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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 13, 2016 to June 7, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples were collected from each treatment and control group 7 and 6 days prior to the start of the test after conditioning the diluter for 1 and 2 days, respectively. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at the end of the test to measure concentrations of the test substance. Samples of the stock solutions being delivered to the diluter were also collected for analysis 6 days prior to the start of the test. Additional samples of test solutions were collected as needed during the test to confirm concentrations of the test substance after a diluter malfunction or suspected error in sample analysis. All water samples (10.0 mL) were collected from mid-depth, placed in glass vials containing 10.0 mL of acetonitrile, while stock solutions (~2 mL) were collected without the addition of solvent. All samples were processed immediately for analysis.
Vehicle:
yes
Remarks:
HPLC-grade dimethylformamide (DMF)
Details on test solutions:
Individual stock solutions were prepared for each of the five concentrations tested, and were prepared five times during the study. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity (97.8%). A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 500 μg a.i./mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 32, 63, 125 and 250 μg a.i./mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and appeared clear and colorless. Stock solutions were stored refrigerated in glass amber bottles, and aliquots of each stock were placed in the syringes every 3 to 4 days during the study.The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 10 μL/minute where they were mixed with dilution water delivered at a rate of 100 mL/minute to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all Naugard 445 treatment groups was 0.1 mL/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group of aquatic invertebrates and were selected for use in the test based upon past history of use in the laboratory. Daphnid neonates used in the test were less than 24 hours old and were obtained from cultures maintained by EAG Laboratories, Easton, Maryland. Identification of the species was verified by the supplier of the original stock culture. Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 20.0 to 20.5°C, measured with a hand-held, digital thermometer. The pH of the water ranged from 8.1 to 8.4, measured with a Thermo Orion Benchtop 4 Star Plus pH meter. Dissolved oxygen concentrations were ≥7.5 mg/L (≥83% of saturation), measured with a Thermo Orion Benchtop 3 Star Plus dissolved oxygen meter. During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), supplemented with a vitamin stock solution and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata. Daphnids were fed two or three times per day through Day 7 of the test and then were fed four times per day until the last day of the test. At each feeding, each test chamber was fed 0.75 mL of YCT, 1.5 mL of algae and 0.50 mL of vitamin solution once daily. This amount of feed is equal to approximately 0.71 mg C/daphnid/day. While this amount of feed exceeds the OECD guideline recommended amount of 0.1 to 0.2 mg C/daphnid/day, an excess amount was fed in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates. The 6 adult daphnids used to supply neonates for the test were held for 20 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress and no ephippia were produced during the holding period. To initiate the test, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained 5 daphnids. Each group of neonates then was impartially assigned to a control or treatment group and the neonates were transferred to the test compartments to initiate the test. All transfers were made below the water surface using wide-bore pipettes.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
No post expoure observation period specified in the study report.
Hardness:
133 - 135 mg/L as CaCO3
Test temperature:
20.3 ± 0.29°C
pH:
8.1 - 8.2
Dissolved oxygen:
8.2 - 8.7 mg/L
Salinity:
177 ± 2 mg/L as CaCO3
Conductivity:
340 - 341 μS/cm
Nominal and measured concentrations:
TEST CONCENTRATIONS:
Nominal Negative Control Mean Measured < LOQ
Nominal Solvent Control Mean Measured < LOQ
Nominal 3.2 μg a.i./L Mean Measured 1.9 μg a.i./L
Nominal 6.3 μg a.i./L Mean Measured 4.0 μg a.i./L
Nominal 12.5 μg a.i./L Mean Measured 11.4 μg a.i./L
Nominal 25 μg a.i./L Mean Measured 21 μg a.i./L
Nominal 50 μg a.i./L Mean Measured 38 μg a.i./L
Details on test conditions:
Test Apparatus

The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance and a negative control (dilution water) to test vessels. Syringe pumps (Harvard Apparatus, Holliston, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The stock solution was diluted with U.V. sterilized well water in the mixing vessels in order to obtain the desired test concentrations prior to delivery to the test vessels. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately 7 volume additions of test water in each test chamber per day. After mixing, the test solution in each mixing chamber was pumped into the appropriate replicate test chamber using a peristaltic pump (Cole-Parmer Instrument Company, Chicago, Illinois).

