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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 13 2003 to August 29 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline.
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
Analytical sampling
Samples were collected from the batches of test solutions prepared for each treatment and control group on Day 0 and Day 1 to measure concentrations of the test substance. Samples also were collected from the 24 hour old test solution in each test chamber at approximately 24 hours and at test temination. All samples were collected at mid-depth, placed in teflon tubes and processed immeadiately for analysis.
Vehicle:
yes
Details on test solutions:
A primary stock solution was prepared on Day 0 by dissolving Phosflex TXP in dimethyl formamide (DMF) at a nominal concentration of 20 mg/mL. Seven secondary stock solutions were prepared in DMF at nominal concentrations of 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 mg/mL by proportional dilution of the primary stock. The stock solutions were mixed by inversion and appeared clear and colourless and were stored refrigerated for use in preparing the test solutions.
The test solutions were prepared on Day 0 and Day 1 at nominal concentrations of 16, 31, 63, 125, 250, 500 and 1000 µg/mL by adding 50 µL of the appropriate stock solution into a total volume of 500 mL of dilution water (Wildlife International Ltd. well water). The solutions were inverted to mix and equal potions placed in each of two test chambers per treatment. The solvent control was prepared at a concentration of 0.1 mL/L by adding 50 µL of DMF into a total volume of 500 mL of dilution water. All test solutions appeared clear and colourless at test initiation and termination, with the exception of the 1000 and 2000 µL/L solutions, which appeared slightly cloudy.
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 and ease of culturing in the laboratory. Daphnid neonates used in the test were less than 24-hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland.

Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. The adult daphnids used to supply neonates for the test were held for 15 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding period. During the 2-week holding period immediately preceding the test, water temperatures ranged from 20.1 to 20.8°C, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.4 to 8.7, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.8 to 9.0 mg/L (>87% of saturation), measured with a Yellow Springs Instruments Model 5 IB dissolved oxygen meter. Daphnids in the cultures were fed daily a mixture of yeast, Cerophyll®, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum. The adults were fed prior to test initiation, but neonates were not fed during the test.
Neonate daphnids were obtained for testing from five individual adult daphnids. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers until each chamber contained ten daphnids. The transfer chambers were indiscriminately assigned to the test chambers and the daphnids were released into the test chambers below the water surface using a wide-bore pipette.
Test type:
other:
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
124-128 mg/l as CaCO3
Test temperature:
20 +/- 2°C
pH:
8.2 - 8.4
Dissolved oxygen:
8.0 - 8.5 mg/l throughout the study.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal: Negative control, solvent control, 16, 31, 63, 125, 250, 500, 1000, 2000 µg/L
Details on test conditions:
During the definitive test, test chambers consisted of 250 mL glass beakers containing approximately 250 mL of test solution. The depth of test solution in a representative test chamber was 7.9 cm. Each test chamber was labelled with the project number, test concentration and replicate. Test chambers were impartially positioned in a temperature-controlled environmental chamber set to maintain the desired test temperature throughout the test period.
Fluorescent lightbulbs that emit wavelengths similar to natural sunlight (Colortone 50) were used for illumination of the culture and test chambers during holding, acclimation and testing. A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. 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 at test initiation was 204 lux at the surface of the water of one representative test chamber.
The target test temperature during the study was 20 +/- 2°C. Temperature was measured continuously during the test in a container of water placed adjacent to the test using a Fulscope ER/C recorder. The recorder was verified prior to test initiation using a liquid in glass thermometer. Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24 hours (before and after renewal) during the test. Hardness, alkalinity, specific conductance and Total Organic Carbon (TOC) were measured in the dilution water at the beginning of the test.
Observations
Observations were made periodically to determine the numbers of dead and immobile organisms. Immobility was defined as lack of movement by the organism except for minor activity of the appendages. The number of individuals exhibiting signs of toxicity or abnormal behaviour also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
60 µg/L
Nominal / measured:
meas. (arithm. mean)
Remarks on result:
other: 95% confidence interval 49 - 107 µg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
11 µg/L
Nominal / measured:
meas. (arithm. mean)
Details on results:
Measurement of test concentrations
Nominal concentrations selected for use in this study were 16, 31, 63, 125, 250, 500, 1000 and 2000 µg/L. Analyses of the test solutions resulted in some variability in measured concentrations, which can be attributed to testing above the limits of solubility. The reported solubility of Phsoflex TXP in purified water was approximately 19 µg/L. Results of analyses to measure concentrations of Phosflex TXP in samples collected from the newly prepared test solutions on Day 0 and Day 1 ranged from approximately 85 to 117% of nominal. Results of analyses to measure concentrations of phosflex TXP in samples collected from 24 hour old test solutions at renewal and at test termination ranged from 48 to 88% of nominal. When the measured concentrations of the test samples collected at test initiation, priot to and after renewal at 24 hours and at test termination were averaged, the mean measured concentrations for the study were 11, 22, 49, 107, 232, 418, 903 and 1597 µg/L, representing 69, 71, 78, 86, 93, 90 and 80% of nominal concentrations, respectively. The results of the study were based on the mean measured test concentrations.

