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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-15 to 2015-06-16
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
according to guideline
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
C4H12NaNO7P2 · 3 H2O
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- 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
- Purity test date: 100.2% (tested as 100%)

- Storage condition of test material: Room temperature in the original container in a dark, ventilated cabinet.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble

- Treatment of test material prior to testing: Creation of a 10,000 mg active ingredient/L stock solution using the prepared water
- Final dilution of a dissolved solid, stock liquid or gel: The stock solution was diluted with prepared water to nominal concentrations of 0.63, 1.3, 2.5, 5.0 and 10 mg active ingredient / L were selected for the definitive exposure.

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

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 0.63, 1.3, 2.5, 5.0 and 10 mg active ingredient / L nominal for the main test,0.063, 0.13, 0.25, 0.50, 1.0 mg active ingredient / L nominal for the preliminary test.
- Sampling method: Prior to the start of the definitive exposure, samples from one replicate of each treatment level and the control solution were collected and analyzed for alendronate sodium. In addition, a sample of the diluter stock solution was also analyzed. Results of the pretest analyses were used to judge whether sufficient quantities of alendronate sodium were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure. During the in-life phase of the definitive study, water samples were removed from a single replicate of each treatment and the control, with the sampled replicate alternating at each successive sampling (replicate B, then C, then D, etc.). Samples were removed on test day 0,4 (hatch), 11, 18, 19, 24 and 32. All exposure samples were removed from the approximate midpoint of each aquarium using a pipette. In addition, a sample of the stock solution was analyzed at each sampling interval during the exposure period.
Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the exposure concentration range and remained with the exposure solution samples throughout the analytical process. Results of the analyses of the QC samples were used to judge the precision and the quality control maintained during the analysis of exposure solution samples.
- Sample storage conditions before analysis: Samples were analyzed immediately following sampling.

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Creation of a concentrated stock solution with total dissolution, then dilution to the needed nominal concentrations.
- 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 vehicle was the only solvent for the solid test material.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None, the test material was totally dissolved in the vehicle.

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
- Common name: Fathead minnow embryos
- Strain: Pimephales promelas
- Source: Pimephales promelas
- Age at study initiation (mean and range, SD): <26 hours old
- Length at study initiation (length definition, mean, range and SD): embryos
- Weight at study initiation (mean and range, SD): embryos

- Acclimation period: 26 hours
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: None
- Feeding frequency during acclimation: None

- Food type: Artemia salina
- Amount: ad libitum
- Frequency: Beginning on day 5, one day post hatch, 3 times a day

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
32 d
Remarks on exposure duration:
4 days into the test, the embryos hatched.
Post exposure observation period:
Larvae were euthanized and then immediately analyzed at the test's conclusion.

Test conditions

60 to 76 mg/L CaCO3
Test temperature:
24 to 26 degrees Centigrade
6.9 to 7.7
Dissolved oxygen:
7.06 to 8.46 mg/L (85.0 to 102% saturation)
18 to 20 mg/L CaCO3
380 to 490 microSiemens/cm
Nominal and measured concentrations:
Nominal concentrations:
Preliminary Test: 0.063, 0.13, 0.25, 0.50, 1.0 mg active ingredient / L
Main Test: 0, 0.63, 1.3, 2.5, 5.0, 10.0 mg active ingredient / L

Measured Concentrations:
Preliminary Test: No measurements of the true concentration were conducted due to a lack of mortality
Main Test: 0, 0.41, 1.1, 1.9, 3.8, and 8.0 mg active ingreident / L respectively
Details on test conditions:
- Test vessel: 2.5 gallon exposure aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Glass and silicone sealant, 5.5 L exposure volume
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Porportional diluter
- Renewal rate of test solution (frequency/flow rate): 7.3 aquarium volumes / 24 h
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: <0.042 g/L

