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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 2013 to April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD, EPA, etc)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
Qualifier:
according to
Guideline:
other: ASTM E 1193-97: Standard Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): XP-7866
- Physical state: solid
- Analytical purity: 99.5 wt%
- Purity test date: 99.5 wt%
- Lot/batch No.: 10188-1 JM
- Expiration date of the lot/batch: February 28, 2015
- Stability under test conditions: Not reported
- Storage condition of test material: Not reported

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Sampling method: All samples were collected from mid-depth, placed in glass vials, acidified with 2-3 drops of 10% phosphoric acid. Samples collected during regularly scheduled sampling intervals.
- Sample storage conditions before analysis: processed immediately or stored refrigerated for possible analysis

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: The cladoceran, Daphnia magna
- Justification for species other than prescribed by test guideline: Not applicable
- Source: cultures maintained by wildlife international, Easton, Maryland.
- Age of parental stock (mean and range, SD):
- Food type: fed with 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.
- Amount: 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. This amount of feed is equal to approximately 0.64 mg C/daphnid/day.
Note: 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 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

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not): The same as used during the test.
- Type and amount of food: The same as used during the test.
- Feeding frequency: see above
- Health during acclimation (any mortality observed): no signs of disease or stress and no ephippia

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Five adult daphnids were 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.

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.

Study design

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

Test conditions

Hardness:
Ranged from 132 to 144 mg/L as CaCO3
Test temperature:
Ranged from 19.3 to 20.5°C
pH:
Ranged from 8.3 to 8.5
Dissolved oxygen:
Ranged from 5.9 to 8.5 mg/L
Salinity:
No data
Nominal and measured concentrations:
* Nominal concentrations: 13, 29, 65, 143, and 314 µg a.i./L + negative control (dilution water) + solvent control (0.225 mL/L HPLC-grade dimethylformamide)
* Measured concentrations: 7.3, 17, 39, 72, and 147 µg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 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.
- Type of flow-through (e.g. peristaltic or proportional diluter): five volume additions of the test water in each test chamber per day.
- No. of organisms per vessel: 10 (20 daphnids for each treatment and control groups)
- No. of vessels per concentration (replicates): 2 (each replicates contained 2 compartments with 5 daphnids)
- No. of vessels per control (replicates): 2 (each replicates contained 2 compartments with 5 daphnids)
- No. of vessels per vehicle control (replicates): 2 (each replicates contained 2 compartments with 5 daphnids)

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 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 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- analyses of pesticide, organics and metals were provided in the original report

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light and 8 hours of darkness
- Light intensity: Ambient laboratory light - wavelenghts similar to natural sunlight

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
* Immobility
* Clinical signs of toxicity (inability to maintain position in the water column, uncoordinated swimming or cessation of feeding)
* The presence of eggs in the brood pouch, aborted eggs, males or ephippia
Note:
*With the onset of reproduction, neonates produced by the first-generation daphnids were counted and then discarded every Monday, Wednesday, Friday and at test termination (Thursday) during the test.
* The body length and the dry weight of each surviving first-generation daphnid were measured at the end of the test.