The syringe pumps used to deliver stock solutions or solvent to the mixing chambers were calibrated once prior to test initiation. The peristaltic pumps used to deliver the test solutions to the test chambers and the rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and calibrated/verified approximately weekly during the test. The proportion of the test solution that was pumped into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 5% of the mean flow rate for the replicates. Delivery of test solutions to the test chambers was initiated eight days prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.

The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target water temperature throughout the test period. Test chambers were 12-L glass aquaria filled with approximately 9 L of test water. The volume in the test chambers was maintained by an overflow port on the side of the test chamber. The daphnids were held in two test compartments suspended in each of two test chambers. Test compartments were 300 mL glass beakers, approximately 6.5 cm in diameter and 12 cm in height. Nylon mesh screens covered two holes on opposite sides of each test compartment to permit test solution to flow in and out of the compartment. The depth of the test water in a representative compartment was approximately 8 cm, while the depth of water in a representative test chamber was approximately 29 cm. All test chambers were labeled with the project number, test concentration and replicate designation.

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 the beginning of the test using a SPER Scientific Model 840006 light meter.

The target test temperature during the test was 20 ± 1°C. Temperature was measured in each test chamber at the beginning of the test, approximately weekly during the test, and at the end of the test using a digital thermometer. Temperature also was monitored continuously in one negative control test chamber using a minimum/maximum thermometer. The recorder was verified prior to test initiation and approximately weekly during the test using a digital thermometer.

Dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test, approximately three times per week during the test, and at the end of the test. Measurements typically rotated between the two replicates in each treatment or control group at each measurement interval.

Measurements of pH were made in one replicate test chamber of each treatment and control group at the beginning of the test, approximately weekly during the test, and at the end of the test. Measurements typically rotated between the two replicates in each treatment or control group at each measurement interval, except on Day 14 of the test the measurements were made in replicate B since the test chambers of replicate A were replaced.

Dissolved oxygen was measured using a Thermo Orion Star A213 dissolved oxygen meter, and measurements of pH were made 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 test concentration at the beginning of the test, approximately 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 an Acorn Series Model CON6 Conductivity-Temperature meter. Total organic carbon (TOC) in the dilution water at the beginning and end of the test was measured using a Shimadzu Model TOC-VCSH total organic carbon analyzer, based on procedures in Standard Methods for the Examination of Water and Wastewater.

Biological Observations and Measurements

Observations of each first-generation daphnid were made daily during the test. At these times, the numbers of immobile daphnids were recorded along with any clinical signs of toxicity (e.g., inability to maintain position in the water column, uncoordinated swimming or cessation of feeding). Those daphnids that are not able to swim within 15 seconds after gentle agitation of the test vessel are considered to be immobilized (even if they can still move their antennae). The presence of eggs in the brood pouch, aborted eggs, males or ephippia also was recorded daily. With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday and Friday during the test. The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.

Statistical Analyses

Test endpoints analyzed statistically for first-generation daphnids were survival, reproduction, and growth (length and dry weight). Reproduction were analyzed using two response variables: 1) the number of live young produced per reproductive day, and 2) the total number of living offspring produced per adult at the beginning of the test. Reproductive days were defined as the number of days that the adult daphnid was alive from the day the first brood was released from any adult daphnid in the test until test termination. If an adult daphnid died, the number of reproductive days, for that adult, ended on the last day it was alive.

Negative control and solvent control data for each parameter were compared using an appropriate statistical test (t-test, α = 0.05). Since no significant differences between the control groups were found in any of the endpoints (p > 0.05), the control data were pooled for comparison with the treatment groups.

