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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:
March 10, 2003 to October 29, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 1984
Deviations:
yes
Remarks:
48 hours test duration (as in OECD 202, adopted 2004), organisms were not randomly assigned to test concentrations
GLP compliance:
yes
Specific details on test material used for the study:
The sample of 3-chloro-o-xylene (T.R. Wilbury Laboratories sample number 1763) used for the study was delivered on March 10, 2003. It was contained in four 50 mL amber bottles that were shipped in a plastic bucket at ambient temperature. The Label attached to the bottles included the following information: "3-Cl-1,2-dimethylbenzene, CAS # 608-23-1, Clariant Lot 26.11.02, 25 g, 98%, combustible". 3-chloro-o-xylene (a colorless liquid) was supplied by General Electric Corp., 1 Research Circle, Niskayuna, New York. Prior to use the test substance was stored at room temperature in the dark. Test concentrations were corrected for the purity of the test substance. The test substance was determined to be stable under test conditions.
Analytical monitoring:
yes
Details on sampling:
Analytical determination of test substance concentration (active ingredient) was performed with samples collected from test solutions prepared at each concentration prior to their distribution to test vessels at the start of the toxicity test, and after 48 hours. Samples collected from replicate test vessels at the end of the test were pooled. Each sample set was accompanied by three matrix spike samples prepared in dilution water and a dilution water blank.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
A series of stock solutions (5 500, 9 100, 15 000, 25 000, and 42 000 mg/L) was prepared for the final test by adding the appropriate amount of test substance to dimethylformamide in 10 mL Class A volumetric flasks, and those solutions were then used to prepare 0.55, 0.91, 1.5, 2.5 and 4.2 mg/L test solutions by transferring 0.025 mL of stock solution into 250 mL of dilution water. Note, the concentration of the solvent in the stock solution was not reported.
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile daphnids, Daphnia magna, less than 24 hours old, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were produced by an in-house culture of adult daphnids that were maintained under test conditions at T.R. Wilbury Laboratories. The original culture was obtained from Aquatic Biosystems Inc., Fort Collins, Colorado, on April 17, 2003.
Prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions, and the test organisms were not treated for disease. No mortality was observed during the 48 hours prior to the beginning of the test and at the beginning of the test organisms were apparently free of disease, injuries, and abnormalities. The culture used to produce neonates for the definitive toxicity test produced young by day 7 and produced an average of at least 3 young/female/day during the 7 days prior to the start of the test.
During the 27 days prior to the beginning of the test, the temperature of the culture ranged from 19.5 to 20.9°C, the pH ranged from 6.9 to 7.4, and the dissolved oxygen concentration was always at least 8.5 mg/L. Test organisms were fed the freshwater algae, Selenastrum capricornutum, and a mixture of yeast and trout chow (Lot number DC04) once each day before the test. Daphnids were not fed during the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
164 mg/L as CaCO3
Test temperature:
20.1 to 20.6°C
pH:
6.7 to 7.0
Dissolved oxygen:
7.7 to 8.5 mg/L
Conductivity:
610 to 620 μmhos/cm
Nominal and measured concentrations:
Nominal: 0.55, 0.91, 1.5, 2.5 and 4.2 mg/L
Measured: 0.479, 0.744, 1.32, 2.22 and 3.65 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 40 mL glass vials
- Type (delete if not applicable): open
- Headspace, fill volume: No headspace (tightly capped), 40 mL
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: loading rate during the definitive toxicity test was <= 40 daphnids per liter

TEST MEDIUM / WATER PARAMETERS
- Dilution water: Water used for the acclimation of test organisms and for all toxicity testing was carbon filtered, deionized water collected at T.R. Wilbury Laboratories in Marblehead, Massachusetts. Water was adjusted to a hardness of 160 to 180 mg/L as CaCO3 and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. Prior to use, the pH of the dilution water was adjusted to <8.0 with 5% H3PO4 (J.T. Baker lot # 40824). A sample of dilution water collected at the start of the definitive test had a hardness of 164 mg/L as CaCO3. Results. Intervals of analytical water quality measurement are 2 times a year.
- Water parameters: Dissolved oxygen (YSI Model 57 meter; instrument number 1), pH (Beckman model 2100 meter; instrument number 209-pHT 19), conductivity (Cole-Parmer conductivity meter; instrument number 94), and temperature (Beckman model 2100 meter; instrument number 209 probe pHT-19) were measured and recorded in each test vessel at the beginning and end of the test and before and after media renewal at 48 hours. The temperature in a representative vessel of water incubated among the test vessels was recorded continuously during the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hour light and 8 hour dark photoperiod
- Light intensity: cool-white fluorescent lights, light intensity of approximately 32 footcandle

TOXICITY TEST / EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Twenty daphnids were randomly distributed among two replicates of each treatment. The test was performed in 40 mL glass vials that were filled to capacity and then tightly sealed to prevent loss of the test substance to the atmosphere. Test vessels were randomly arranged during the test (a random numbers table was used). A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 32 footcandles. A 15 minute transition period was provided between dark and light. Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels. The numbers of surviving organisms, the occurrence of immobility and sublethal effects, and the presence of insoluble material were determined visually and recorded after 0, 24, and 48 hours. Dead test organisms were not removed when first observed because test vessels remained tightly sealed throughout the exposure period.

