Anisole

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study performed per OECD Guideline 201 (1998) following GLP. However, two deviations were reported during the study: 1) 2 of 4 shakers had malfunctioned between day 0 and 1; 2) 3 day old cultures were not of sufficient cell density, so 7 day old cultures were used. Additionally to these deviations, the algal biomass in each flask has been determined at 24h and 72h only, although the guideline requires that it should be performed at least daily. No justification of the absence of measurement at 48h is reported. This can have an impact on the validity of the study because daily measurements are required for the determination of the section-by-section growth rates and the evaluation of the validity criterion for the daily growth rate. Therefore, there are uncertainties on how this validity criterion of has been checked. Moreover, based on Kruskal Wallis Test, no significant reduction in growth rate was observed in any of the treatment levels tested, therefore the NOEC was determined to be 100 a.i. mg/L. However, since 100% inhibition of growth rate was observed at this treatment level (100 a.i. mg/L), it is very surprising that the statistical analysis do not conclude on a significant difference from control. Therefore, there are uncertainties on the validity/suitability of the control, and consequently on the results. Based on the above points, there are uncertainties on the validity of this study. Therefore, it is assigned a reliability score 4 (not assignable) and it is used in a weight of evidence approach with a published data.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Two deviations were reported during the study: 1) 2 of 4 shakers had malfunctioned between day 0 and 1; 2) 3 day old cultures were not of sufficient cell density, so 7 day old cultures were used. Neither deviation had a negative impact on the study.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

At test initiation (0 hour), 24 hours and test termination (72 hours), a single sample was removed from each test concentration and the controls and analyzed for Anisole. Samples analyzed at 0 hour were removed from the replicate vessels established for this purpose. Samples analyzed at 24 and 72 hours were collected prior to performing cell counts from the replicate vials established for this purpose.

Since the sampling intervals were not equally spaced (e.g., 0, 24 and 72 hours), a time weighted average was calculated for each treatment level.
At 72 hours of exposure, a sample was removed from one of the two replicate vessels of the 16 mg a.i./L test concentration, which did not contain algae. The result of this analysis was compared with that obtained for the 72-hour analyses of the 16 mg a.i./L solution containing algae to assess the impact of algae on the test substance concentration.

Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.

Vehicle:

no

Details on test solutions:

Based on the results of preliminary testing conducted at Springborn Smithers and in consultation with the Study Sponsor, the nominal concentrations selected for the definitive test were 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L.

A 100 mg a.i./L primary stock solution was prepared prior to test initiation by placing 0.2060 g (0.1999 g as active ingredient) of Anisole in a 2000 mL volumetric flask and bringing it to volume with AAP medium. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following mixing using a Teflon®- coated stir bar and magnetic plate for 10 minutes. Nominal test concentrations were prepared from dilutions of the 100 mg a.i./L primary stock solution.

Replicate approximately 60-mL Volatile Organic Analysis (VOA) vials, 11 for the 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L treatment levels, and 21 for the control were conditioned prior to use by rinsing with untreated algal medium. Each vial was filled to approximately 90% of the final volume with algal medium. Exposure solutions were individually prepared by adding the appropriate amount of the 100 mg a.i./L stock solution to achieve the desired nominal concentration. Additional untreated AAP medium was used for the control. Algal inoculum was then added to yield an initial cell density of approximately 1.0 x 104 cells/mL. Additional AAP media was added to fill the vial to capacity (i.e., zero headspace) and then shaken, after which all solutions were observed to be clear and colorless with no visible undissolved test substance. After each vial was completely filled, the total volume was 65 mL. The replicate vials for the control were maintained under the same conditions as the treatment level vials but contained no anisole. Each test vial was labeled with the test concentration, replicate, test species and study number. One additional replicate for each treatment level was established to serve as a replacement in the event that any of the vessels were cracked or broken during exposure due to increased pressure in the test vessels. All test vials were covered with Teflon®-lined screw caps to minimize the potential for volatilization of the test substance. Three replicates per treatment level and six replicates for the control were opened and discarded after daily observation intervals. The remaining two replicates per treatment level and controls were used for analytical and water quality measurements at test initiation.

