3 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 3. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by BADGEDA concentration. Light intensity at test initiation was 683 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. Fathead minnow embryos in the control and treatment replicates began hatching on Day 3 of the test and the majority of fathead minnow embryos in the control and treatment replicates hatched on Day 4 of the test and all viable embryos hatched by Day 5 of the test. The mean time to hatchfor 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 the 0.033, 0.061, 0.11, 0.25 and 0.49 mg/L treatment groups was 4.2, 4.0, 4.2, 4.1, 4.2 and 4.1 days, respectively. There were no statistically significant difference in mean time to hatch in any of the BADGEDA treatment group when compared to the negative control (Dunnett’s one-tailed test, p >0.05. Consequently, the NOEC for mean time to hatch was 0.49 mg/L and the LOEC was > 0.49 mg/L.

 

Majority of fathead minnow embryos in the control and treatment replicates hatched on Day 4 of the test and all of the viable embryos in the controls and treatment groups hatched by Day 5 of the test, when the surviving larvae were released into their test chambers and the post-hatch period began. 

Hatching success in the negative control and the 0.033, 0.61, 0.11, 0.25 and 0.49 mg/L treatment groups was 99, 98, 96, 99, 98 and 96%, respectively (Table 5). No statistically significant reductions in percent hatching success were noted in any of the treatment groups from the negative control (Fisher’s Exact test, p > 0.05). Consequently, the NOEC for hatching success was reported as 0.49 mg/L and the LOEC was >0.49 mg/L. Because the data for hatching success did not meet the conditions specified by the guideline for the determination of ECx (i.e., less than 10% differences were noted from the pooled control resulted in the ECx values outside of the data used for the calculation), the EC10and EC20were not estimated but empirically estimated to be > 0.49 mg/L, the highest concentration tested (Table 7).

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 post-hatch survival in the negative control and the 0.033, 0.061, 0.11, 0.25 and 0.49 mg/L treatment groups was 95, 95, 91, 94, 90 and 96%, respectively (Table 5). Fisher’s Exact test indicated no statistically significant decreases in post-hatch survival in any of the treatment groups in comparison to the negative controls (p > 0.05). Consequently, the NOEC for post-hatch survival was 0.49 mg/L and the LOEC was > 0.49 mg/L.

 

Overall survival in the negative control and the 0.033, 0.061, 0.11, 0.25 and 0.49 mg/L treatment groups was 94, 93, 88, 93, 88 and 93%, respectively (Table 5). Fisher’s Exact test indicated no statistically significant decreases in overall survival in any of the treatment groups in comparison to the negative controls (p > 0.05). Consequently, the NOEC for larval survival was 0.49 mg/L and the LOEC was > 0.49 mg/L.

 

Because there were less than 10% reduction in post-hatch survival, the EC10and EC20values for post-hatch survival and overall survival were empirically estimated to be >0.49 mg/L, the highest concentration tested (Table 7).

 

In general, the majority of the fish in the control group and in the 0.033, 0.062, 0.11, 0.25 and 0.49 mg/L treatment groups appeared normal throughout the test. There were some observations of organisms that appeared smaller in comparison to the fish in the control replicates, weak, curled, crooked spine, discolored (pale), lying on the bottom of the test chamber, swimming erratically 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,and the individual replicate data for total length, wet weight and dry weight are presented in Appendices 14, 15 and 16, respectively. A statistically significant difference in mean total length was noted at 0.49 mg/L treatment group when compared to the negative control (Dunnett’s one-tailed test, p = 0.05). However, the mean total length of the 0.49 mg/L treatment group did not follow a dose-response pattern (it was the same as that of the 0.25 mg/L treatment group) and therefore was not considered to be biologically meaningful. Dunnett’s one-tailed test indicated statistically significant differences in mean wet and dry weight at 0.062, 0.25 and 0.49 mg/L treatment groups from the negative control (p = 0.05). However, the mean wet weight in the 0.062 and 0.25 mg/L treatment groups did not follow a dose response pattern, the statistically significant reductions noted in these levels were not considered to be biologically meaningful. Consequently, the NOEC for growth was 0.25 mg/L and the LOEC was 0.49 mg/L. The IC10and IC20values for growth endpoints, total length, wet weight and dry weight, were reported in Table 7.

 

Percent Inhibition

Stimulate or a greater response in the test substance treatment than the pooled control is reported as negative %I. The percent inhibitions are summarized in Table 6.

 

Conditionsfor 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 = 95% (7.8 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 from23.62 to 24.90°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 4.9 to 6.3% 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 99%. At test termination, post-hatch larval survival in the negative control was 95%.

