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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 10, 2003 - October 30, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
shorter acclimatisation period (3 days)
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. Test concentrations were corrected for the purity of the test substance. Prior to use the test substance was stored at room temperature in the dark. The stability of the test substance was determined under test conditions by the analysis of test solutions during the definitive toxicity test.
Analytical monitoring:
yes
Details on sampling:
Analytical determination of test substance concentration (active ingredient) was performed with samples collected from the first replicate test vessel of each concentration at the start of the toxicity test and before media renewal at 48 hours, and with samples collected from the second replicate test vessel of each concentration at the end of the toxicity test and after media renewal at 48 hours. 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:
The test substance was used to prepare 70 000, 42 000, 25 000, 15 000, and 9 100 mg/L dimethylformamide stock solutions in 10 mL Class A volumetric flasks, and those stock solutions were then used at 0 and 48 hours to prepare 7.0, 4.2, 2.5, 1.5, and 0.91 mg/L test solutions by transferring 0.20 mL of stock solution into 2.0 L of dilution water. The control received only dilution water and the solvent control received 0.1 mL/L dimethylformamide, the same concentration of solvent that was delivered to test vessels containing 3-chloro-o-xylene.
Test organisms (species):
Pimephales promelas
Details on test organisms:
Juvenile fathead minnow, Pimephales promelas, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were procured from a commercial supplier (Aquatic Biosystems, Fort Collins, Colorado) on May 16, 2002. Prior to testing, the fish were maintained under flow-through conditions in a 20 L tank. During acclimation, fish were not treated for disease and they were free of apparent disease, injuries, and abnormalities at the beginning of the test. Fish were fed dry commercial fish food (Tetra-Min® Staple Food, Lot # TM09) once per day except during the 48 hours preceding the test and they were not fed during the toxicity test.
As the test was performed in sealed containers with no head space, the biomass was reduced as much as possible by using young fathead minnows and reducing the acclimation period from 14 days to three days to limit the growth of the fish during acclimation. During the 3 day period before the start of the test, the acclimation temperature range was 21.2 to 22.3°C and the dissolved oxygen concentration was always at least 8.6 mg/L. Mortality during the acclimation period was 0%.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
48 mg/L as CaCO3
Test temperature:
21.1 to 22.5°C
pH:
7.0 to 7.4
Dissolved oxygen:
7.5 to 9.1 mg/L
Conductivity:
160 to 170 µmhos/cm
Nominal and measured concentrations:
Nominal: 0.91, 1.5, 2.5, 4.2 and 7.0 mg/L
Measured: 0.757, 1.37, 2.41, 4.06, and 6.71 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 2-L glass jars
- Type (delete if not applicable): closed
- Headspace, fill volume: No headspace (tightly capped), 2L
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 48 hours
- 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: 0.01 g/L

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 40 to 48 mg/L as CaCO3 and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. A sample of dilution water collected at the start of the definitive test had a hardness of 48 mg/L as CaCO3. 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 54 footcandls