Survival data was considered to be discrete-variable data, while reproduction and growth data were considered continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (α = 0.05) from the control. All continuous-variable data were evaluated for normality using Shapiro-Wilk’s test and for homogeneity of variance using Levene’s test (α = 0.01). When the data passed the assumptions of normality and homogeneity, those treatments that were significantly different from the control means were identified using Dunnett’s test (α = 0.05). When the reproduction data (measured as the mean number of live neonates produced per reproductive day) did not meet the assumptions for homogeneity of variance and data transformation did not improve the conditions, a non-parametric step-down trend test (e.g., Jonckheere-Terpstra trend test, α = 0.05) was used to identify the treatment level that was different from the control. All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and MATC. However, scientific judgment was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. The NOEC was defined as the highest test concentration that produced no significant treatment-related effects on survival, reproduction or growth. The LOEC was defined as the lowest test concentration that produced a significant treatment-related effect on survival, reproduction or growth. The MATC was calculated as the geometric mean of the NOEC and LOEC. EC10 and EC50 values based on reproduction and EC50 value based on immobility observed in the first-generation daphnids at the end of the test were also determined.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
38 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 38 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 38 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
Measurement of Test Concentrations

Nominal concentrations selected for use in the study were 3.2, 6.3, 12.5, 25 and 50 μg a.i./L. During the course of the test, the appearance of the test solutions at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. The test solutions in the mixing chambers and test chambers appeared clear and colorless at test initiation, with no evidence of precipitation observed in any control or treatment solution. At test termination, test solutions appeared clear and light green due to algal feed. Microbial growth was also noted in the mixing chambers of all treatment concentrations at test termination.

Samples of the stock solutions being delivered to the diluter system had measured concentrations that ranged from 94.7 to 100% of nominal concentrations, confirming the concentrations being delivered to the diluter system. The measured concentrations of samples collected to verify the diluter system prior to the test ranged from 51.8 to 72.1% of nominal concentrations in samples collected on Day -7 and from 45.2 to 67.0% of nominal concentrations in samples collected on Day -6. Samples of the test solutions collected during the test had measured concentrations that ranged from 45.9 to 118% of nominal concentrations. Due to an unusually high analytical recovery for the 6.3 μg a.i./L treatment level on Day 7, the backup sample for this level on Day 7 and an additional sample collected from this level on Day 8 of the test were analyzed to confirm the original measurement on Day 7. Analytical results of the backup sample from Day 7 and additional sample collected on Day 8 were used in the calculation of mean measured test concentration. When the measured concentrations of test solution samples collected on Days 0, 7, 14 and 21 of the test were averaged for each treatment group, the mean measured test concentrations were 1.9, 4.0, 11.4, 21 and 38 μg a.i./L, which representing 59, 64, 91, 84 and 76% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Water Chemistry Measurements

Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥81% of saturation (7.3 mg/L). Measurements of pH ranged from 8.0 to 8.3 during the test. Measurements of specific conductance, hardness and alkalinity were comparable between the control and treatment group and did not appear to be influenced by Naugard 445 concentration. TOC in the dilution water at test initiation and termination was <1 mg C/L. Light intensity at test initiation was 1032 lux at the surface of the water of one representative test chamber.

Survival and Clinical Observations

After 21 days of exposure, survival in the negative control and solvent control groups was 100 and 95%, respectively. There was no significant difference in survival between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Survival in the pooled control and the 1.9, 4.0, 11.4, 21 and 38 μg a.i./L treatment groups at test termination was 98, 100, 100, 95, 90 and 95%, respectively. The NOEC for survival was 38 μg a.i./L and the LOEC was >38 μg a.i./L. Because there was less than 50% immobility in any of the treatment groups during the test which precluded the statistical calculation of EC50 value, the 21-day EC50 value was estimated to be >38 μg a.i./L, the highest concentration tested.

Daphnids in the 1.9, 4.0, 11.4, 21 and 38 μg a.i./L treatment groups that survived to test termination generally appeared normal. No clinical signs of toxicity were noted in any of the treatment and control groups.

Reproduction

The first day of brood production in the negative and solvent control replicates and in all Naugard 445 treatment replicates was Day 8 of the test, indicating that there was no apparent delay in the onset of production at any Naugard 445 concentration tested. With the exception of two and three immobile neonates in the negative control and 4.0 μg a.i./L treatment on Day 17 and 13 of the test, respectively, no immobile neonates were noted in the control or treatment groups during the test. On Day 13 of the test, two and one aborted or shed eggs were present in one replicate of the 4.0 and 38 μg a.i./L treatment groups. No males or ephippia were produced during the test.

