Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2005 to 18 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. Environmental Protection Agency Series 850-Ecological Effects Test Guidelines OPPTS Number 850.1010 (public draft)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ASTM Standard E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphibians

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analytical Sampling
Samples were collected from each batch of test solution at the beginning of the test and from pooled test solution from the replicates of each test concentration at 48 hours to measure concentrations of total organic carbon (TOC). The samples were collected from mid-depth into glass vials, acidified with 2N HCl, and stored under refrigeration prior to analysis.

Vehicle:

no

Details on test solutions:

Preparation of Test Concentrations
A primary stock solution was prepared at a nominal concentration of 10 mg/L, the highest concentration tested, by mixing 30.0 mg of alkenyl sulfolane into 3 L of dilution water (UV sterilized well water). The stock solution was sonicated for one minute and mixed with a top-down stainless steel mixer. Aliquots of the stock were proportionally diluted with dilution water to prepare 500 mL of test solution at nominal concentrations of 0.31, 0.63, 1.3, 2.5 and 5.0 mg/L. The primary stock solution was mixed continuously during preparation of the test solutions. The solutions were mixed by inversion and appeared clear and colourless at test initiation and termination.

Observations
Observations were made periodically to determine the numbers of dead and immobile organisms. Immobility was defined as a lack of movement by the organism except for minor activity of the appendages. The numbers of individuals exhibiting signs of toxicity or abnormal behaviour also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation.

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. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 18.9 to 20.4ºC, measured with a hand-held liquid-in-glass thermometer. The pH of the water ranged from 8.3 to 8.6, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 7.9 to 9.0 mg/L (≥88% of saturation), measured with a Yellow Springs Instruments Model 51B 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.

The seven adult daphnids used to supply neonates for the test were held for 21 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 Wildlife International, Ltd. Project Number 264A-108 7-day period prior to the test. The adults showed no signs of disease or stress during the holding period. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred one or two at a time to transfer chambers (e.g., 10 mL glass beakers) until each chamber contained 10 daphnids. Each group of daphnids then was transferred to an indiscriminately assigned test chamber. All transfers were made below the water surface using wide-bore pipettes.

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 is characterised as moderately-hard water. The specific conductance, hardness, alkalinity and pH of the well water during the four-week period immediately preceding the test are presented in attached Appendix 1.

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 ultra violet (UV) sterilizer. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water used by Wildlife International, Ltd. are presented in attached Appendix 2.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3. Hardness, alkalinity, specific conductance and total organic carbon (TOC) were measured in the dilution water at the beginning of the test. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4).

Test temperature:

The target water temperature during the study was 20 ± 1ºC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24 hours using a liquid-in-glass thermometer. Temperature also was measured continuously during the test in a container of water placed adjacent to the test chambers, using a Fulscope ER/C Recorder. The recorder was verified prior to test initiation with a liquid-in-glass thermometer. (see attached table 2)

pH:

Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24 hours. (see attached table 2)

Dissolved oxygen:

Dissolved oxygen was measured in each test chamber at the beginning and end of the test and at approximately 24 hours. (see attached table 2)

Salinity:

Freshwater used.

Nominal and measured concentrations:

Nominal test concentrations were selected in consultation with the Sponsor based on the results of an exploratory range finding toxicity test. Nominal test concentrations selected were 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg alkenyl sulfolane/L.

Details on test conditions:

Test Apparatus
Test chambers were 250-mL glass beakers containing 250 mL of test water. The depth of the test water in a representative chamber was 5.9 cm. Test chambers were labelled with the project number, test concentration and replicate, and were indiscriminately positioned in a temperature controlled environmental chamber set to maintain the desired test temperature.

Environmental Conditions
Fluorescent light bulbs that emit wavelengths similar to natural sunlight (Colortone® 50) were used for illumination of the culture and test chambers. 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 475 lux at the surface of the water of one representative test chamber. The target water temperature during the study was 20 ± 1ºC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24 hours using a liquid-in-glass thermometer. Temperature also was measured continuously during the test in a container of water placed adjacent to the test chambers, using a Fulscope ER/C Recorder. The recorder was verified prior to test initiation with 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. Hardness, alkalinity, specific conductance and total organic carbon (TOC) were measured in the dilution water at the beginning of the test.

Light intensity was measured using a SPER Scientific Model 840006C light meter. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Model 525Aplus meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater. TOC was measured using a Shimadzu Model TOC-5000 total organic carbon analyzer.

Conditions for the Validity of the Test
The following criteria used to judge the validity of the test were met:
1. immobility of the daphnids in the negative control group did not exceed 10% by the end of the test, and
2. the dissolved oxygen concentration was at least 60% of the air saturation value throughout the test.