TOXICITY TEST / EFFECT PARAMETERS MEASURED :
Twenty fathead minnows were randomly distributed among two replicates of each treatment. The test was performed in 2-L glass jars that contained 2 liters of test solution (water depth was approximately 21 cm). Test vessels were randomly arranged in a water bath during the 96-hour test (random numbers tables were used to add test organisms to test vessels, to select the location of each vessel, as well as the assignment of test concentrations). A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a light intensity of approximately 54 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 sublethal effects, and the presence of insoluble material were determined visually and recorded after 0, 24, 48, 72, and 96 hours. Because test vessels were not opened at 24 or 72 hours, dead test organisms were removed only after 48 and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.67
- Range finding study: yes
- Test concentrations: 1.0, 10, 100, 500 and 1 000 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.43 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CI: 2.05 - 2.87
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.757 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
Exposure of fathead minnows to 3-chloro-o-xylene resulted in a 96 hour median lethal concentration (LC50) of 2.43 mg/L, with a 95% confidence interval of 2.05 to 2.87 mg/L. The 96 hour no observed effect concentration (NOEC) was 0.757 mg/L 3-chloro-o-xylene, the lowest tested concentration (this concentration allowed 100% survival with no sublethal effects). Initial mortality occurred after 48 hours at a concentration of 1.37 mg/L and 2.41, and after 24 hours at concentrations of >=4.06 mg/L. Sublethal effects were observed after 96 hours at a concentration of 1.37 mg/L and after 24 hours in concentrations of >= 2.41 mg/L. Affected fathead minnows were e.g. immobilized, swimming erratically, lethargic, and/or exhibiting a loss of equilibrium (the nature of the sublethal effects at 96 hours was not recorded in the raw data).
Exposure of fathead minnows, Pimephales promelas, to 3-chloro-o-xylene resulted in the following LC50 values (mean measured concentrations):
24 hour LC50 = 3.24 mg/L (95% confidence interval = 2.41 to 4.06 mg/L)
48 hour LC50 = 2.56 mg/L (95% confidence interval = 2.17 to 3.03 mg/L)
72 hour LC50 = 2.43 mg/L (95% confidence interval = 2.05 to 2.87 mg/L)
96 hour LC50 = 2.43 mg/L (95% confidence interval = 2.05 to 2.87 mg/L)

No insoluble material was observed during the definitive toxicity test. Ninety to 95% survival occurred and no sublethal effects were observed in the control and solvent control.

Note: Biological data generated by the acute toxicity test are given in any other information on results.
Reported statistics and error estimates:
Results of the toxicity test were interpreted by standard statistical techniques (Stephan, 1983). The LC50 values were calculated using the moving average or binomial method and mean measured concentrations of test substance. The slope of the 96 hour concentration-response curve was calculated by the probit method. The slope of the 96 hour concentration-response curve was 5.2.
The no observed effect concentration is the highest tested concentration at which there was no observed mortality or sublethal effect.

Survival and sublethal effect data from the toxicity test with 3-chloro-o-xylene and the fathead minnow, Pimephales promelas.

Measured test item concentration
(mg/L)
Rep. Number Alive Number Affected*
0 hr 24 hr 48 hr 72 hr 96 hr 0 hr 24 hr 48 hr 72 hr 96 hr
Control 1 10 10 9 9 9 0 0 0 0 0
  2 10 10 9 9 9 0 0 0 0 0
Solvent Control 1 10 10 9 9 9 0 0 0 0 0
  2 10 10 10 10 10 0 0 0 0 0
0.757 1 10 10 10 10 10 0 0 0 0 0
  2 10 10 10 10 10 0 0 0 0 0
1.37 1 10 10 8 8 8 0 0 0 0 0
  2 10 10 8 8 8 0 0 0 0 1
2.41 1 10 10 8 7 7 0 2 1 2 3
  2 10 10 10 9 9 0 4 3 3 6
4.06 1 10 0 0 0 0 0 - - - -
  2 10 1 1 1 1 0 - 1 1 1
6.71 1 10 0 0 0 0 - - - - -
  2 10 0 0 0 0 - - - - -

* Affected fathead minnows were immobilized, swimming erratically, lethargic, and/or exhibiting a loss of equilibrium (the nature of the sublethal effects at 96 hours was not recorded in the raw data).

Measured concentrations of 3-chloro-o-xylene during the toxicity test with the fathead minnow, Pimephales promelas.

Nominal
concentration of the test item (mg/L)

Measured Concentration of the test item (mg/L)

Percent of nominal

0 hr

48 hr1

48 hr2

96 hr

Mean

0 (control)

ND3

ND

ND

ND

ND

-

0.91

0.811

0.714

0.780

0.721

0.757

83

1.5

1.40

1.31

1.35

1.41

1.37

91

2.5

2.49

2.38

2.45

2.33

2.41

96

4.2

4.17

3.83

4.28

3.95

4.06

97

7.0

6.8

6.62

-

-

6.71

96

Matrix Spike Samples4

 

 

 

 

 

 

2.5 replicate 1

2.64

-

2.47

2.57

 

 

replicate 2

2.94

-

2.62

8.175

 

 

replicate 3

3.19

-

3.56

6.155

2.86

114

 

1 Sample collected before media renewal.

2 Sample collected after media renewal.

3 ND = none detected at or above the limit of quantitation (0.0245 mg/L).

4 Standards prepared in water from a control solution from the toxicity test.

5 This sample was assumed to have been prepared incorrectly and this value was excluded from the calculation of the mean.

