Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-05 to 2014-11-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MSD International GMBH, Clonmel, Ireland and CTA-553
- Expiration date of the lot/batch: 2016-01-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the original container in a dark, ventilated cabinet
- Solubility and stability of the test substance in the solvent/vehicle: Completely soluble

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 10 mg test ingredient / L stock solution was prepared using dilution water
- Preliminary purification step (if any): The dilution water was filtered for contaminants and fortiifed to a set standard for hard water.
- Final dilution of a dissolved solid, stock liquid or gel: The 10 mg test ingreident / L stock solution was diluted down to the test concentrations by addition of more dilution water and two minutes of stirring with a glass rod.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Concentrations of 0.63, 1.3, 2.5, 5.0, and 10 mg test substance / L

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg test substance/L
- Sampling method: During the definitive exposure, a sample of newly prepared test solutions (test days 0, 2, 16 and 19) was removed from each treatment level and control prior to division into replicate test vessels and analyzed for alendronate sodium concentration. Additionally, aged test solutions were sampled and analyzed on test days 2, 5, 19 and 21 for determination of alendronate sodium concentration. Samples from aged test solutions were removed from a composite of all available replicate vessels.

- Sample storage conditions before analysis: Samples were analyzed immediately.

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Dilution of a primary stock solution and then stirring with a glass rod for two minutes
- Controls: A negative vehicle control was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): The water was the only solvent.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnids
- Strain/clone: Daphnia magna
- Source: Laboratory cultures maintained at Smithers Viscient
- Age of parental stock (mean and range, SD): <24 hours old
- Feeding during test: Yes
- Food type: Varied depending on the age of the daphnid
- Amount: 200 µL of algal suspension (Ankistrodesmus falcatus; 4 x 10^7 cells/mL), and 50 µL of a yeast, cereal leaves and digested flaked fish food (YCT) suspension
- Frequency: Once daily

ACCLIMATION
- Acclimation period: <24 hours
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: unicellular green algae, Ankistrodesmus falcatus (4 x 10^7 cells/mL) in addition to a suspension of YCT (yeast, cereal leaves and flaked fish food
- Feeding frequency: Once
- Health during acclimation (any mortality observed): None

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Offspring were removed, counted, and discarded at each observation interval. In addition, the number of immobilized offpsring and the time to first brood release were recorded for each treatment level and control.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
21 d
Post exposure observation period:
None

Test conditions

Hardness:
180 to 210 mg/L CaCO3
Test temperature:
20-21 degrees centigrade
pH:
7.8-8.9
Dissolved oxygen:
6.5 to 11 mg/L
Salinity:
68-90 mg/L CaCO3
Conductivity:
480-730 uS/cm
Nominal and measured concentrations:
Nominal concentrations:
Preliminary Test: 0, 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg test substance/L
Main Test: 0, 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg test substance/L

Measured Concentrations:
Preliminary Test: No concentration measurements were taken during this test.
Main Test: 0.26, 0.56, 1.2, 2.3, 4.7 and 9.1 mg test ingredient/L
Details on test conditions:
TEST SYSTEM
- Test vessel: clear beakers
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass, 100 mL, 20 mL, 80 mL
- Renewal rate of test solution (frequency/flow rate): 48 or 72 hours for each renewal
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates):10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated Town of Wareham well water passed through a carbon canister and a deionization canister prior to fortifying
- Total organic carbon: 0.36 mg/L
- Particulate matter: None of these
compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Metals: None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Pesticides: None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Chlorine: None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Alkalinity: 70-90 mg/L CaCO3
- Ca/mg ratio: None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM (2002) standard practice.
- Conductivity: 530-890 microSiemens/cm
- Culture medium different from test medium: They are the same.
- Intervals of water quality measurement: Measured on each up to 14 day old batch of water prior to use