In order to estimate the impact that the presence of algal biomass had on the test substance concentration, two additional replicates (L and N) of the 16 mg a.i./L nominal treatment level were prepared. These replicates were not inoculated with algae. One of the replicates was analyzed after 72 hours of exposure for Anisole concentration. The results of this analysis was compared with the 72-hour results for the 16 mg a.i./L solution containing algae. The second replicate was designed to serve as a replacement, if necessary.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The alga used in this toxicity test was the freshwater green alga Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), strain 1648, Class Chlorophyceae. The alga was obtained from University of Texas, Austin, Texas and was maintained in stock culture at Springborn Smithers Laboratories culture facility. The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.
If necessary, the pH of the culture medium was adjusted to approximately pH 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide. Stock cultures were grown in 250 mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.

The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 degrees C and continuous illumination at the surface of the medium with an intensity range of 4500 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber.

The inoculum used to initiate the toxicity test with anisole was taken from a stock culture that had been transferred to fresh medium seven days before testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23-24 degrees C

pH:

7.9-8.0

Nominal and measured concentrations:

Nominal: 1.0, 2.6, 6.4, 16, 40 and 100 mg a.i./L
Time-Weighted Average Concentrations: 0.84, 2.0, 4.9, 12, 30 and 75 mg a.i./L

Details on test conditions:

The test was conducted in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 23 ± 2 degrees C, continuous light intensity of 4400 to 5900 lux (420 to 550 footcandles) and Photosynthetically-Active Radiation (PAR) range of 60 to 120 µE/m2/s. Four orbital shaker tables provided a shaking rate of 100 ± 10 rpm.

Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test vials in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured at four locations around the perimeter of the test area with a VWR Traceable light meter at 0-hour and at each 24 hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-Active Radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189 photometer and sensor model LI-190SA. Test vessels were randomly placed on the shaking tables at test initiation based on computer-generated random numbers. Following each observation interval, the test vessels were assigned new random positions based on computer-generated random numbers.

Water quality parameters (pH and conductivity) were measured at test initiation, and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the replicate test solutions established for this purpose. At test termination, after cell counts were completed, replicate solutions for each treatment and control were respectively composited for pH and conductivity measurements. Test solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 pH meter, and conductivity was measured with a YSI Model No. 33 salinity-conductivity-temperature meter.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. (20-22)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. (29-32)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.I. (44-50)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I.(14-16)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I. (18-20)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% C.I. (28-33)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

The results of the analysis of the exposure solution for Anisole concentration established that measured concentrations at each sampling interval (i.e., 0, 24 and 72 hours) were generally stable throughout the exposure and followed the expected concentration-gradient. Since the time intervals were not equally spaced (e.g., 0, 24 and 72 hours) the mean measured concentrations were calculated as a time weighted average. The mean measured concentrations (as time weighted average) ranged from 74 to 84% of nominal concentrations and defined the treatment levels tested as 0.84, 2.0, 4.9, 12, 30 and 75 mg a.i./L.

The analytical results of the 72-hour sample from the 16 mg a.i./L nominal treatment level, with algae present, was 12 mg a.i./L. The equivalent test solution without algae present resulted in a recovery of 14 mg a.i./L at the 72-hour sample, indicating that the presence of algae had a slight impact on the concentration of Anisole in the test solution.

Percent recoveries of 8 of the 9 QC samples were consistent with the predetermined recovery range and ranged from 84.5 to 111% of the nominal concentrations (1.00, 24.5 and 105 mg a.i./L). Based on these results, it was determined that the appropriate quality control was maintained during the analyses of the exposure solutions. One of the QC samples at the 72-hour sampling interval had a recovery that was outside of the acceptable limits for this study (i.e., 124%). QC samples can be out of range due to a number of factors, some of which are spiking, handling and instrument errors.
Cell densities were determined at each observation interval. At test termination, cells exposed to the tested treatment levels and the control were observed to be normal. The 72 hour cell density in the control averaged 29.50 x 104 cells/mL. Cell densities in the 0.84, 2.0, 4.9, 12, 30 and 75 mg a.i./L treatment levels averaged 31.42, 30.58, 35.08, 33.42, 16.08 and 1.00 x 104 cells/mL, respectively.