VEHICLE CONTROL PERFORMED:
Stock solution prepared with HPLC-grade dimethylformamide (DMF). A solvent control was used in the test.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: survival
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth
Remarks:
measured as length and dry weight
Duration:
21 d
Dose descriptor:
other: MATC: Maximum Acceptable Toxicant Concentration
Effect conc.:
< 147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: Reproduction, survival and growth measured as lenght and dry weight
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: Reproduction, survival and growth measured as lenght and dry weight
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Remarks on result:
other: Not calculable
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 147 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: Not calculable
Details on results:
- Test concentrations:
No visible precipitate was noted in the mixing chambers at the 13, 29 and 65 μg a.i./L test concentrations. A small amount of white precipitate was noted on the sides of the mixing chambers for the 143 and 314 μg a.i./L test concentrations.
- Survival and clinical observations:
After 21 days of exposure, survival in the negative control and solvent control groups were 95 and 100%, 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. The treatment group data were also evaluated further by comparison to the negative control and solvent control group data. Survival in the 7.3, 17, 39, 72 and 147 µg a.i./L treatment groups at test termination was 100, 95, 100, 100 and 100%, respectively. Fisher’s Exact test indicated that there were no statistically significant differences in survival in any of the XP-7866 treatment groups in comparison to the negative control, solvent control or pooled controls (p > 0.05). Consequently, the NOEC for survival was 147 µg a.i./L and the LOEC was >147 µg a.i./L. Based on the mortalities observed in the treatment groups, the 21-day EC50 value was >147 µg a.i./L, the highest concentration tested.
- Reproduction:
Adult daphnids in the negative and solvent control groups produced an average of 12.1 and 11.0 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. The treatment group data were also evaluated further by comparison to the negative control and solvent control group data. Adult daphnids in the 7.3, 17, 39, 72 and 147 µg a.i./L treatment groups produced an average of 8.9, 10.9, 10.6, 11.7 and 10.4 live young per reproductive day, respectively. Dunnett’s test indicated there was a statistically significant decrease in mean neonate production in the 7.3 µg a.i./L treatment group in comparison to the negative control group, solvent control group and pooled control data (p ≤ 0.05). However, the mean neonate production in the 7.3 µg a.i./L treatment group did not follow a dose-response pattern and was not considered to be treatment related. Dunnett’s test also indicated a statistically significant difference in mean neonate production in the 147 µg a.i./L treatment group in comparison to the negative control group (p ≤ 0.05). However, since the difference was slight (5.5%) and not statistically significant when compared to the solvent control group or pooled control groups, it was not considered biologically meaningful. Consequently, the NOEC for reproduction was 147 µg a.i./L and the LOEC was >147µg a.i./L, the highest concentration tested. Based on the reproduction observed in the treatment groups, the 21-day EC50 value was >147 µg a.i./L, the highest concentration tested.
- Growth:
* Daphnids in the negative control group averaged 4.6 mm in length and 0.99 mg in dry weight, while daphnids in the solvent control group averaged 4.4 mm in length and 0.93 mg in dry weight, respectively. There were no significant differences in dry weight between the negative and solvent control groups. Therefore, the control data for dry weight were pooled for comparison with the treatment groups. However, since significant differences were found between negative and solvent control total length, the treatment total length data were compared to the negative and solvent control data separately. Further evaluation of the growth data was done by comparing dry weight data for the treatment groups to negative and solvent control groups separately, and by comparing length data for the treatment groups to the pooled control.

*Daphnids in the 7.3, 17, 39, 72 and 147 µg a.i./L treatment groups had mean lengths of 4.5, 4.6, 4.5, 4.5 and 4.5 mm, respectively, and mean dry weights of 1.0, 0.94, 0.83, 0.85 and 0.87 mg, respectively. Dunnett’s test indicated there were statistically significant decreases in mean length in the 7.3, 39, 72 and 147 µg a.i./L treatment groups in comparison to the negative control (p ≤ 0.05), but no statistically significant differences were found in any of the treatment total length when compared to the solvent control (p > 0.05). Based upon the lack of significant difference from the solvent control group and the accuracy of the length measurement (nearest 0.1 mm), the differences between the mean length of the negative control and the 7.3, 39, 72 and 147 µg a.i./L treatment groups were not considered to be treatment related or biologically meaningful.

*Dunnett’s test indicated there were statistically significant decreases in mean dry weight in the 39, 72 and 147 µg a.i./L treatment groups in comparison to the negative control and pooled controls (p ≤ 0.05), but no statistically significant differences in mean dry weight were observed when compared to the solvent controls in any of the treatment groups (p > 0.05). Based on comparison with the pooled and negative controls only the NOEC for growth (measured as dry weight) was 17 µg a.i./L, and the LOEC was 39 µg a.i./L. The MATC was calculated to be 26 µg a.i./L,.However, no clear dose response was observed at these concentration levels. This fact, together with the lack of statistical significant differences compared to the solvent controls makes a treatment related effect unlikely. Therefore, the NOEC for the dry weight parameter was > 147 µg a.i./L, which is consistent with the NOEC for the length parameter.

Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Test endpoints analyzed for first-generation daphnids were: survival, reproduction, and growth (length and dry weight).
* Reproduction was based on the number of live young produced per reproductive day. 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.
- Statistical comparisons of treatment group data for all endpoints were made to the negative control group, solvent control group and pooled control data.
- Survival data were 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 (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).
- All statistical tests were performed using a personal computer with SAS (5) software.

Any other information on results incl. tables

Summary of Survival, Reproduction and Growth of Daphnia magna Exposed to XP-7866 for 21 Days

Mean measured conc. (µg a.i./L) % Adult survival (1,2)  Mean No. Neonates per reproductive day +/- Std. Dev. (3) Mean Length +/- Std. Dev. (mm) (5) Mean Dry Weight +/- Std. Dev. (mg) 
Negative control 95 12.1 +/-0.73  4.6 +/-0.05  0.99 +/-0.03 
Solvent control (2) 100 11.0 +/-0.87  4.4 +/-0.12  0.93 +/-0.06 
Pooled control  98  11.5 +/-0.96  0.96 +/-0.06 
7.3  100  8.9 +/-0.75 (4, a, b, c) 4.5 +/- 0.05 (a, 5) 1.0 +/- 0.09 
17  95  10.9 +/- 1.1  4.6 +/- 0.06  0.94 +/- 0.03 
39  100  10.6 +/- 1.1  4.5 +/- 0.08 (a, 5) 0.83 +/-0.06 (a, c)
72  100  11.7 +/- 0.81  4.5 +/- 0.10 (a, 5) 0.85 +/- 0.01 (a, c) 
147  100  10.4 +/- 0.90 (a) 4.5 +/- 0.06 (a, 5) 0.87 +/- 0.08 (a, c) 

(a) Indicates statistically significant differences from the negative control.

(b) Statistically significant differences from the solvent control.

(c) Statistically significant differences from the pooled controls.

(1) There were no statistically significant differences in survival from the negative control, solvent control or pooled controls (Fisher’s Exact test, p > 0.05).

(2) 21-day EC50 for survival: >147 µg a.i./L, the highest concentration tested.

(3) 21-day EC50 for reproduction : > 147 µg a.i./L, the highest concentration tested.

(4) The statistically significant differences in reproduction noted at the 17 µg a.i./L treatment level was not considered to be treatment related since the reduction did not follow a dose response pattern.

(5) Since there were no significant reductions compared to the solvent control or pooled control, the difference was slight (5.5%) and the total length measurement was made to the nearest 0.1 mm, the significant differences found at the 7.3, 39, 72 and 147 µg a.i./L treatment groups from the negative control were not considered treatment related or biologically meaningful.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to XP-7866 at mean measured concentrations of 7.3 to 147 µg a.i./L under flow-through conditions for 21 days. There were no significant treatment-related effects on survival or reproduction or growth measured as length and dry weight at concentrations ≤147 µg a.i./L, the highest achievable test concentration, when compared to the ., solvent controls .

Based on the results, the NOEC was 147 µg a.i./L for the survival, the reproduction and growth measured as length and dry weight. The LOEC was >147 µg a.i./L and the MATC was determined to be ≤147 µg a.i./L. The 21-day EC50 value for adult immobility was >147 µg a.i./L, the highest concentration tested, and the 21-day EC50 value for reproduction was >147 µg a.i./L, the highest concentration tested.

Thus it can be concluded that no adverse effect on daphnids growth or reproduction was observed in this study, when compared to the solvent control, at the highest tested concentration of 147 µg a.i./L, which is the maximum concentration that can be obtained in the test medium under the conditions of the test.