Adult daphnids in the negative and solvent control groups produced an average of 13.6 and 13.8 live young per reproductive day, respectively. There was no significant difference in reproduction between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. On Day 13 of the test, all neonates from the 21 μg a.i./L treatment group were lost when the test compartment was inadvertently spilled. Therefore, the reproductive days (11, 12 and 13) were excluded from the calculation of reproduction per reproductive day for this treatment concentration. Adult daphnids in the pooled control and the 1.9, 4.0, 11.4, 21 and 38 μg a.i./L treatment groups produced an average of 13.7, 13.6, 14.4, 13.7, 11.0 and 14.0 live young per reproductive day, respectively.

Mean number of live neonates produced per adult at the beginning of the test in the negative control, solvent control, pooled control and the 1.9, 4.0, 11.4, 21 and 38 μg a.i./L treatment groups was 190, 187, 188, 191, 195, 191, 113 and 192 neonates, respectively.The NOEC for reproduction was 38 μg a.i./L and the LOEC was >38 μg a.i./L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >38 μg a.i./L, the highest concentration tested. The 21-day EC10 value based on the mean number of live neonates produced per adult at the beginning of the test was 16 μg a.i./L. Since the 95% confidence interval was outside of the data range used for the calculation, it was not reported. However, the EC10 value was not meaningful because the mean number of live neonates produced per adult at the beginning of the test in the 21 μg a.i./L did not follow a dose-response pattern. The EC50 value based on the mean number of live neonates produced per adult at the beginning of the test was >38 μg a.i./L, the highest concentration tested.

Growth

Daphnids in the negative control group averaged 5.0 mm in length and 1.17 mg in dry weight, while daphnids in the solvent control group averaged 5.0 mm in length and 1.25 mg in dry weight. There were no significant differences in the growth parameters between the negative and solvent control groups. Therefore, the control data were pooled for comparison with the treatment groups. Daphnids in the pooled control, 1.9, 4.5, 11.4, 21 and 38 μg a.i./L treatment groups had mean lengths of 5.0, 5.0, 4.9, 4.9, 5.0 and 5.0 mm, respectively, and had mean dry weights of 1.21, 1.20, 1.20, 1.26, 1.27 and 1.28 mg, respectively. The NOEC for growth (length and dry weight) was 38 μg a.i./L, and the LOEC was >38 μg a.i./L.
Results with reference substance (positive control):
Reference substance not utilised in this study.
Reported statistics and error estimates:
Fisher’s Exact test indicated that there were no statistically significant decreases in survival in any of the Naugard 445 treatment groups in comparison to the pooled control (p > 0.05). There were no statistically significant decreases in mean neonate production per reproductive day in any of the Naugard 445 treatment groups in comparison to the pooled control (Jonckheere-Terpstra trend test, p > 0.05). Dunnett’s one-tailed test found a statistically significant reduction in mean number of live neonates produced per adult at the beginning of the test in the 21 μg a.i./L treatment group in comparison to the pooled control (p ≤ 0.05). However, the mean number of neonates in this treatment level did not follow a dose response pattern and was not considered to be biologically meaningful.Dunnett’s one-tailed test indicated there were no statistically significant decreases in mean length or mean dry weight in any of the Naugard 445 treatment group in comparison to the pooled control (p > 0.05).

Measured Concentrations of Naugard 445 in Stock solution Samples

Nominal Concentration (μg a.i./L)

Sample Number (616A-136C-)

Sampling Time (Day)

Measured Concentration (μg a.i./L)1

Percent of Nominal

32000

63000

125000

250000

500000

PT-S-1

PT-S-2

PT-S-3

PT-S-4

PT-S-5

-6

-6

-6

-6

-6

32100

59600

121000

246000

484000

100

94.7

96.5

98.5

96.8

1Results were generated using Analyst version 1.6.2 Manual calculations may differ slightly.