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 light / 8 dark
- Light intensity: 6.8 to 20 µE m^-2 s^-1 at the surface of the exposure solutions was provided by fluorescent bulbs.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnid survival, reproduction, and total body length.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on preliminary study.
- Range finding study: Yes
- Test concentrations:
Nominal concentrations:
Preliminary Test: 0, 0.00010, 0.0010, 0.010, 0.10 and 1.0 mg test substance/L
Main Test: 0, 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg test substance/L
Measured Concentrations:
Preliminary Test: No concentration measurements were taken during this test.
Main Test: 0.26, 0.56, 1.2, 2.3, 4.7 and 9.1 mg test ingredient/L
- Results used to determine the conditions for the definitive study: 100% survival was observed at all test conditions in the preliminary study.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 4.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: Reproduction, mortality, and body weight
Key result
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
ca. 9.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
ca. 5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
other: EC20
Effect conc.:
ca. 5.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
ca. 6.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: None
- Observations on body length and weight: Total body length (Table 10) at exposure termination among control daphnids averaged 4.99 mm. Total body length among daphnids exposed to the 0.26, 0.56, 1.2, 2.3 and 4.7 mg/L treatment levels averaged 5.15, 4.96, 4.96, 5.03 and 5.04 mm, respectively. No length data is available for the 9.1 mg/L exposure level, as 100% of parental daphnids were immobilized by day 21. Statistical analysis (Dunn’s Test with Bonferroni-Holm Adjustment) determined no significant difference in total length among daphnids exposed to all treatment levels compared to the control (4.99 mm). Therefore, the NOEC for total body length was determined to be 4.7 mg/L and the LOEC was determined to be > 4.7 mg/L. The 21-day EC10, EC20, and EC50 values for length were not able to be determined.
- Other biological observations: Following 21 days of exposure, the organisms exposed to the 0.26, 0.56, 1.2, 2.3 and 4.7 mg/L treatment levels had released a mean cumulative offspring per female of 227, 204, 185, 206 and 217, respectively. The cumulative offspring per female exposed to the 1.2 mg/L treatment level
tested was statistically significant, however, the following two treatment levels (2.3 and 4.7 mg/L) were not statistically significant. Therefore, the effect observed with the 1.2 mg/L treatment level was due to biological variability and not toxicant related. No reproduction data is
available for the 9.1 mg/L exposure level, as 100% of parental daphnids were immobilized by day 21. First brood release by daphnids occurred on day 7 for all treatment levels, with the exception of the 9.1 mg/L treatment level where no offspring were released. Statistical analysis (Dunn’s Test with Bonferroni-Holm Adjustment) determined no significant difference in offspring per female among daphnids exposed all treatment levels compared to the control (212 offspring per female).
The mean number of offspring released per female organism in the dilution water control during the 21-day test was 212 offspring per female. The coefficient of variation for daphnid reproduction in the control was 11%.
- Mortality of control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
No positive control was used.
Reported statistics and error estimates:
Significant differences in the percent survival were determined using Fisher’s Exact Test (US EPA, 2002). Daphnid survival data were analyzed before reproduction and length; if there were concentrations that caused significant survival effects, then those levels were excluded from the analysis of daphnid reproduction and growth. For the purpose of determining survival effects, immobilized organisms were considered to be dead. Offspring produced by adults that died during the test which were inadvertent or not toxicant related were excluded.
Prior to analysis of the reproduction data, daphnid survival data was analyzed with Cochran-Armitage Trend Step-Down Test to determine whether or not
the mortality followed a concentration-response pattern. If the mortality did not follow a concentration-response pattern, then those replicates with parental mortality were excluded from the analysis of reproduction. If the mortality did follow a concentration-response pattern, then replicates for parental mortality were included. For this study, survival did follow a concentration-response pattern.
Shapiro-Wilks' Test for normality (U.S. EPA, 2002) was used to assess the observed sample distribution for reproduction and length data. If the data
were normally distributed, then a parametric procedure was used for subsequent analyses. If the data were not normally distributed, than a nonparametric
procedure was used for subsequent analyses. As a check on the assumption of homogeneity of variance, data for reproduction and length were analyzed using Bartlett’s Test (U.S. EPA, 2002). For this study, reproduction data did not meet the assumptions for normal distribution and homogeneity of variance. Length data did not meet the assumption for normality, however it did meet the assumption for homogeneity of variance. Therefore, both these data were evaluated using Dunn’s Test with Bonferroni-Holm Adjustment (U.S. EPA, 2002), a nonparametric procedure, to establish treatment effects.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
This study determined the 21 day NOEC, LOEC, EC10, EC20, and EC50 for the test substance validly according to the specified guideline.