- Source/preparation of dilution water: A mixture of unadulterated water from a 100-meter bedrock well and de-chlorinated Town of Wareham well water.
- Total organic carbon: 0.51 mg/L - 1.2 mg/L
- Particulate matter: Not in toxic concentrations according to U.S. EPA standard method (1997)s in agreement with ASTM (2007) standard practice.
- Metals: Not in toxic concentrations according to U.S. EPA standard method (1997)s in agreement with ASTM (2007) standard practice.
- Pesticides: Not in toxic concentrations according to U.S. EPA standard method (1997)s in agreement with ASTM (2007) standard practice.
- Chlorine: Not in toxic concentrations according to U.S. EPA standard method (1997)s in agreement with ASTM (2007) standard practice.
- Alkalinity: 20-21 mg/L CaCO3
- Ca/mg ratio: Not in toxic concentrations according to U.S. EPA standard method (1997)s in agreement with ASTM (2007) standard practice.
- Culture medium different from test medium: No
- Intervals of water quality measurement: Weekly for hardness/alkalinity/conductivity, monthly for TOC, bi-annually otherwise

- Photoperiod: A 16-hour light photoperiod and 8 hours darkness with a 15- to 30-minute transition period was provided at the exposure solutions' surface.
- Light intensity: 81 to 110 footcandles (870 to 1200 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Effect parameters measured: Stage of embryonic development reached; embryonic mortality; live, normal larvae at hatch; larval survival rate; total length of larvae; wet weight of larvae.
- Observation Intervals: Embryo mrotality, success rate, were both measured at 4 days (day of hatch), while other parameters were measured at study conclusion (32 days)

- Test concentrations: 0.063, 0.13, 0.25, 0.50, 1.0 mg active ingredient / L
- Results used to determine the conditions for the definitive study:
Nominal Concentration (mg/L) Mean Embryo Hatching Success (%) Live, Normal Larvae at Hatch (%) Mean Larval Survival
Control 92 90 95
0.063 90 94 94
0.13 88 96 89
0.25 96 94 89
0.50 89 91 94
1.0 87 93 88

(CONT.) Nominal Concentration (mg/L) Mean Larval Length (mm) Mean Larval Wet Weight (g)
Control 15.02 0.0282
0.063 15.39 0.0311
0.13 16.07 0.0348
0.25 16.01 0.0338
0.50 15.47 0.0303
1.0 16.07 0.0348
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
Key result
32 d
Dose descriptor:
Effect conc.:
ca. 1.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Length and wet weight
Most sensitive to toxicity
Key result
32 d
Dose descriptor:
Effect conc.:
ca. 1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Length and wet weight
Most sensitive to toxicity
Details on results:
- Fish weights (individual and mean values) on day 28: see Table #4
- Type of and number with morphological / behavioural abnormalities:
- Other biological observations: At exposure initiation, a subsample of 30 embryos was collected and the embryonic stage of development was determined (Table 3). All embryos were determined to be at stage 20 (otic vesicle, 16 somite pairs)
At the completion of hatch (day 4), hatching success in the control averaged 100%. Embryo hatching success in the 0.41, 1.1, 1.9, 3.8 and 8.0 mg/L treatment levels averaged 97, 98, 100, 97 and 98%, respectively. Fisher’s Exact Test with Bonferroni-Holm’s Adjustment determined no significant difference in embryo hatching success in any of the treatment levels compared to the control.
At exposure termination (32 days, 28-days post-hatch), larval survival in the control averaged 100%. Larval survival in the 0.41, 1.1, 1.9, 3.8 and 8.0 mg/L treatment levels averaged 99, 95, 100, 93 and 79%, respectively. Cochran-Armitage Trend Step-Down Test determined a significant difference in larval survival among organisms exposed to treatment levels 3.8 and 8.0 mg/L compared to the control (100%). Due to significant survival effects in the 3.8 and 8.0 mg/L treatment levels, these concentrations were excluded from length and weight analysis.
- Effect concentrations exceeding solubility of substance in test medium: No
- Incidents in the course of the test which might have influenced the results: None
Results with reference substance (positive control):
No positive control.
Reported statistics and error estimates:
Determination of NOEC and LOEC
Following termination of the early life-stage exposure, data obtained on embryo hatching
success, the percentage of embryos producing live, normal larvae at hatch, larval survival and
larval growth at exposure termination (total length and wet weight) were statistically analyzed to
establish treatment level effects. Statistical analyses of the data essentially followed the
procedures described in the document, Current Approaches in the Statistical Analysis of
Ecotoxicity Data: A Guidance to Application (OECD, 2006). Analysis were done to a 95% confidence level.

Applicant's summary and conclusion

Validity criteria fulfilled:
This study validly assesed the chronic aquatic toxicity to fish during the early life growth stage according to the stated guideline.