 

 Measured Concentrations of Naugard 445 in Pretest Diluter Verification Samples

Nominal Concentration (μg a.i./L)

Sample Number (616A-136C-)

Sampling Time (Day)

Measured Concentration (μg a.i./L)1,2

Percent of Nominal2

Negative control

 

 

Solvent Control

 

 

3.2

 

 

6.3

 

 

12.5

 

 

25

 

 

50

PT-1

PT-8

 

PT-2

PT-9

 

PT-3

PT-10

 

PT-4

PT-11

 

PT-5

PT-12

 

PT-6

PT13

 

PT-7

PT-14

-7

-6

 

-7

-6

 

-7

-6

 

-7

-6

 

-7

-6

 

-7

-6

 

-7

-6

<LOQ

<LOQ

 

<LOQ

<LOQ

 

2.31

1.86

 

3.26

2.85

 

6.85

7.38

 

15.6

14.5

 

33.3

33.5

-

-

 

-

-

 

72.1

58.2

 

51.8

45.2

 

54.8

59.1

 

62.5

57.9

 

66.6

67.0

1The limit of quantification (LOQ) was 1.00 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2Results were generated using Analyst version 1.6.2. Manual calculations may differ slightly.

 

Measured Concentrations of Naugard 445 in Test Solution Samples

Nominal Test Concentration

(μg a.i./L)

Sample Number (616A-136C-)

Sampling Time (Days)

Measured Concentration (μg a.i./L)1,2

Percent of Nominal2

Mean Measured Concentration (μg a.i./L)

Mean Measured Percent of Nominal

Negative Control

1

8

16

25

0

7

14

21

<LOQ

<LOQ

<LOQ

<LOQ

--

--

--

--

<LOQ

--

Solvent Control

2

9

17

26

0

7

14

21

<LOQ

<LOQ

<LOQ

<LOQ

--

--

--

--

<LOQ

--

3.2

3

10

18

27

0

7

14

21

2.15

1.73

1.54

2.06

67.2

54.0

48.0

64.5

1.9 ± 0.285

CV = 15.2%

59

6.3

4

11-BU3

153

19

28

0

7

8

14

21

3.10

6.48

4.25

2.89

3.33

49.2

103

67.4

45.9

52.8

4.0 ± 1.48

CV = 36.8%

64

12.5

5

12

20

29

0

7

14

21

10.1

14.8

11.0

9.70

81.2

118

87.7

77.6

11.4 ± 2.33

CV = 20.4%

91

25

6

13

21

30

0

7

14

21

25.0

17.9

17.1

23.0

100

71.4

68.5

92.2

21 ± 3.85

CV = 18.6%

84

50

7

14

22

234

244

31

0

7

14

16

16

21

40.1

43.7

51.8

28.6

35.5

27.8

80.3

87.4

104

57.2

71.1

55.6

38 ± 9.23

CV = 24.3%

76

1The limit of quantification (LOQ) was 1.00 μg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.500 μg a.i./L) and the dilution factor of the matrix blank samples (2.00).

2Results were generated using Analyst version 1.6.2. Manual calculations may differ slightly.

3Because the original sample analysis on Day 7 was unusually high and considered to be suspect, the back up sample from Day 7 and an additional sample collected on Day 8 were analyzed. The Day 7 back up and Day 8 sample results were used in the calculation of the mean measured test concentration.

4Additional samples were collected when the diluter malfunction was found and after the problem was corrected and the diluter resumed its normal operation. The results were used in the calculation of the mean measured test concentration. 

 

Means and Ranges of Water Quality Measurements Taken During the 21-Day Exposure to Naugard 445

Mean Measured Concentration

(μg a.i./L)

Mean ± Std. Dev. and Range of Measured Parameters1

Temperature2(°C)

Dissolved Oxygen3(mg/L)

pH

Hardness4(mg/L as CaCO3)

Alkalinity4

(mg/L as CaCO3)

Conductivity4(μS/cm)

Negative Control

20.3 ± 0.29

(20.1 – 20.8)

8.7 ± 0.27

(8.4 – 9.1)

8.1 ± 0.05

(8.0 – 8.1)

133 ± 4

(128 – 136)

175 ± 1

(174 – 176)

340 ± 12

(330 – 358)

Solvent Control

20.3 ± 0.23

(20.1 – 20.7)

8.5 ± 0.41

(7.8 – 9.1)

8.2 ± 0.06

(8.1 – 8.2)