Reference substance (positive control):

not specified

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL of 2.5 to 10 mg/l

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation, reproduction and growth

Remarks on result:

other: 95% CL not stated

Duration:

48 h

Dose descriptor:

other: No-Mortaility:Immobility Concentration

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Details on results:

Measurement of TOC in Test Solutions
Results of analyses for TOC in the test solutions are presented in attached Table 1. Measurements of TOC in the newly prepared test solutions on Day 0 ranged from <1 to 2.9 mg C/L. Measurements of TOC in the 48-hour old solutions on Day 2 ranged from <1 to 4.1 mg C/L. TOC content generally increased with increasing test substance concentration.

Observations and Measurements
Measurements of temperature, dissolved oxygen and pH of the water in each test chamber are presented in attached Table 2. Water temperatures were within the 20 ± 1°C range established for the test. Dissolved oxygen concentrations remained ≥8.3 mg/L (≥92% of saturation) throughout the test. Measurements of pH ranged from 8.2 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 (attached in Table 3).
Daily observations for mortality, immobility and signs of toxicity during the test are presented in attached Table 4. All daphnids in the negative control group and in the 0.31 and 0.63 mg/L treatment groups appeared normal during the study. Percent mortality/immobility in the 1.3, 2.5, 5.0 and 10 mg/L treatment groups at test termination was 0, 0, 60 and 100%, respectively. While no mortality/immobility occurred in the 1.3 and 2.5 mg/L treatment groups, 25 and 75% of the daphnids, respectively, appeared lethargic at test termination. Surviving daphnids in the 5.0 mg/L treatment group also appeared lethargic at test termination. The no-mortality/immobility concentration was 2.5 mg/L and the NOEC was 0.63 mg/L. EC50 values at 24 and 48 hours were calculated from the mortality and immobility data and are presented in attached Table 5. A graph of the concentration-response
curve at test termination is presented in attached Figure 1.

Results with reference substance (positive control):

- Mortality:
Percent mortality/immobility in the 1.3, 2.5, 5.0 and 10 mg/L treatment groups at test termination was 0, 0, 60 and 100%, respectively.

- EC50/LC50:
The 48-hour EC50 value was 4.6 mg/L, with a 95% confidence interval of 2.5 to 10 mg/L and the NOEC was 0.63 mg/L.

Reported statistics and error estimates:

Statistical Analyses
The mortality and immobility data were analysed using the computer program of C. E. Stephan (5). 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 nonlinear interpolation (6, 7, 8). Based on the pattern of mortality/immobility in this study, probit analysis was used to calculate the 24-hour EC50 value and binomial probability was used to calculate the 48-hour EC50 value. The no-mortality/immobility concentration and the no-observed-effect concentration were determined by visual interpretation of the mortality, immobility and observation data. The results of the study were based on the nominal test concentrations.

Validity criteria fulfilled:

yes

Conclusions:

The cladoceran, Daphnia magna, was exposed for 48 hours under static conditions to six nominal concentrations of alkenyl sulfolane ranging from 0.31 to 10 mg/L. The 48-hour EC50 value was 4.6 mg/L, with a 95% confidence interval of 2.5 to 10 mg/L. The no-mortality/immobility concentration was 2.5 mg/L and the NOEC was 0.63 mg/L.

Executive summary:

SPONSOR: American Chemistry Council

TITLE: Alkenyl Sulfolane: A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna)

WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 264A-108

TEST DATES: Experimental Start (OECD & EPA): November 15, 2005 Biological Termination: November 17, 2005 Experimental Termination: November 18, 2005

LENGTH OF EXPOSURE: 48 Hours

TEST ORGANISMS: Cladoceran (Daphnia magna)

SOURCE OF TEST ORGANISMS: Wildlife International, Ltd. Cultures Easton, Maryland 21601

AGE OF TEST ORGANISMS: Neonates <24 hours at test start

TEST CONCENTRATIONS: NOMINAL: Negative control, 0.31 mg/l, 0.63 mg/l, 1.3 mg/l, 2.5 mg/l, 5.0 mg./l, 10 mg/l

RESULTS & CONCLUSION:

Based on nominal test concentrations 48 -Hour

EC50 : 4.6 mg/l

95% Confidence Interval: 2.5 - 10 mg/l

No-Mortaility/Immobility Concentration: 2.5 mg/l

NOEC: 0.63 mg/l

Description of key information

48h EC50 = 4.6 mg/L, NOEC = 0.63 mg/L; study performed in line with OECD Guideline 202, EPA OPPTS 850.1010 and ASTM Standard E729-96; Palmer and Krueger (2008).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.6 mg/L

Additional information

In a GLP compliant short-term toxicity to aquatic invertebrates study conducted in line with standardised guidelines, the toxicity of the test substance to Daphnia magna was determined following 48 hour exposure to the test substance at 0.31, 0.63, 1.3, 2.5, 5.0 and 10 mg/L. Based on nominal concentrations, the 48 hour EC₅₀ was determined to be 4.6 mg/L (95% C.I. 2.5-10 mg/L). The NOEC was determined to be 0.63 mg/L and the no-mortality/immobility concentration to be 2.5 mg/L.