Validity criteria fulfilled:
yes
Conclusions:
Fathead minnows (Pimephales promelas) were exposed to BADGEDA at time-weighted mean measured concentrations of 0.033, 0.061, 0.11, 0.25 and 0.49 mg/L under flow-through conditions for 33 days (a 5-day hatching period plus a 28-day post-hatch growth period). There were no statistically significant treatment related effects on hatching success, mean time to hatch, post-hatch/larvae survival or overall survival at concentrations = 0.49 mg/L. No biologically significant treatment-related effects on mean total length, wet or dry weight at concentrations = 0.25 mg/L. Growth (measured as total length, wet and dry weight) was the most sensitive biological endpoint measured in this study. Fathead minnows exposed to BADGEDA at concentrations = 0.25 mg/L exhibited reductions in growth in comparison to the negative control. Consequently, the NOEC, based on growth was 0.25 mg/L and the LOEC was 0.49 mg/L. The EC10 and EC20 values for hatching success, post-hatch survival, overall survival and mean total length, dry weight and the EC20 value for wet weight were empirically estimated to be > 0.49 mg/L. The EC10 value for wet weight was 0.43 mg/L with the 95% confidence interval of 0.14 to > 0.49 mg/L.
Executive summary:

LENGTH OF EXPOSURE: 33 Days (5-Day Hatch and 28-Day Post-Hatch)

TEST ORGANISMS:  Fathead Minnow (Pimephales promelas)

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

TEST CONCENTRATIONS:                                

Nominal                                                               

Negative Control

0.063 mg/L

0.13 mg/L

0.25 mg/L

0.50 mg/L

1.0 mg/L

 

Mean Measured

<LOQ

0.033 mg/L

0.061 mg/L

0.11 mg/L

0.25 mg/L

0.49 mg/L

 

RESULTS: Based on mean measured concentrations.

The most sensitive endpoint was growth (wet and dry weight).

No-Observed-Effect Concentration: 0.25 mg/L

Lowest-Observed-Effect Concentration: 0.49 mg/L

Percent Inhibition of Each Endpoint from the Negative Control

 

Time-Weighted

Mean Measured Concentration

(mg/L)

Percent Hatching

Success

% Inhibition

From

Control

Mean Time

to Hatch

(Day)

% Inhibition

From

Control

Percent

Post-Hatch

Survival

% Inhibition

From

Control

Percent

Overall

Survival

% Inhibition

From

Control

Negative Control

99

--

4.2

--

95

--

94

--

0.033

98

1.0

4.0

4.8

95

0.0

93

1.1

0.061

96

3.0

4.2

0.0

91

4.2

88

6.4

0.11

99

0.0

4.1

2.4

94

1.1

93

1.1

0.25

98

1.0

4.2

0.0

90

5.3

88

6.4

0.49

96

3.0

4.1

2.4

96

-1.1

93

1.1

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

Time-Weighted

Mean Measured Concentration

(mg/L)

Mean Total

Length

(mm)

% Inhibition

From

Control

Mean Wet

Weight

(mg)

% Inhibition

From

Control

Mean Dry

Weight

(mg)

% Inhibition

From

Control

Negative Control

22.1

--

77.2

--

15.8

--

0.033

21.8

1.4

74.0

4.1

15.1

4.4

0.061

21.7

1.8

71.4

7.5

14.4

8.9

0.11

21.9

0.90

74.8

3.1

15.2

3.8

0.25

21.6

2.3

71.7

7.1

14.6

7.6

0.49

21.6

2.3

68.0

12

14.2

10

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

 

 

Parameter

EC/IC10(95% Confidence Interval)

(mg/L)

EC/IC20(95% Confidence Interval)

(mg/L)

Hatching Success

>0.491

>0.491

Post-Hatch Larval Survival

>0.491

>0.491

Overall Larval Survival

>0.491

>0.491

Total Length

>0.491

>0.491

Wet Weight

0.43 (0.14 – >0.492)

>0.491

Dry Weight

>0.491

>0.491

1  Because there were less than 10% and 20% reductions in the treatment endpoints from the pooled controls, the EC/IC10and EC/IC20were estimated to be > 0.49 mg/L, the highest concentration tested.

2   Because the upper limit of the 95% confidence interval was outside of the highest test concentration, the upper limit was reported as > 0.49 mg/L, the highest concentration tested.


Description of key information

The chronic toxicity of the test substance to Pimephales promelas was determined in a flow-through test performed in accordance with the OECD 210 test guideline and GLP requirements.

No significant effects on hatching, survival and length were detected up to the highest tested concentration (0.49 mg/L, TWA measured concentrations). Inhibitory effects were detected at the highest tested concentration for weight (dry and wet weight). The 33d-NOEC based on wet and dry weight was equal to 0.25 mg/L and the 33d-EC10 based on wet weight inhibition was equal to 0.43 mg/L (both TWA measured concentrations).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.25 mg/L

Additional information