--

--

--

--

--

--

1.9

20.3 ± 0.23

(20.1 – 20.7)

8.4 ± 0.47

(7.8 – 9.1)

8.2 ± 0.05

(8.1 – 8.2)

--

--

--

--

--

--

4.0

20.3 ± 0.23

(20.1 – 20.8)

8.4 ± 0.54

(7.5 – 9.1)

8.1 ± 0.05

(8.1 – 8.2)

--

--

--

--

--

--

11.4

20.3 ± 0.19

(20.1 – 20.6)

8.3 ± 0.54

(7.5 – 9.1)

8.2 ± 0.06

(8.1 – 8.2)

--

--

--

--

--

--

21

20.3 ± 0.19

(20.1 – 20.6)

8.2 ± 0.57

(7.3 – 9.1)

8.2 ± 0.08

(8.1 – 8.3)

--

--

--

--

--

--

38

20.3 ± 0.27

(20.1 – 20.8)

8.2 ± 0.54

(7.4 – 9.1)

8.2 ± 0.08

(8.1 – 8.3)

135 ± 2

(132 – 136)

177 ± 2

(132 – 136)

341 ± 16

(330 – 364)

1Total organic carbon measured in the dilution water at test initiation and termination was <1 mg C/L.

2Temperature monitored continuously during the test ranged from approximately 19.5 to 20.9°C

3A dissolved oxygen concentration of 5.4 mg/L represents 60% saturation at 20°C in freshwater.

4-- = no measurements scheduled.

 

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to Naugard 445 for 21 Days

Mean Measured Concentration

(μg a.i./L)

Percent Adult Survival1

Mean No. Neonates Per Reproductive Day ± Std. Dev.1

Mean No. Neonates Per Adult At Test Start ± Std. Dev.1

Mean Length ± Std. Dev. (mm)1

Mean Dry Weight ± Std. Dev. (mg)1

Negative Control

Solvent Control

Pooled Control

1.9

4.0

11.4

21

38

100

95

98

100

100

95

90

95

13.6 ± 0.32

13.8 ± 0.39

13.7 ± 0.34

13.6 ± 0.29

14.4 ± 0.95

13.7 ± 0.67

11.0 ± 1.4

14.0 ± 0.19

190 ± 4.5

187 ± 7.1

188 ± 5.7

191 ± 4.6

195 ± 11

191 ± 9.0

113 ± 18Δ

192 ± 7.8

5.0 ± 0.082

5.0 ± 0.050

5.0 ± 0.064

5.0 ± 0.050

4.9 ± 0.050

4.9 ± 0.050

5.0 ± 0.050

5.0 ± 0.050

1.17 ± 0.11

1.25 ± 0.024

1.21 ± 0.086

1.20 ± 0.041

1.20 ± 0.082

1.26 ± 0.019

1.27 ± 0.057

1.28 ± 0.11

EC10 & 95% CI (μg a.i./L)

EC50 & 95% CI (μg a.i./L)

NA

>38

>38

>38

*

>38

NA

NA

NA

NA

1There were no statistically significant reductions in survival (Fisher’s Exact test, p>0.05), in growth (mean total length and dry weight (Dunnett’s one-tailed test, p>0.05) or in mean number of neonates produced per reproductive day (Jonckheere-Terpstra step down trend test, p>0.05) from the pooled control.

ΔA statistically significant difference in mean number of neonates produced per adult at the start of the test. However, the mean number of neonates in this treatment level did not follow a dose response pattern and was not considered to be biologically meaningful.

* The EC10 was calculated to be 16.0 μg a.i./L, with the 95% confidence interval outside of the data range used for the calculation. However, the EC10 value was not meaningful because the mean number of live neonates produced per adult at the beginning of the test in the 21 μg a.i./L did not follow a dose-response pattern.

NA = not applicable, ECx values were not required for the growth endpoints.

