Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Results derived from a valid read across, with adequate and reliable documentation / justification.
Justification for type of information:
The read across justification is presented in the overall Aquatic Endpoint Summary because in that document short-term toxicity to aquatic invertebrates and toxicity to aquatic algae is also included. The corresponding documentation file is also attached there.
Reason / purpose:
read-across source
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.069 mg/L
Basis for effect:
reproduction
Remarks on result:
other: Based on read across information
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.107 mg/L
Basis for effect:
immobilisation
Remarks on result:
other: Based on read across information
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.063 mg/L
Basis for effect:
other: growth and reproduction
Remarks on result:
other: Based on read across information
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.157 mg/L
Basis for effect:
immobilisation
Remarks on result:
other: Based on read across information
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.157 mg/L
Basis for effect:
reproduction
Remarks on result:
other: Based on read across information
Validity criteria fulfilled:
yes
Remarks:
Justification for read across is presented in the overall aquatic endpoint summary.
Conclusions:
For Acalea a 21-day EC10 value of 0.069 mg/L has been derived for Daphnia based on read across information from Hexyl Cinnamic Aldehyde.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 March 2011 to 29 April 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed according to the OECD Guideline 211, the US EPA OPPTS 850.1300 and ASTM E 1193-97, with GLP statement. All validity criteria were fulfilled. However, a solvent was used in this study which leads to confounding factors, and one deviation was observed: this amount of feed is equal to approximately 0.5 to 0.7 mg C/daphnid/day. This amount of feed exceeds the OECD guideline recommended amount.
Justification for type of information:
Information used for read across to Acalea.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
. The amount of feed during the test (in order to maintain sufficient feed in the flow-through system to support acceptable reproduction rates)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Flashpoint: 212°F (100°C) according to MSDS
- Vapour pressure: 0.00hPa (0.000 mm Hg) according to MSDS
- Relative density (20°C): 0.955 to 0.960 according to MSDS
Analytical monitoring:
yes
Details on sampling:
Samples were collected from each treatment and control group one day prior to the start of the test after conditioning the diluter for one day. 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. A second set of water samples was collected and stored at the end of the test for potential analysis to confirm analytical results. Samples of the stock solutions being delivered to the diluter were collected for analysis on Day 0 to confirm proper delivery of the test substance to the test system. All samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis or stored for possible future analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Individual stock solutions were prepared for each of the five concentrations tested, and were prepared twice during the study. All test solutions were adjusted to 100% active ingredient during preparation, based on the test substance purity. A primary stock solution was prepared by mixing a calculated amount of test substance into HPLC-grade dimethylformamide (DMF) at a nominal concentration of 2500 µg a.i./mL. Four secondary stock solutions were prepared in DMF at nominal concentrations of 64, 160, 400 and 1000 µg a.i./mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion, and ranged in appearance from clear and colorless to light yellow in color. Stock solutions were stored refrigerated in glass amber bottles with Teflon®-lined lids, and aliquots of each stock were placed in the syringe pump every two days during the test.
- Controls: 2 (negative and solvent controls). 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.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): HPLC-grade dimethylformamide
- Concentration of vehicle in test medium: The concentration of DMF in the solvent control and all Hexylcinnamaldehyde (101-86-0) treatment groups was 0.1 mL/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): 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.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: The cladoceran, Daphnia magna
- Source: obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland.
- Age of parental stock (mean and range, SD): Daphnid neonates used in the test were less than 24 hours old at initiation
- Feeding during test: yes
- Food type: During culture and testing, daphnids were fed a mixture of yeast, cereal grass media, and trout chow (YCT), as well as a suspension of the freshwater green alga, Pseudokirchneriella subcapitata.
- Amount: At each feeding, each test chamber was fed 0.75 mL of YCT and 1.5 mL of algae. This amount of feed is equal to approximately 0.5 to 0.7 mg C/daphnid/day based on a Non-GLP analyses for carbon content of feed. 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.
- Frequency: Daphnids were fed two or three times per day through Day 8 of the test and then were fed four times per day until the last day of the test. Daphnids were fed once on the day of the test termination.