Cell biomass was expressed as yield. The 0- to 72-hour yield in the control averaged 28.50 x 104 cells/mL. The 0- to 72-hour yield in the 0.84, 2.0, 4.9, 12, 30 and 75 mg a.i./L treatment levels averaged 30.42, 29.58, 34.08, 32.42, 15.08 and 0.00 x 104 cells/mL, respectively. Based on Kruskal Wallis’ Test, no significant reduction in yield was observed in any of the treatment levels tested, therefore the NOEC was determined to be 100 mg a.i./L. However, since 100% inhibition of yield was observed at this treatment level (100 mg a.i./L), a more conservative estimate of the NOEC is the EyC10 value (i.e., 15 mg a.i./L) as recommended by the OECD 201 Guideline. The 72 hour EyC10 value was determined to be 15 mg a.i./L with 95% confidence intervals of 14 to 16 mg a.i./L. The 72-hour EyC20 value was determined to be 19 mg a.i./L with 95% confidence intervals of 18 to 20 mg a.i./L. The 72-hour EyC50 value was determined to be 30 mg a.i./L with 95% confidence intervals of 28 to 33 mg a.i./L.

The 0- to 72-hour growth rate in the control averaged 1.13 days-1. The 0- to 72 hour growth rate in the 0.84, 2.0, 4.9, 12, 30 and 75 mg a.i./L treatment levels averaged 1.16, 1.15, 1.19, 1.18, 0.93 and -0.03 days 1, respectively. Based on Kruskal Wallis’ Test, no significant reduction in growth rate was observed in any of the treatment levels tested, therefore the NOEC was determined to be 100 mg a.i./L. However, since 100% inhibition of growth rate was observed at this treatment level (100 mg a.i./L), a more conservative estimate of the NOEC is the ECr10 value (i.e., 21 mg a.i./L) as recommended by the OECD 201 Guideline. The 72 hour ErC10 value was determined to be 21 mg a.i./L with 95% confidence intervals of 20 to 22 mg a.i./L. The 72-hour ErC20 value was determined to be 30 mg a.i./L with 95% confidence intervals of 29 to 32 mg a.i./L. The 72-hour ErC50 value was determined to be 47 mg a.i./L with 95% confidence intervals of 44 to 50 mg a.i./L.

Validity criteria fulfilled:

yes

Conclusions:

With respect to yield, the NOEC was determined to be 100 mg a.i./L; however, since 100% inhibition was observed at this treatment level, a more conservative estimate is the the EC10 value of 15 mg a.i./L. The EC50 value for yield is 30 mg a.i./L. With respect to growth rate, the NOEC was determined to be 100 mg a.i./L; however, as mentioned above, due to the 100% inhibition, a more conservative estimate is the EC10 value of 21 mg a.i./L. The EC50 value for growth rate is 47 mg a.i./L.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to standardised method, but details on results are not reported.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

conductivity of water, without incidence

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Anisole was solubilized by mixing with an equal amount of acetone and then dispersed in test medium using an ultrasound water bath.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): culture collection of the Norwegian Institute of Water Research (NIVA)
Algal material for inoculation in growth tests and for incubation in 14C-assimilation tests was taken from exponentially growing growth test control cultures (pre-cultures) and inoculated/incubated at a cell density of 1-1.1x10E4 cells/mL as calculated from the cell number of the precultures and the dilution. Cell numbers were determined by means of a Coulter Counter model FN with capillary diameter of 70µ.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

4.1 mg/L CaCO3

Test temperature:

20°C

pH:

Adjusted to 7 - 7.5

Nominal and measured concentrations:

6 concentrations, geometrically spaced.

Details on test conditions:

TEST SYSTEM
- Test vessel: 1-L Kluyver flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: no additionnal data
- Aeration: yes, continuously with 0.8% CO2 in air (filtered through a ceramic filter), the air flow rate was 0.5 L/min
- Initial cells density: 1 - 1.1x10E4 cells/mL
- Control end cells density: The test system allowed exponential growth in control cultures for 4 days and sustained growth for 6 days.

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: 10% Z8 medium modified by the Norwegian Institute for Water Reasearch (NIVA)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The medium was made up using high quality deionized water obtained fresh from a Millipore Super Q system and autoclaved before use.
It contains 0.37 mg/L of Na2EDTA.2H2O and is a softwater medium with a hardness of 4.1 mg/LCACO3 equivalents. It is prepared with 2.1 mg/L of Na2CO3.

OTHER TEST CONDITIONS
- Sterile test conditions: no data
- Adjustment of pH: yes, once a day, and did never exceed pH 8.
- Light intensity and quality: the light source was a rack of horizontally arranged Philips TL 20 W/33 cool white flourescent lamps. The incident light intntensities at the surface of test vessels are measuredwith a LI-COR LI-185 quantameter and a LI-192S underwater sensor (Lambda Instruments) were 414 ± 27 µE/m²/s (approx. 15 klux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Biomass was measured once a day as optical density at 600 nM in 5 cm cuvettes.
The response was expressed as the specific growth rate calculated by linear regression of the initial (4 days) linear part of the growth curve, as presented in a semilogarithmic plot.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: yes, geometric, no more data

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

162 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC90

Effect conc.:

400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The specific growth rate of control cultures in growth inhibition tests was 1.23 +/- 0.006 d-1.