 

Validity criteria fulfilled:
yes
Remarks:
The test material does not display toxicity at the limit of solubilty in water.
Conclusions:
There were no significant treatment-related effects on survival, reproduction or growth at concentrations ≤38 μg a.i./L. Consequently, the NOEC, based on growth, was 38 μg a.i./L. The 21-day EC50 value for adult immobility was >38 μg a.i./L, the highest concentration tested. The 21-day EC10 and EC50 values for reproduction based on mean number of live neonates produced per reproductive day were both >38 μg a.i./L.
Executive summary:

The objective of this study was to determine the effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline (Naugard 445) on the survival, growth and reproduction of the cladoceran, Daphnia magna, during a 21-day exposure period under flow-through test conditions.

 

The study was conducted according to the procedures outlined in the OECD Guidelines for Testing of Chemicals, Guideline 211: Daphnia magna Reproduction Test (1); the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1300: Daphnid Chronic Toxicity Test; and ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests.

 

Daphnids were exposed to a geometric series of five test concentrations, a negative control (dilution water) and a solvent control (0.1 mL/L HPLC-grade dimethylformamide). Two replicate test chambers were tested for each treatment and control group. Each replicate contained two compartments with five daphnids, resulting in a total of 20 daphnids in each treatment and control group. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range finding toxicity data. The nominal test concentrations were 3.2, 6.3, 12.5, 25 and 50 μg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at test initiation, at approximately weekly intervals during the test and at test termination.

Delivery of the test substance to the test chambers was initiated eight days prior to the introduction of the daphnids to the test water in order to achieve equilibrium of the test substance in the test chambers. At test initiation, neonate daphnids were impartially assigned to exposure chambers. First-generation daphnids were observed daily during the test for immobility, the onset of reproduction, and clinical signs of toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were counted three times per week (e.g., Monday, Wednesday and Friday), and at test termination (Day 21). Body lengths and dry weights of the surviving first-generation daphnids were measured at the end of the exposure period. Observations of the effects of Naugard 445 on survival, reproduction and growth were used to determine the no-observed-effect concentration (NOEC), the lowest-observed-effect concentration (LOEC), and the maximum acceptable toxicant concentration (MATC). An EC10 value based on reproduction and EC50 values based on immobility and reproduction of first-generation daphnids were determined at test termination. 

 

The cladoceran, Daphnia magna, was exposed to Naugard 445 at mean measured concentrations of 1.9, 4.5, 11.4, 21 and 38 μg a.i/L under flow-through conditions for 21 days. There were no significant treatment-related effects on survival, reproduction or growth at concentrations ≤38 μg a.i./L. Consequently, the NOEC for reproduction was 38 μg a.i./L and the LOEC was >38 μg a.i./L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >38 μg a.i./L, the highest concentration tested. The 21-day EC10 value based on the mean number of live neonates produced per adult at the beginning of the test was 16 μg a.i./L. Since the 95% confidence interval was outside of the data range used for the calculation, it was not reported. However, the EC10 value was not meaningful because the mean number of live neonates produced per adult at the beginning of the test in the 21 μg a.i./L did not follow a dose-response pattern. The EC50 value based on the mean number of live neonates produced per adult at the beginning of the test was >38 μg a.i./L, the highest concentration tested.

Description of key information

Key value determined in a GLP accredited laboratory study performed in accordance with OECD Guideline 211 & US EPA Procedure OPPTS 850.1300.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
38 µg/L

Additional information

The cladoceran, Daphnia magna, was exposed to the test substance at mean measured concentrations of 1.9, 4.5, 11.4, 21 and 38 μg a.i/L under flow-through conditions for 21 days. There were no significant treatment-related effects on survival, reproduction or growth at concentrations ≤38 μg a.i./L. Consequently, the NOEC for reproduction was 38 μg a.i./L and the LOEC was >38 μg a.i./L. Based on the mean number of live neonates produced per reproductive day observed in the treatment groups, the 21-day EC10 and EC50 values were both >38 μg a.i./L, the highest concentration tested. The 21-day EC10 value based on the mean number of live neonates produced per adult at the beginning of the test was 16 μg a.i./L. Since the 95% confidence interval was outside of the data range used for the calculation, it was not reported. However, the EC10 value was not meaningful because the mean number of live neonates produced per adult at the beginning of the test in the 21 μg a.i./L did not follow a dose-response pattern. The EC50 value based on the mean number of live neonates produced per adult at the beginning of the test was >38 μg a.i./L, the highest concentration tested.