ACCLIMATION
The four adult daphnids used to supply neonates for the test were held for 15 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:
no
Total exposure duration:
21 h
Remarks on exposure duration:
none
Post exposure observation period:
none
Hardness:
The dilution water is characterized as moderately-hard water.
Hardness = 144 mg/L as CaCO3
Test temperature:
The test chambers were placed in a temperature-controlled water bath to maintain the target water temperature throughout the test period.
The target test temperature during the test was 20 ± 1°C.
pH:
Measurements of pH ranged from 8.0 to 8.2 during the test.
Dissolved oxygen:
Dissolved oxygen concentrations remained ≥67% of saturation (6.0 mg/L).
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: Negative control; Solvent control; 6.4; 16; 40; 100 and 250 µg a.i./L
- Mean measured concentrations:
Details on test conditions:
TEST SYSTEM
- Test vessel: Test chambers with two compartments
- Material, size, headspace, fill volume: Test chambers were 25 L Teflon®-lined stainless steel aquaria filled with approximately 22 L of test water. 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.
- Aeration: no data
- Type of flow-through / Renewal rate of test solution: Syringe pumps (Harvard Apparatus, Massachusetts) were used to deliver test substance stock solutions or solvent to impartially assigned mixing chambers. The flow of dilution water into each mixing chamber was controlled using rotameters and was adjusted to provide approximately five volume additions of test water in each test chamber per day. The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 15.5 µL/minute where they were mixed with dilution water delivered at a rate of 155 mL/minute to achieve the desired test concentrations.
- No. of organisms per vessel: Each replicate contained two compartments with five daphnids, resulting in a total of 20 daphnids in each treatment and control group.
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: no data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- TOC: <1 mg C/L
- Specific conductance: 359-375 µS/cm
- Alkalinity: 174-176 mg/L as CaCO3
- Hardness: 144 mg/L as CaCO3
- pH: 8.0
- Pesticides, Organics and Metals: See Appendix in "Attached background material".
- Salinity: not applicable
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 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.
- Light intensity: Ambient laboratory light was used to illuminate the test systems. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006C light meter: 366 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : First-generation daphnids were observed daily during the test for mortality, 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 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.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
Nominal test concentrations were selected in consultation with the sponsor based on exploratory range finding toxicity data.
No other information.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.069 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: 95%CL = 20-89 µg a.i./L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
0.107 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.063 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: growth and reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.157 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.157 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
- Survival and Clinical Observations:
After 21 days of exposure, survival in both the negative control group and the solvent control group was 89 and 85%, respectively.
One daphnid in the negative control group was inadvertently killed during transfer on Day 2 of the test and was excluded from calculation of negative control survival.
Since there were no statistically significant differences in survival between the negative and solvent control, the control data were pooled for comparison with the treatment data.
Survival in the 3.8, 12, 25, 63 and 157 µg a.i./L treatment groups at test termination was 95, 100, 100, 90 and 75%, respectively.
Fisher’s Exact test indicated that there were no statistically significant decreases in survival in any of the treatment groups in comparison to the pooled control (p > 0.05).
In the 157 µg a.i./L treatment group, all daphnids were observed to be lethargic, discolored and small or discolored and small on Day 2 of the test. The majority of the daphnids in the 157 µg a.i./L treatment group were noted as small through Day 16 of the test and four daphnids were noted as small at test termination.

- Reproduction:
The first day of brood production in the negative and solvent control replicates and in the 3.8, 12, 25 and 63 µg a.i./L was Day 9 or 10 of the test, indicating that there was no apparent delay in the onset of production at these test substance concentration tested. Brood production in the 157 µg a.i./L treatment replicates initiated on Days, 11, 12 or 13 of the test. Immobile neonates were noted in all treatment groups during the test. The number of immobile neonates in the treatment groups was generally comparable to the number of immobile neonates in the control groups and were not considered to be treatment related. No aborted eggs, males or ephippia were observed during the test.
Adult daphnids in the negative and solvent control groups produced an average of 8.6 and 9.8 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. Adult daphnids in the 3.8, 12, 25, 63 and 157 µg a.i./L treatment groups produced an average of 10.5, 10.2, 8.4, 9.5 and 5.9 live young per reproductive day, respectively. Dunnett’s test indicated there were no statistically significant difference in mean neonate production in the 3.8, 12, 25 and 63 µg a.i./L treatment groups in comparison to the pooled control (p > 0.05). Dunnett’s test indicated there was a statistically significant decrease in mean neonate production in the 157 µg a.i./L treatment group in comparison to the pooled control (p ≤ 0.05).

- Growth:
Daphnids in the negative and solvent control groups averaged 4.5 and 4.5 mm in length, and 1.22 and 1.31 mg in dry weight, respectively. 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 3.8, 12, 25, 63 and 157 µg a.i./L treatment groups had mean lengths of 4.5, 4.5, 4.5, 4.5 and 4.0 mm, respectively, and mean dry weights of 1.22, 1.21, 1.17, 1.20 and 0.91 mg, respectively. Dunnett’s test indicated there were no statistically significant differences in mean length or mean dry weight in the 3.8, 12, 25 and 63 µg a.i./L treatment groups in comparison to the pooled control (p > 0.05). Dunnett’s test indicated there was a statistically significant decrease in both mean length and mean dry weight in the 157 µg a.i./L treatment group in comparison to the pooled control (p ≤ 0.05).