The study presented a direct comparison of the performance of a short term 14C-assimilation test (6 hours) with the longer term growth test (here 4 days) using the same biological material, Pseudokirchneriella subcapitata, and similar test conditions.The results of the 14C-assimilation test were the following:
EC50 (6h): 230 mg/L
EC10 (6h): 110 mg/L
It is seen that the growth inhibition test is consistently more sensitive that the short term 14C-assimilation test.

Validity criteria fulfilled:

not specified

Conclusions:

Anisole was not harmful to algae.

Executive summary:

The effect of the test item anisole on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 96h static test according to a method similar to the standardised EU method C.3.

The response was expressed as the specific growth rate calculated by linear regression of the initial (4 days) linear part of the growth curve, as presented in a semilogarithmic plot. The test system allowed exponential growth in control cultures for 4 days and sustained growth for 6 days.

 

The results obtained with nominal concentrations (no measurement of the concentrations were performed) were as follows:

96h-ErC10 = 36 mg/L (growth rate)

96h-ErC50 = 162 mg/L (growth rate)

 

The study presented a direct comparison of the performance of a short term 14C-assimilation test (6 hours) with the longer term growth test (here 4 days) using the same biological material, Pseudokirchneriella subcapitata, and similar test conditions.The results of the 14C-assimilation test were the following:
EC50 (6h): 230 mg/L
EC10 (6h): 110 mg/L
It is seen that the growth inhibition test is consistently more sensitive that the short term 14C-assimilation test.

 

 

Based on these results, anisole is considered as not harmful to the tested organisms.

 

Description of key information

Anisole is considered as harmful to algae with a 72hErC50 of 47 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

47 mg/L

EC10 or NOEC for freshwater algae:

21 mg/L

Additional information

Two studies are available for this endpoint.

 

The first one has been performed according to the OECD TG 201 and GLP, and showed an EC10 value for growth rate of 21 mg/L and an EC50 value for growth rate of 47 mg/L. However, two deviations were reported during the study: 1) 2 of 4 shakers had malfunctioned between day 0 and 1; 2) 3 day old cultures were not of sufficient cell density, so 7 day old cultures were used. Additionally to these deviations, the algal biomass in each flask has been determined at 24h and 72h only, although the guideline requires that it should be performed at least daily. No justification of the absence of measurement at 48h is reported. This can have an impact on the validity of the study because daily measurements are required for the determination of the section-by-section growth rates and the evaluation of the validity criterion for the daily growth rate. Therefore, there are uncertainties on how this validity criterion of has been checked. Moreover, based on Kruskal Wallis Test, no significant reduction in growth rate was observed in any of the treatment levels tested, therefore the NOEC was determined to be 100 a.i. mg/L. However, since 100% inhibition of growth rate was observed at this treatment level (100 a.i. mg/L), it is very surprising that the statistical analysis do not conclude on a significant difference from control. Therefore, there are uncertainties on the validity/suitability of the control, and consequently on the results.

Based on the above points, there are uncertainties on the validity of this study. Therefore, it is assigned a reliability score 4 (not assignable) and it is used in a weight of evidence approach with the second data.

 

The second data is a published study (Nyholm N. and Damgaard B.M., 1990), which investigated the effect of the test item anisole on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a 96‑hour static test according to a method similar to the standardized EU method C.3.

 

The response was expressed as the specific growth rate calculated by linear regression of the initial (4 days) linear part of the growth curve, as presented in a semi-logarithmic plot. The test system allowed exponential growth in control cultures for 4 days and sustained growth for 6 days.

The results obtained with nominal concentrations were as follows:

96h-ErC10 = 36 mg/L (growth rate)

96h-ErC50 = 162 mg/L (growth rate)

 

Based on the first data, anisole is considered as harmful to algae, whereas the second data showed that it is not harmful to the tested organisms. As a worst case approach, the results of the first study are considered for the classification and labeling of the substance, and for the chemical safety assessment.

 

In conclusion, anisole is considered as harmful to algae with a 72hErC50 of 47 mg/L.