Observations and measurements
Water temperatures were within the 20 +/- 2°C range established for the test. Dissolved oxygen concentrations remained > 7.8 mg/L (87% of saturation) throughout the test. Measurements of pH ranged from 8.0 to 8.6. The measurements of hardness, alkalinity, specific conductance and TOC in the dilution water at test initiation were typical of Wildlife International Ltd. well water.
During sampling of the test chambers for analytical measurements at 24 hours, one daphnid inadvertently was removed from one test chamber of each of the solvent and negative control groups. These daphnids were excluded from mortality calculations. With the exception of one dead daphnid in the solvent control group by test termination, all other control daphnids appeared normal during the study. In the 11 µg/L treatment group, two immobile daphnids were observed by test termination. The 10% immobility was equivalent to the acceptable control immobility criteria, and therefore, was not considered to be treatment related. Consequently, the NOEC was considered to be 11 µg/L. Percent mortality/immobility in the 22, 49, 107, 232, 418, 903 and 1597 µg/L treatment groups at test termination was 15, 25, 100, 100, 95, 100 and 95%, respectively. EC50 values at 24 and 48 hours were estimated from mortality/immobility data.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
Statistical analyses
There was <50% mortality/immobility in any treatment group at 24 hours, which precluded the statistical calculation of an EC50 value. The 48 hour mortality/immobility data were analyzed using the computer program of E. C. Stephan. The program was designed to calculate the EC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with a non-linear interpolation. In this study, binomial probability was used to calculate the 48 hour EC50 value. The NOEC was determined by visual interpretation of the mortality, immobility and observation data.
Validity criteria fulfilled:
yes
Conclusions:
The cladoceran, Daphnia magna, was exposed to eight concentrations of Phosflex TXP under static-renewal conditions for 48 hours. Based on the mean, measured test concentrations, the 48 hour EC50 value was 60 µg/L, with a 95% confidence interval of 49 to 107 µg/L. The no observed effect concentration (NOEC) was 11 µg/L
Executive summary:

The cladoceran, Daphnia magna, was exposed to eight concentrations of Phosflex TXP under static-renewal conditions for 48 hours. Based on the mean, measured test concentrations, the 48 hour EC50 value was 60 µg/L, with a 95% confidence interval of 49 to 107 µg/L. The no observed effect concentration (NOEC) was 11 µg/L.

Classification as "very toxic to aquatic organisms" is applicable.

Description of key information

Short-term toxicity to aquatic invertebrates

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
60 µg/L

Additional information

The cladoceran, Daphnia magna, was exposed to eight concentrations of Phosflex TXP under static-renewal conditions for 48 hours. Based on the mean, measured test concentrations, the 48 hour EC50 value was 60 µg/L, with a 95% confidence interval of 49 to 107 µg/L. The no observed effect concentration (NOEC) was 11 µg/L.

Classification as "very toxic to aquatic organisms" is applicable.