Anisole

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed per OECD Test Guideline 202 (1998) following GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The pH, dissolved oxygen concentration and temperature were measured at 0 and 48 hours. Measurements at 0 hour were taken in new solutions prior to division into replicates and measurements at 48 hours were taken from a composite sample of each of the four replicates from the treatment levels and the control.

Vehicle:

no

Details on test solutions:

A preliminary test was conducted during which daphnids were exposed to nominal concentrations of 0.010, 0.10, 1.0, 10 and 100 mg a.i./L and a control in an open exposure system. Four 250-mL test vessels, each containing five daphnids and 200 mL of test solution, were established for each treatment level and a control. Following 48 hours of exposure, 100% immobilization was observed among daphnids exposed to the 100 mg a.i./L treatment level. One daphnid was not accounted for from the 10 mg a.i./L treatment level. No immobilization or adverse effects were observed in the remaining treatment levels or control.

Based on these results, an initial definitive test was conducted during which daphnids were exposed to nominal concentrations of 6.3, 13, 25, 50 and 100 mg a.i../L and a control. Four test vessels, each containing five daphnids, were established for each treatment level and control. Following 48 hours of exposure, 100% immobilization was observed among daphnids exposed to the 100 mg a.i./L treatment level. Several daphnids exposed to the 25 mg a.i./L treatment level were observed to be on the surface of the test vessel and several were observed to be lethargic. No mortality or adverse effects were observed among daphnids exposed to the remaining treatment levels and control. The analytical recoveries of anisole were low during this exposure and it was determined that volatilization of the test substance was occurring in an open test system at these test concentrations. Therefore, preliminary and definitive exposures were repeated in a closed exposure system (with no head space).

The second preliminary test was conducted during which daphnids were exposed to nominal concentrations of 0.0010, 0.010, 0.10, 1.0 and 10 mg a.i./L and a control in a closed system (with no head space). Four test vessels, each containing five daphnids, were established for each treatment level and control. Following 48 hours of exposure, 5% immobilization was observed among daphnids exposed to the 0.010 mg a.i./L treatment level. No immobilization or adverse effects were observed among daphnids exposed to the remaining treatment levels and control. Based on these results, nominal concentrations of 4.3, 9.4, 21, 45 and 100 mg a.i./L were selected for the closed system definitive exposure study.

Test organisms (species):

Daphnia magna

Details on test organisms:

The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers Laboratories. The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD 7 resin column to remove any potential organic contaminants. This water had a total hardness and a total alkalinity range as calcium carbonate (CaCO3) of 180 mg/L and 86 to 88 mg/L, respectively, a pH range of 7.7 to 8.2, a temperature range of 20 to 21 degrees C, a dissolved oxygen concentration range of 6.7 to 9.7 mg/L, and a specific conductivity of 600 to 650 microohms per centimeter (µohms/cm).
The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light at an intensity of 49 to 67 footcandles (530 to 720 lux) at the surface of the culture solutions was provided by fluorescent bulbs. Daphnids were fed a unicellular green algae, Ankistrodesmus falcatus (4 x 10^7 cells/mL), in addition to a suspension of YCT (yeast, cereal leaves and flaked fish food) daily. Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds have been detected in the food samples at concentrations considered toxic in any of the samples analyzed. Based on these analyses, the food sources were considered to be of acceptable quality since all analyte concentrations were below limits of concern.

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L

Test temperature:

20-21 degrees C

pH:

8.3

Dissolved oxygen:

6.7-9.7 mg/L

Nominal and measured concentrations:

4.3, 9.4, 21, 45, and 100 mg a.i./L (nominal)
3.4, 7.8, 18, 38, and 85 mg a.i./L (measured)

Details on test conditions:

The number of immobilized daphnids in each replicate test vessel was recorded at 0, 24 and 48 hours of exposure. Immobilization was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel. Biological observations and observations of the physical characteristics of each replicate test solution were also made and recorded at 0, 24 and 48 hours.