A summary of survival, reproduction and growth of Daphnia magna exposed to the test substance for 21 days is presented in Table 6.1.4/2 in "Any other information on results incl. tables".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Negative control and solvent control data for each parameter were compared using an appropriate statistical test. Performance by the negative and solvent control groups was comparable, indicating no impact from the solvent. No significant differences between the control groups were found for any parameter tested (p > 0.05). Therefore, 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 (p <= 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 (p = 0.01). All data passed the assumptions of normality and homogeneity, and those treatments that were significantly different from the control means were identified using one tailed Dunnett’s test (p ≤ 0.05). All statistical tests were performed using a personal computer with or SAS software.

Table 6.1.4/1: Measured concentrations of the test substance in test solution samples

Nominal Test Concentration

(mg a.i./L)

Sample Number (558A-118-)

Sampling

Time

(Days)

Measured

Concentration1

(mg a.i./L)

Percent

of

Nominal2

Mean

Measured

Concentration

(mg a.i./L)

Mean

Percent

of

Nominal

Negative Control

(0.0)

 

1

8

15

22

 

0

7

14

21

< LOQ

< LOQ

< LOQ

< LOQ

--

--

--

--

--

--

Solvent Control

(0.0)

 

2

9

16

23

 

0

7

14

21

< LOQ

< LOQ

< LOQ

< LOQ

--

--

--

--

--

--

6.4

3

10

17

 24

 

0

7

14

21

4.54

4.02

3.61*

3.03*

70.9

62.8

56.5

47.3

3.8

59

16

4

11

18

25

 

0

7

14

21

17.6

9.12

12.0

8.44

110

57.0

74.7

52.8

12

75

40

5

12

19

26

 

0

7

14

21

31.0

23.8

23.1

22.7

77.4

59.6

57.7

56.8

25

63

100

6

13

20

27

 

0

7

14

21

72.0

68.5

57.8

54.4

72.0

68.5

57.8

54.4

63

63

250

7

14

21

28

0

7

14

21

195

156

126

151

78.0

62.5

50.3

60.2

157

63

1   The limit of quantitation (LOQ) was 3.75mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (3.00mg a.i./L) and the dilution factor of the matrix blank samples (1.25).

2   Results were generated using Excel 2000 in the full precision mode. Manual calculations may differ slightly.

*  While the measured concentration falls below the LOQ, the reported value was extrapolated since a peak was noted at the proper retention time of the test substance and the recovery was generally consistent with other analytical recoveries for this interval.

Table 6.1.4/2: Summary of survival, reproduction and growth of Daphnia magna exposed to the test substance for 21 days

Mean Measured

Concentration

(µg a.i./L)

 

Percent Adult

Survival1,2

Mean No. Neonates

Per Reproductive Day

± Std. Dev.3

Mean Length

± Std. Dev.

(mm)

Mean Dry Weight

± Std. Dev.

(mg)

Negative Control

894

8.6 ± 0.88

4.5 ± 0.10

1.22 ± 0.153

Solvent Control

85

9.8 ± 0.94

4.5 ± 0.10

1.31 ± 0.133

Pooled Control

874

9.2 ± 1.08

4.5 ± 0.09

1.27 ± 0.141

3.8

95

10.5 ± 0.53

4.5 ± 0.10

1.22 ± 0.062

12

100

10.2 ± 0.91

4.5 ± 0.08

1.21 ± 0.090

25

100

8.4 ± 0.77

4.5 ± 0.13

1.17 ± 0.084

63

90

9.5 ± 1.51

4.5 ± 0.12

1.20 ± 0.207

157

75

5.9 ± 1.43*

4.0 ± 0.18*

0.91 ± 0.112*

* Indicates a statistically significant decrease in comparison to the pooled control (p ≤ 0.05).

1  There were no statistically significant decreases in survival in comparison to the pooled control using one-tailed Dunnett’s test (p > 0.05).

2  21-day EC50 for survival: >157 µg a.i./L

3  21-day EC50 for reproduction (95% CI): >157 µg a.i./L (NA).