Validity criteria fulfilled:
yes
Conclusions:
96h-LC50 (Pimephales promelas, mortality) = 2.43 mg/L (mean measured)
Executive summary:

The purpose of this study was to determine the acute toxicity of 3-chloro-o-xylene to the fathead minnow, Pimephales promelas. This was accomplished by determining the 24, 48, 72, and 96 hour LC50s and 95% confidence intervals, and the 96 hour NOEC. The test was performed according to OECD 203 and in compliance with GLP. The purity of the test item was 98.3% active ingredient. The test was performed under semi-static conditions with a control, a solvent control (0.10 mL/L dimethylformamide), and five concentrations of test substance at a temperature of 22 ± 1°C. Nominal concentrations of 3-chloro-o-xylene were: 0 mg/L (control and solvent control), 0.91, 1.5, 2.5, 4.2, and 7.0 mg/L. Mean measured concentrations were 0.757, 1.37, 2.41, 4.06, and 6.71 mg/L. No insoluble material was observed during the definitive toxicity test.

Mean measured concentrations were used for all toxicity determinations. Fathead minnows used in the test were obtained from a commercial supplier and acclimated to test conditions for 3 days (a shortened acclimation period was used, because small fish were required). As the test was performed in sealed containers with no head space, the biomass was reduced as much as possible by using young fathead minnows and reducing the acclimation period from 14 days to three days to limit the growth of the fish during acclimatisation. Sealed vessels were necessary because of the volatile characteristics of the test item.

All test organisms were in good condition at the beginning of the study. After 96 hours of exposure, the control organisms had an average wet weight (blotted dry) of 0.002 g and they ranged in length from 0.61 to 0.99 cm. Exposure of fathead minnows to 3-chloro-o-xylene resulted in a 96 hour median lethal concentration (LC50) of 2.43 mg/L, with a 95% confidence interval of 2.05 to 2.87 mg/L. The 96 hour no observed effect concentration (NOEC) was 0.757 mg/L 3-chloro-o-xylene, the lowest tested concentration (this concentration allowed 100% survival with no sublethal effects). Initial mortality occurred after 48 hours at a concentration of 1.37 mg/L and 2.41 mg/L, and after 24 hours at concentrations of >=4.06 mg/L. Sublethal effects were observed after 96 hours at the concentration of 1.37 mg/L and after 24 hours in concentrations of >= 2.41 mg/L. Affected fathead minnows were e.g. immobilized, swimming erratically, lethargic, and/or exhibiting a loss of equilibrium (the nature of the sublethal effects at 96 hours was not recorded in the raw data).

The validity criteria of OECD 203 were fulfilled, and the study was considered reliable and adequate for the environmental hazard assessment.

Description of key information

96h-LC50 (Pimephales promelas, mortality) = 2.43 mg/L (mean measured, OECD 203, 2003)

Key value for chemical safety assessment

LC50 for freshwater fish:
2.43 mg/L

Additional information

A reliable key study was performed according to OECD 203 and in compliance with GLP (RL1, 2003). The test was performed under semi-static conditions with a control, a solvent control and five concentrations of test substance. Nominal concentrations of 3-chloro-o-xylene were: 0 mg/L (control and solvent control), 0.91, 1.5, 2.5, 4.2, and 7.0 mg/L. Mean measured concentrations for the test concentrations were: 0.757, 1.37, 2.41, 4.06, and 6.71 mg/L. Exposure of fathead minnows to 3 -chloro-o-xylene resulted in a 96h-LC50 of 2.43 mg/L, with a 95% confidence interval of 2.05 to 2.87 mg/L. The 96h-NOEC was 0.757 mg/L 3 -chloro-o-xylene, the lowest tested concentration (the concentration allowed 100% survival with no sublethal effects).