The test vessels were placed in a temperature-controlled water bath designed to maintain exposure solution temperatures at 20 ± 1 °C. Due to the volatility of this material, the toxicity test was conducted in a closed exposure system with 250-mL glass flasks, each containing approximately 290 mL of test solution to avoid headspace. Four replicate test vessels were established for each concentration and the control. Each test vessel was labeled with the study number, concentration and replicate designation. The test area was illuminated with fluorescent bulbs at an intensity range of 67 to 88 footcandles (720 to 950 lux) at the solutions’ surface. Light intensity was measured with a VWR Traceable light meter. The photoperiod during the test was the same as in the culture area. Sudden transitions from light to dark and vice versa were avoided.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

27 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% C.I. of 18 to 38 mg a.i./L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

7.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: Highest concentration producing 0% toxicant related immobilization was 18 mg a.i./L; the lowest concentration producing 100% immobilization was 85 mg a.i./L

Details on results:

The water quality parameters were unaffected by the concentrations of Anisole tested and remained within acceptable ranges for the survival of daphnids. Daily measurement of the temperature in the test solutions and continuous temperature monitoring established that the exposure solution temperature ranged from 20 to 21 degrees C during the definitive study.

Following 48 hours of exposure, 0, 0, 0, 95 and 100% immobilization was observed among daphnids exposed to the 3.4, 7.8, 18, 38 and 85 mg a.i./L treatment levels, respectively. No immobilization or adverse effects were observed among daphnids exposed to the control. Surviving daphnids exposed to the 18 and 38 mg a.i./L treatment levels were observed to be on the bottom of the test vessel.

Based on mean measured concentrations of Anisole, the 48-hour EC50 value for daphnids was determined by binomial probability to be 27 mg a.i./L with 95% confidence intervals of 18 to 38 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be 7.8 mg a.i./L. The highest concentration producing 0% toxicant related immobilization was 18 mg a.i./L and the lowest concentration producing 100% immobilization was 85 mg a.i./L.

Results with reference substance (positive control):

A 24-hour reference test was conducted with daphnids (< 24 hours old) from the laboratory culture. The reference test is used to evaluate the sensitivity of the daphnid population at Springborn Smithers. The reference toxicant used, potassium dichromate, was purchased from a commercial supplier and is, therefore, characterized by its label. The 24 hour reference test results established that the 24-hour EC50 value for Daphnia magna and potassium dichromate was 3.1 mg/L. This result was within the expected range for Daphnia magna exposed to potassium dichromate.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations, the 48-hour EC50 value for Daphnia magna and Anisole was determined to be 27 mg a.i./L, with 95% confidence intervals of 18 to 38 mg a.i./L.  The No-Observed-Effect Concentration (NOEC) was determined to be 7.8 mg a.i./L.  The highest concentration producing 0% toxicant related immobilization was 18 mg a.i./L.  The lowest concentration producing 100% immobilization was 85 mg a.i./L.

Description of key information

48h EC50 (daphnia magna) = 27 mg/L (OECD TG202, GLP)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

27 mg/L

Additional information

One reliable key study is available for Anisole for this endpoint. In this acute toxicity study (Springborn Smithers Laboratories, 2010), the acute immobilization (EC50) of the test item to daphnia magna was determined according to the OECD Guideline 202 and the GLP. The study was conducted under static conditions in closed exposure system over a duration of 48 hours. 20 test organisms were exposed to each test concentration and control. The nominal tested concentration were 4.3, 9.4, 21, 45 and 100 mg/L of test item. The stability of these concentration were checked and the mean measured concentrations obtained were , respectively, 3.4, 7.8, 18, 38 and 85 mg/L of test item. The water quality parameters were unaffected by the concentrations of Anisole tested and remained within acceptable ranges for the survival of daphnids.

Following 48 hours of exposure, 0, 0, 0, 95 and 100% immobilization was observed among daphnids exposed to the 3.4, 7.8, 18, 38 and 85 mg a.i./L treatment levels, respectively. No immobilization or adverse effects were observed among daphnids exposed to the control. Surviving daphnids exposed to the 18 and 38 mg a.i./L treatment levels were observed to be on the bottom of the test vessel.

Based on mean measured concentrations of Anisole, the 48-hour EC50 value for daphnids was determined by binomial probability to be 27 mg a.i./L with 95% confidence intervals of 18 to 38 mg a.i./L. The No-Observed-Effect Concentration (NOEC) was determined to be 7.8 mg a.i./L. The highest concentration producing 0% toxicant related immobilization was 18 mg a.i./L and the lowest concentration producing 100% immobilization was 85 mg a.i./L.

Based on the results of this study, Anisole is considered as harmful to the aquatic organisms tested.