RANGE-FINDING STUDY
- Range finding test: two range finding tests were performed
- Test concentrations: (1st) 1.0, 10, 50, 100 and 1,000 mg/L, (2nd) 1.0, 5.0, 10, 50 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.05 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 0.744 to 1.32 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1.52 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 1.32 to 2.22 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.744 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
Mortality and immobilisation were first observed at 1.32 mg/L after 48 hours and 24 hours respectively. Exposure of daphnids to 3-chloro-o-xylene resulted in a 24 hour LC50 of 3.19 mg/L (95% confidence interval = 2.22 to 3.65 mg/L) and a 48 hour LC50 of 1.52 mg/L (95% confidence interval = 1.32 to 2.22 mg/L). This exposure also resulted in a 24 hour EC50 of 1.51 mg/L (95% confidence interval = 1.32 to 2.22 mg/L) and a 48 hour EC50 of 1.05 mg/L (95% confidence interval = 0.744 to 1.32 mg/L). The 48 hour no observed effect concentration (NOEC) was 0.744 mg/L 3-chloro-o-xylene (100% survival, 0% affected (immobilised)).

No insoluble material was observed during the definitive toxicity test. Ninety-five to one hundred percent survival occurred in the control and solvent control, and no sublethal effects were observed.

Note 1: Biological data generated by the acute toxicity test are given in any other information on results.

Note 2: The definitive toxicity test was repeated once, because the measured test item concentrations were less than 70% of nominal concentrations.
After 48 hours there was 95 to 100% survival in the control and solvent control, and at 0.91 mg/L, and there was 90% survival at 1.5 mg/L, 10% survival at 2.5 mg/L, and 0% survival at 4.2 and 7.0 mg/L. Sublethal effects (immobilization) were observed in 44% of the surviving daphnids exposed to 1.5 mg/L and 100% of the surviving daphnids exposed to 2.5 mg/L. No other sublethal effects were observed at any tested concentration at the end of the test and no insoluble material was observed. Measured concentrations of 3-chloro-o-xylene were less than 70% of nominal concentrations alter 48 hours, and the test was repeated.
Reported statistics and error estimates:
Results of the toxicity test were interpreted by standard statistical techniques (Stephan, 1983). The LC50 and EC50 values were calculated using the binomial method (the slope of the concentration-response curve could not be calculated using this method). The no observed effect concentration is the highest tested concentration of test substance that did not cause toxicant-related mortality or sublethal effects.
Validity criteria fulfilled:
yes
Remarks:
(acc. to OECD 202, adopted 2004)
Conclusions:
48h-EC50 (Daphnia magna, immobilisation) = 1.05 mg/L (mean measured)
Executive summary:

The purpose of this study was to determine the acute toxicity of 3-chloro-o-xylene to the daphnid, Daphnia magna. This was accomplished by determining the 24 and 48 hour LC50 values and 95% confidence intervals, the 24 and 48 hour EC50 values and 95% confidence intervals, and the 48 hour NOEC. The test was performed according to OECD 202 (1984) and in compliance with GLP. The purity of the test item was 98.3% active ingredient. The test was performed under static conditions with a control, a solvent control, and five concentrations of test substance at a temperature at 20 ± 1°C. The test was performed in glass vials that were filled to capacity and then tightly sealed to prevent loss of the test substance to the atmosphere (in fact, the definitive test was repeated once, because the measured test item concentrations were less than 70% of nominal concentrations). The dilution water was deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 160 to 180 mg/L as CaCO3 and a pH less than 8.0. Nominal concentrations of 3-chloro-oxylene were: 0 mg/L (control and solvent control), 0.55, 0.91, 1.5, 2.5, and 4.2 mg/L. Mean measured test concentrations were 0.479, 0.744, 1.32, 2.22, and 3.65 mg/L. No insoluble material was observed during the definitive toxicity test. Mean measured concentrations were used for all toxicity determinations.

Organisms used in the test were less than 24 hours old. They were obtained from an in-house culture that was acclimated to test conditions at T.R. Wilbury Laboratories. All animals were in good condition at the beginning of the toxicity test. Exposure of daphnids to 3-chloro-o-xylene resulted in a 48 hour median lethal concentration (LC50) of 1.52 mg/L (95% confidence interval = 1.32 to 2.22 mg/L) and a 48 hour median effective concentration (EC50) of 1.05 mg/L (95% confidence interval = 0.744 to 1.32 mg/L). The 48 hour no observed effect concentration (NOEC) was 0.744 mg/L 3-chloro-o-xylene.

Deviation from the test guideline (test organims were not randomly assigned to test concentrations) were considered to not affect the outcome of the toxicity test. The validity criteria of OECD 202 (2004) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment despite of minor deficiencies in reporting.

Description of key information

48h-EC50 (Daphnia magna, immobilisation) = 1.05 mg/L (mean measured, OECD 202, 2003)

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.05 mg/L

Additional information

A reliable key study was performed according to OECD 202 and in compliance with GLP (RL1, 2003). The test was performed under static conditions with a control, a solvent control, and five concentrations of test substance. The test was performed in glass vials that were filled to capacity and then tightly sealed to prevent loss of the test substance to the atmosphere. Nominal concentrations of 3-chloro-oxylene were: 0 mg/L (control and solvent control), 0.55, 0.91, 1.5, 2.5, and 4.2 mg/L. Mean measured test concentrations were: 0.479, 0.744, 1.32, 2.22, and 3.65 mg/L. No insoluble material was observed during the definitive toxicity test. Exposure of daphnids to 3-chloro-o-xylene resulted in a 48h-EC50 of 1.05 mg/L (95% confidence interval = 0.744 to 1.32 mg/L). The 48h-NOEC was 0.744 mg/L 3 -chloro-o-xylene. Deviation from the test guideline were considered to not affect the outcome of the toxicity test. The validity criteria of OECD 202 (2004) were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.