4  The single negative control mortality caused by handling during transfer on Day 2 was excluded from calculations.

Validity criteria fulfilled:
yes
Conclusions:
The 21d-NOEC, based on growth and reproduction, was 63 µg a.i./L and the LOEC was 157 µg a.i./L. The 21-day EC10 value for adult immobility was 107 µg a.i./L and the 21-day EC10 value for reproduction was 69 µg a.i./L. The 21-day EC50 value for adult immobility and the 21‑day EC50 value for reproduction were >157 µg a.i./L, the highest concentration tested.
Executive summary:

This study was performed according to the OECD Guideline 211, the US EPA OPPTS 850.1300 and ASTM E 1193-97, with GLP statement. The objective of this study was to determine the effects of the test substance on the survival, growth and reproduction of the cladoceran Daphnia magna during a 21-day exposure period under flow-through test conditions.

Daphnids (neonates < 24 hours old at initiation) 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 (DMF)). 

While it is preferable to avoid the use of a solvent during test solution preparation, solvent was required to prepare the concentrated stock solutions required for this study. The solvent concentration did not exceed the guideline recommended concentration of 0.1 mL/L. Additionally, DMF has been proven to have no impact on the test organism at the concentration used in the test. There were no significant differences observed between the negative control and solvent control group performance in this test.

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 were6.4, 16, 40, 100 and 250µg active ingredient (a.i.)/L. 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 3.8, 12, 25, 63 and 157µg a.i./L, which represented 59, 75, 63, 63 and 63% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

First-generation daphnids were observed daily during the test for mortality, 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 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. 

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤ 63 µg a.i./L. Growth, measured as length and dry weight, and reproduction were the most sensitive biological endpoints measured in this study. Daphnids exposed to the test substance at a concentration of 157 µg a.i./L had statistically significant reductions in growth and reproduction in comparison to the pooled control. 

Consequently, the NOEC, based on growth and reproduction, was 63 µg a.i./L and the LOEC was 157 µg a.i./L. The 21-day EC10 value for adult immobility was 107 µg a.i./L and the 21-day EC10 value for reproduction was 69 µg a.i./L. The 21-day EC50 value for adult immobility and the 21‑day EC50 value for reproduction were >157 µg a.i./L, the highest concentration tested.

All validity criteria were fulfilled: in the control(s), the mortality of the parent animals does not exceed 20% at the end of the test and the mean number of live offspring produced per parent animal surviving at the end of the test is > 60.

Description of key information

For Acalea a 21-day EC10 value of 0.069 mg/L has been derived for Daphnia based on read across information from Hexyl Cinnamic Aldehyde.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.069 mg/L

Additional information

The experimental infomation from the source is presented here. The read across justification is presented in the overall Aquatic Endpoint Summary.

Hexyl Cinnamic Aldehyde and its long-term toxicity to Daphnia (copied from the Lead Registrant):

"This study was performed according to the OECD Guideline 211, the US EPA OPPTS 850.1300 and ASTM E 1193-97, with GLP statement. The objective of this study was to determine the effects of the test substance on the survival, growth and reproduction of the cladoceran Daphnia magna during a 21-day exposure period under flow-through test conditions.

Daphnids (neonates < 24 hours old at initiation) 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 (DMF)). 

While it is preferable to avoid the use of a solvent during test solution preparation, solvent was required to prepare the concentrated stock solutions required for this study. The solvent concentration did not exceed the guideline recommended concentration of 0.1 mL/L. Additionally, DMF has been proven to have no impact on the test organism at the concentration used in the test. There were no significant differences observed between the negative control and solvent control group performance in this test.

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 6.4, 16, 40, 100 and 250µg active ingredient (a.i.)/L. 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 3.8, 12, 25, 63 and 157µg a.i./L, which represented 59, 75, 63, 63 and 63% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

First-generation daphnids were observed daily during the test for mortality, 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 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. 

There were no statistically significant treatment-related effects on survival, reproduction or growth at concentrations ≤ 63 µg a.i./L. Growth, measured as length and dry weight, and reproduction were the most sensitive biological endpoints measured in this study. Daphnids exposed to the test substance at a concentration of 157 µg a.i./L had statistically significant reductions in growth and reproduction in comparison to the pooled control. 

Consequently, the NOEC, based on growth and reproduction, was 63 µg a.i./L and the LOEC was 157 µg a.i./L. The 21-day EC10 value for adult immobility was 107 µg a.i./L and the 21-day EC10 value for reproduction was 69 µg a.i./L. The 21-day EC50 value for adult immobility and the 21‑day EC50 value for reproduction were >157 µg a.i./L, the highest concentration tested.

All validity criteria were fulfilled: in the control(s), the mortality of the parent animals does not exceed 20% at the end of the test and the mean number of live offspring produced per parent animal surviving at the end of the test is > 60".